Functional and Structural Study of Pannexin1 Channels

Transcription

1 University of Miami Scholarly Repository Open Access Dissertations Electronic Theses and Dissertations Functional and Structural Study of Pannexin1 Channels Junjie Wang University of Miami, Follow this and additional works at: Recommended Citation Wang, Junjie, "Functional and Structural Study of Pannexin1 Channels" (2009). Open Access Dissertations This Open access is brought to you for free and open access by the Electronic Theses and Dissertations at Scholarly Repository. It has been accepted for inclusion in Open Access Dissertations by an authorized administrator of Scholarly Repository. For more information, please contact

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3 UNIVERSITY OF MIAMI FUNCTIONAL AND STRUCTURAL STUDY OF PANNEXIN1 CHANNELS By Junjie Wang A DISSERTATION Submitted to the Faculty of the University of Miami in partial fulfillment of the requirements for the degree of Doctor of Philosophy Coral Gables, Florida May 2009

4 2009 Junjie Wang All Rights Reserved

5 UNIVERSITY OF MIAMI A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy FUNCTIONAL AND STRUCTURAL STUDY OF PANNEXIN1 CHANNELS Junjie Wang Approved: Gerhard Dahl, M.D. Professor of Physiology and Biophysics Terri A. Scandura, Ph.D. Dean of the Graduate School Kenneth Muller, Ph.D. Professor of Physiology and Biophysics Wolfgang Nonner, M.D. Professor of Physiology and Biophysics John Bixby, Ph.D. Professor of Molecular and Cellular Pharmacology Hong-Bo Zhao, Ph.D. /M.D. Associate Professor of Surgery University of Kentucky

6 JUNJIE WANG (Ph.D., Physiology and Biophysics) Functional and Structural Study of Pannexin1 (May 2009) Channels Abstract of a dissertation at the University of Miami. Dissertation supervised by Professor Gerhard Dahl No. of pages in text. (85) Pannexins are vertebrate proteins with limited sequence homology to the invertebrate gap junction proteins, the innexins. However, in contrast to innexins and the vertebrate connexins, pannexins do not form gap junction channels. Instead they appear to solely function as unpaired membrane channels allowing the flux of molecules, including ATP, across the plasma membrane. We provided additional evidence for their ATP release function by demonstrating that the connexin mimetic peptides, which were thought to inhibit ATP release through connexin channels, do not inhibit their host connexin channels but instead inhibit pannexin1 channels by a mechanism of steric block. Therefore, the inhibitory effects of mimetic peptides on ATP release may represent supporting evidence for a role of pannexin1 in ATP release. We also analyzed the pore structure of pannexin1 channels with the Substituted Cysteine Accessibility Method. The thiol reagents MBB and MTSET reacted with several positions in the external portion of the first transmembrane segment and the first extracellular loop. In addition, MTSET reactivity was found in the internal portion of TM3. These data suggest that portions of TM1, E1 and TM3 line the pore of pannexin1 channels. Thus, the pore structure of pannexin1 is similar to that of connexin channels.

7 Acknowledgements I would like to give my sincere thanks to the people who made this thesis possible: my wife Haoming Liu, my advisor Dr. Gerhard Dahl, my committee members, including Dr. John Bixby, Dr. Kenneth Muller, Dr. Wolfgang Nonner, Department of Physiology and Biophysics. iii

8 TABLE OF CONTENTS Page LIST OF FIGURES... LIST OF TABLES... vi viii Chapter 1 BACKGROUND Gap Junction Function of unpaired connexons and innexons Calcium Wave Propagation Pannexin1 Acts as an ATP Release Channel Connexin Mimetic Peptide Pore Structure MATERIALS AND METHODS Plasmid Mimetic Peptides Mutagenesis Synthesis of mrna Preparation of Oocytes Electrophysiological techniques Thiol Reagent Dye uptake Data Analysis ATTENUATION OF PANNEXIN1 CHANNELS BY MIMETIC PEPTIDES Do Connexin Mimetic Peptides Inhibit Connexin Currents? Do Connexin Mimetic Peptides Inhibit Pannexin1 Currents? Do Pannexin Mimetic Peptides Inhibit Pannexon Currents? Do Mimetic Peptides Inhibit Pannexon Currents by Steric Block? Do Mimetic Peptides Inhibit Dye Uptake Through Pannexons? THE PANNEXIN1 PORE: PORE LINING SEGMENTS AND RESIDUES Cysteine at Position 426 in Pannexin1 are Reactive to MBB Several Positions in TM1 and E1 are Reactive to MBB No Positions in TM2 are Reactive to MBB No Positions in TM3 are Reactive to MBB No Positions in TM4 are Reactive to MBB Several Positions in TM1 and E1 are also Reactive to MTSET No Positions in the Inner Portion of TM2 are Reactive to MTSET iv

9 4.8 Two Positions in the Inside Portion of TM3 are Reactive to MTSET No Positions in the Inner Portion of TM4 are Reactive to MTSET Several Positions at the Carboxyl Terminus are Reactive to MBB Summary CONCLUSION AND DISCUSSION The Inhibitory Effect of Mimetic Peptide does not Indicate the Involvement of Connexons but Instead Pannexin1 Channels in ATP Release Mimetic Peptides Work on Pannexin1 Channels through Steric Block Pannexin1 Channels Share Similar Pore Structure with Connexons The end of CT May form the Plug in the Vestibule in the Density Map General Assumptions Used in Cysteine Scanning APPENDIX I SITE DIRECTED MUTAGENESIS APPENDIX II TRANSFORMATION OF SUPER COMPETENT CELLS APPENDIX III PURIFICATION OF PLASMID DNA FROM E. coli CULTURE 76 APPENDIX IV TRANSCRIPTION OF mrna REFERENCES v

10 LIST OF FIGURES Figure 1.1 Formation of a gap junction channel Figure 1.2 Molecular organization of a C terminus truncated Cx 43 gap junction channel Figure 1.3 Topology of connexins or innexins... 4 Figure 1.4 Alignment of human pannexin 3 with leech innexin Figure 1.5 Gating properties of gap junction channels by voltage... 7 Figure 1.6 Immunofluorescent localization of Cx Figure 1.7 Immunofluorescent staining of pannexin1 in cultured tracheal epithelial cells 9 Figure 1.8 A general hypothesis about the function of gap junctional proteins Figure 1.9 Pannexin1 channel mediated calcium wave propagation Figure 1.10 Connexin peptide Gap27 reduced calcium wave propagation induced by mechanical stimulation in airway epithelial cells Figure 2.1 The chemical reaction between MBB and the thiol group of a cysteine Figure Gap26 inhibited the formation of Cx32E 1 43 gap junction channels Figure Gap26 did not acutely inhibit Cx32E 1 43 currents Figure Gap24 did not acutely inhibit Cx32E 1 43 currents Figure Gap24 acutely inhibited pannexin1 currents Figure Gap27 acutely inhibited pannexin1 currents Figure 3.6 Pannexin mimetic peptides acutely inhibited pannexin1 currents Figure panx1 inhibited Cx46 channels Figure 3.8 Size dependent inhibitions of pannexin1 currents by PEGs Figure 3.9 PEG1500 acutely inhibited pannexin1 currents Figure 3.10 The inhibition level of 1mM PEG1500 is similar to that of 200µM 32 Gap Figure 3.11 The inhibition effects of scrambled versions of 32 Gap24 and 10 panx1 on pannexin1 currents Figure 3.12 Dose response curves of pannexon inhibition by peptides and PEG Figure 3.13 Effect of peptides and PEG molecules on dye uptake in oocytes expressing pannexin1 and Cx32E Figure 4.1 Predicted topology of mouse Pannexin vi

11 Figure 4.2 MBB effects on WT and C426S pannexin1 currents Figure 4.3 Inhibitory effects of MBB on membrane currents carried by TM1 cysteine mutant channels Figure 4.4 Quantitative analysis of inhibition of MBB on membrane currents carried by channels formed by cysteine mutants in TM Figure 4.5 Quantitative analysis of inhibition effect of MBB on TM3 mutant currents 45 Figure 4.6 Quantitative analysis of inhibition of MBB on TM4 mutant currents Figure 4.7 Schematic demonstration of binding between cysteine and thiol reagents 47 Figure 4.8 Inhibitory effects of MTSET on membrane currents carried by channels formed by TM1 cysteine mutants Figure 4.9 the effects of various thiol reagents on F54C currents Figure 4.10 Quantitative analysis of inhibition of membrane currents carried by channels formed by cysteine mutants in the inside portion of TM2 by MTSET Figure 4.11 Inhibitory effects of MTSET on membrane currents carried by channels formed by cysteine mutants at the inside portion of TM Figure 4.12 Quantitative analysis of inhibition of membrane currents carried by channels formed by cysteine mutants in the inside portion of TM4 by MTSET Figure 4.13 Quantitative analysis of inhibition of membrane currents carried by channels formed by cysteine mutants in the Carboxyl terminus by MBB Figure 4.14 Summary of quantitative analysis of inhibition of membrane currents carried by channels formed by cysteine mutants in all four transmembrane domains by MBB and MTSET Figure 4.15 Summary of the positions with thiol reagent reactivity or loss of function 56 Figure 5.1 The structural views of gap junction channels composed by Cx43L263Δ and Cx26M34A Figure 5.2 Mapping the thiol reagent reactivity into pore structure vii

12 LIST OF TABLES Table 3.1 The sizes of effective mimetic peptides Table 5.1 List of publications where gap junction mimetic peptides were used in channel identification viii

13 Chapter 1 Background 1.1 Gap Junction channels Gap junction channels are pores interconnecting two neighboring cells. Each gap junction channel is composed of two hemichannels residing in apposing cell membranes, which interact to form a hydrophilic path between two cells. Gap junctions are ubiquitous in multicellular animals and establish direct intercellular communication between neighboring cells. Molecules with mass up to 1000 daltons can be exchanged by diffusion through gap junction channels. These molecules include metabolites, ions, second messengers and water (Kumar and Gilula 1996; Alexander and Goldberg 2003). Through gap junctions, electrical and metabolic activities in the neighboring cells are synchronized. A well-known function of gap junction channels is the electrical synchronization of myocardial cells in heart. Gap junctions also mediate strongly synchronized activity of the myometrical smooth muscle cells in the expulsion of the fetus during birth. Gap junctions are also reported to be involved in tissue inflammation and repair, cross presentation in the immune system, neocortical GABAergic neuron networking, neuronal synchronization in the mammalian olfactory bulb, and hearing sensitivity in the inner ear (Chanson et al. 2005; Neijssen et al. 2005; Hestrin and Galarreta 2005; Zufall 2005; Zhao et al. 2005). Gap junction channels are important for multicellular communication. Knockout of gap junction proteins in vertebrate (mouse) and invertebrate (C. elegans and fruit fly) induces a variety of defects in the heart, nervous system, liver, lens and muscles (Spray et al. 2000; Phelan and Starrich 2001). Vertebrate gap junctions are formed by a family of proteins, called connexins. There are 21 connexins in the human genome and each is 1

14 2 identified by a number, which stands for the predicted molecular mass in kilo daltons (kda). For example, the major gap junction protein in heart with a mass of 43 kda is called connexin 43 (Cx43). In humans, the mutations of gap junction proteins Cx26 or Cx30 are linked with deafness and epidermal disease, Cx32 mutations with congenital demyelinating neuropathy, and Cx50 mutations with congenital cataracts (Kelsell et al. 2001) Structural Basis of Gap Junctions Six subunits form a hemichannel (also known as a connexon) in the cell membrane that can dock to another hemichannel in the plasma membrane of an adjacent cell. The joined hemichannels form a contiguous intercellular path, the gap junction channel (Figure 1.1). Individual hemichannels typically are closed in physiological conditions. The whole gap junction channel opens when two hemichannels dock to each other. Figure 1.1 Formation of a gap junction channel. One hemichannel is composed of six subunits, of which only 3 are shown in this cutaway diagram. Typically the undocked hemichannels are closed. When two hemichannels meet each other, the interaction of extracellular loops changes the conformation of the proteins and forms an open gap junction channel.

15 3 Figure 1.2 Molecular organization of a C terminus truncated Cx 43 gap junction channel. (a) A full side view is shown, and (b) the density has been cropped to show the channel interior. M represents the membrane bilayers; E represents extracellular gap; C represents cytoplasmic space. The white arrows identify the locations of the cross sections (c) that are parallel to the membrane bilayers. The red asterisk in (b) marks the narrowest part of the channel (Unger et al. 1999). A three dimensional structure of a C-terminus truncated Cx43 gap junction was derived from electron cryomicroscopy data (Figure 1.2 Unger et al. 1999). The resolution is 7.5 Ångstroms in the membrane plane and 20 Ångstroms in the normal direction. Figure 1.2 a and b show the side views of a gap junction channel composed of two opposing Cx43 connexons (hemichannels). Figure 1.2c shows the cross sections parallel to the membrane plane, consisting of 24 transmembrane domains contributed by 6 subunits. The narrowest part of the pore is marked by the red asterisk and is located just outside the membrane. It has a diameter of about 15 Å. The pore is wider in the central position, corresponding to the extracellular part of the proteins where a tight seal excludes the exchange of substances between the interior pore and the extracellular milieu. The pore is the widest in the cytoplasmic mouth. The three dimensional structure of Cx26 constructed by similar methods suggested a similar structure for Cx26 gap junctions (Oshima et al. 2007).

16 4 Figure 1.3 Topology of connexins or innexins. E1: extracellular loop 1; E2, extracellular loop 2; CL, cytoplasmic loop; NT, amine terminus; CT, carboxyl terminus Molecular Composition of Gap Junctions: Connexins and Innexins Two families of proteins have been identified to be gap junction subunits: connexins and innexins. There is no significant sequence similarity between connexins and innexins. Connexins are confined to chordate lineages. There are no connexin homologs in the genomes of the arthropod Drosophila and the nematode C. elegans (Phelan 2005). In these organisms, as in other protostomes, gap junctions instead are formed by innexins (Eiberger et al. 2001; Phelan and Starich 2001). About 22 connexins and 25 innexins are identified to form gap junctions in vertebrates and in invertebrates. Connexins have 4 transmembrane domains and two extracellular loops with cytoplasmic carboxyl terminus and amine terminus (Figure 1.3; Yeager and Harris 2007). Connexins have three conserved cysteines in each of the two extracellular loops. Based on hydrophobicity plots, innexins are thought to have a similar membrane topology as connexins. This includes conserved cysteines in the two extracellular loops of which there are, however, only two in each loop.

17 5 Innexins had been thought to be confined to invertebrates, but this assertion was challenged by the identification of pannexins in the human and mouse genomes (Panchin et al. 2000). Like innexins, pannexins have four transmembrane domains and 2 pairs of conserved cysteines in two putative extracellular loops predicted by hydrophilicity analysis. Further sequence analysis showed that three conserved motifs were shared by innexins and pannexins (Figure 1.4 Ren and Saier 2007). Motif I showed 37% identity and 58% similarity between leech innexin2 and human pannexin3; motif II showed 39% identity and 61% similarity; and motif III showed 20% identity and 45% similarity. The extracellular localization of the first loop and the intracellular localization of the carboxyterminus of pannexin1 were corroborated by immunohistochemistry with peptide-specific antibodies (Locovei et al. 2006b). Furthermore, the extracellular localization of the predicted second extracellular loop was confirmed by identification of glycosylation site in this segment of pannexin1 (Boassa et al. 2007). Thus, the topology of pannexin1 is confirmed. Together, these data supported the idea that innexins in invertebrates and pannexins in vertebrates belong to the same family. Originally, innexins were named from invertebrate connexins. Once they were also found in vertebrates rather than confined to invertebrates, the name pannexin was proposed to cover the invertebrate and vertebrate branches of the protein family. However, in this report, we still use innexins in invertebrate and pannexins in vertebrate because of functional differences between the two branches. Mainly, in contrast to innexins, pannexins do not form gap junction channels in vivo.

18 6 Figure 1.4 Alignment of human pannexin 3 (top sequence) with leech innexin 2 (bottom sequence). Numbers indicate residue positions in the proteins. I, identity; :, close similarity;., distant similarity. (Ren and Saier 2007) Regulation of Gap Junctions Homotypic gap junctions are defined as composed of two identical hemichannels, each containing identical subunits, heterotypic gap junction channels as composed of two different hemichannels, each containing identical subunits, and heteromeric gap junctions as composed of hemichannels that contain more than one type of subunit. The extent of transfer across gap junctions depends on the conductance state and the number of gap junction channels in the junction. Three variables known to affect conductance states of all gap junction channels are: transjunctional voltage, intracellular

19 7 ph and intracellular calcium. However, the sensitivity of the various connexins to these variables differs. Thus, due to the various types of gap junction subunits and various combinations like homotypic, heterotypic or heteromeric, the gap junction channels can display a wide range of gating properties and selectivity properties. For example, the conductance of frog Cx38 gap junctions decreases when the transjunctional voltage increases, while human Cx43 gap junctions show no dependence on transjunctional voltage (Figure 1.5A). Frog Cx38 and human Cx43 form heterotypic gap junctions with rectified transjunctional voltage dependence (Figure 1.5B). Figure 1.5 Gating properties of gap junction channels by voltage. (A) Normalized g -V (conductance VS voltage) relation of human Cx43 homotypic gap junction ( ) and Xenopus Cx38 homotypic junction ( ). (B) Normalized g V relation of Cx43/Cx38 heterotypic junction (Werner et al. 1989). Normalization is to the conductance at minimal transjunctional voltage. Polarity refers to the potential in the Cx38 expressing cell. ph sensitivity has been tested in various cell types during the past two decades. In virtually all cells cytosolic acidification decreases gap junction conductance although the sensitivity to ph varies among connexins or innexins. A convenient way to induce cytoplasmic acidification is to expose cells to solutions gassed with 100% CO 2. This procedure uncouples cells, including insect gland cells, pancreatic acinar cells, crayfish axons, embryonic chick lens and cardiac cells (Peracchia 2004).

20 8 Like cytosolic acidification, intracellular calcium (> 1 M) can decrease connexin gap junction conductance and uncouple the cells. The role of calcium in cell uncoupling is also documented for many types of cells, including cardiac cells, amphibian embryonic cells, rat lacrimal cells, crayfish giant axons, Novikoff hepatoma cells, astrocytes, lens cultured cells, pancreatic cells and acinar cells, osteoblasts, and cochlear supporting cells (Peracchia 2004). However, several connexins have been shown to be insensitive to changes in cytoplasmic calcium. 1.2 Function of unpaired connexons and innexons Connexons or innexons are composed of six connexins or innexins in the cell membrane. They are also called hemichannels, because two of them can form a complete gap junction channel by docking to each other. Connexins form gap junctions but not functional membrane channels (hemichannels) under physiological conditions. Cx43 connexons were reported to open in extreme conditions when the cells were depolarized to +50mV in low calcium conditions (Contreras et al. 2003). Lens specific connexins Cx46 and Cx50 do form membrane channels when expressed in Xenopus oocytes that can be opened by moderate depolarization. However, Cx46 and Cx50 apparently do not form open membrane channels in other cells (Li et al. 1996; Ebihara 1996). Innexins form not only gap junctions, but also membrane channels. The leech innexins (inx1, inx2, inx3 and inx6) were reported to form membrane channels that open in response to moderate depolarization (Bao et al. 2007). The channels close at resting membrane potential of -40 mv and open when the membranes are depolarized to -20 mv in oocytes.

21 9 Pannexins do not form gap junction channels. Instead, they form open pannexons on the plasma membrane. Pannexins have three members, pannexin1, pannexin2 and pannexin3. Pannexin3 was reported to form membrane channels in 293T cells but not gap junction channels (Penuela et al. 2007; Bruzzone et al. 2004). Pannexin1 was reported to form membrane channels on the surface of oocytes and to form gap junctions between paired oocytes (Bruzzone et al. 2003). The capability of pannexin1 to form membrane channels is well documented in many papers (e.g. Bao et al. 2004, Locovei et al. 2006), while its capability to form gap junctions under physiological conditions is challenged by many observations. Figure 1.6 Immunofluorescent localization of Cx32 in frozen section of rat liver (Paul 1986). Figure 1.7 Immunofluorescent staining of pannexin1 (green) in cultured tracheal epithelial cells of the mouse (Ransford et al. 2007). Cell nuclei are stained by a fluorescent dye DAPI that binds strongly to DNA (blue).

22 10 First, the expression of pannexin1 is far different from the expression of normal gap junctions. The appearance of connexin or innexin gap junctions is punctate in areas of cell-cell contact, indicating the clusterings of gap junctions between two neighboring cells. Figure 1.6 shows the punctate staining of cx32 in the rat liver (Paul 1986). The appearance of pannexin1 is diffuse rather than punctate. Figure 1.7 shows pannexin1 in cultured tracheal epithelial cells. Pannexin1 is not found between neighboring cells but in the luminal side of epithelial cells exposed to the air, indicating that pannexin1 does not form gap junctions. Second, the formation of pannexin1 gap junction channels in paired oocytes is 100 times less efficient than the formation of connexin gap junctions. The formation of pannexin1 gap junction in oocytes required 24 to 48 hours, much longer than the pairing time for connexins. For Cx46, the formation of gap junctions is detectable in 2 hours and a large number of gap junction channels form in 6 hours (Boassa et al. 2007; Boassa et al. 2008). Third, pannexin1 is glycosylated in the lumen of the Endoplasmic Reticulum, corresponding to the extracellular portion of the protein. The gycosylation of pannexin1 at its extracellular surface makes it unlikely that two pannexons dock to form gap junctions, because artificial glycosylation of connexins prevents gap junction formation. Deglycosylation of the cell surface by using N-Glycosidase F, which removes glycans from proteins, significantly enhanced the formation of pannexin1 gap junction (Boassa et al. 2008). All these lines of evidence indicate that pannexin1 may only form membrane channels rather than gap junctions in vivo. A general hypothesis about the evolution of gap junctional proteins has been proposed (Figure 1.8). In the early times of evolution, innexins in invertebrates not only formed membrane channels to help communication between the outside and the inside of cells, but

23 11 also formed gap junctions to help communication between neighboring cells. In the long history of evolution, innexins were replaced by connexins to form gap junctions, while they were retained in the form of pannexins for their ability to form membrane channels due to an indispensable physiological function. Figure 1.8 A general hypothesis about the function of gap junctional proteins. Innexins form not only membrane channels but also gap junctions in invertebrates, while the innexins homolog pannxins form membrane channels and a different family of protein connexins form gap junctions in vertebrates (Bao et al. 2007). 1.3 Calcium Wave Propagation In many tissues, a variety of stimuli, including mechanical and metabolic stress, can elicit a calcium response in one cell that can propagate from this cell to neighboring cells at the speed of about 30 m/s. This phenomenon is known as a calcium wave and its function is to coordinate the activities induced by calcium among a group of cells. Calcium wave propagation has been reported to control ciliary beating in airway epithelial cells, modulate synaptic transmission between neurons, coordinate metabolism by glial cells,

24 12 and control vascular perfusion (Sanderson et al. 1990; Newman 2001; Charles 2005; Sigurdson et al. 1993). Calcium waves have been reported to propagate through two complementary pathways: an intercellular pathway and an extracellular pathway. In the extracellular pathway, ATP is released from the excited cell and diffuses to a second cell extracellularly. ATP then binds to P2Y receptors in the second cell, which results in cytoplasmic IP3 production. This event then increases the cytoplasmic calcium concentration (Osipchuk and Cahalan 1992; Hassinger et al. 1996). In the intercellular pathway, IP3 molecules are thought to diffuse directly to the neighboring cells through gap junctions and lead to calcium responses (Sanderson et al. 1990). In calcium wave propagation, the importance of ATP release from the cytoplasm of the cells is generally recognized. ATP degradation by apyrase or purinergic receptor blockers largely reduce the propagation (Frame and de Feijter 1997). However, how ATP is released from the cell is controversial. A vesicular release mechanism has been proposed because calcium wave propagation is sensitive to Brefeldin A, which inhibits transport in the Golgi complex, and nocodazole, which interferes with vesicle trafficking to the cell surface (Maroto and Hamill 2001). ATP was also proposed to be released through channels, such as Cx43 channels, CFTR (cystic fibrosis transmembrane conductance regulator), a volume regulated channel (VRAC), and the purinergic receptor P2X7 (Bal-Price et al. 2002; Maroto and Hamill 2001; Reisin et al. 1994; Cotrina et al. 1998; Hisadome et al. 2002; Parpura et al. 2004). However, these proposed mechanisms are based on weak experimental evidence. ATP release is observed in vesicle-free erythrocytes (Bergfeld and Forrester, 1992); Cx43 does not form open hemichannels under

25 13 physiological conditions (Li et al. 1996) and astrocytes from Cx43 knockout mice do not affect calcium wave propagation (Scemes et al. 2000); CFTR is not ATP permeable (Grygorczyk et al. 1996); P2X7 alone does not lead to formation of an ATP permeable large pore in oocytes unless co-injected with macrophage mrna (Petrou et al. 1997). In the next section, I will discuss the probability of pannexin1 working as an ATP release channel in calcium wave propagation. 1.4 Pannexin1 Acts as an ATP Release Channel Many papers attribute ATP release to connexin membrane channels based on the evidence that ATP release is reduced by gap junction blockers, such as α-glycyrrhetinic acid, FFA and connexin mimetic peptides (Braet et al. 2003; Stout et al. 2002). However, there is no evidence that connexin membrane channels exist under physiological conditions. Discovery of pannexin1 membrane channels provides another possibility that pannexin1 membrane channels rather than connexin membrane channels play the role of ATP release channels. The characteristics of pannexin1 membrane channels support this argument. Pannexin1 membrane channels are opened easily by depolarization and mechanical stress; pannexin1 membrane channels are permeable to big molecules, such as ATP and carboxyfluorescein (Bao et al. 2004); pannexin1 membrane channels can be activated by cytoplasmic calcium (Locovei et al. 2006a); pannexin1 membrane channels can be blocked by general gap junction blockers, such as carbenoxolone and α-glycyrrhetinic acid (Bruzzone et al. 2005). In addition, application of ATP induces the opening of pannexin1 channels in oocytes coexpressing P2Y receptors and pannexin1, mimicking the ATP-induced ATP release in calcium wave propagation (Locovei et al. 2006a). Furthermore, pannexin1 is expressed where calcium wave propagation occurs.

26 14 Pannexin1 was found in erythrocytes and vascular endothelial cells where ATP is released from erythrocytes to signal the surrounding endothelial cells, which leads to backward calcium wave propagation in capillary to relax smooth muscle in the precapillary sphincter region to increase blood flow (Locovei et al. 2006b). Pannexin1 was also found in tracheal epithelial cells where ciliary beating is controlled by calcium wave propagation (Ransom et al. 2009). The characteristics of pannexin1 membrane channels are consistent with those of an ATP release channel in calcium wave propagation. Figure 1.9 Pannexin1 channel mediated calcium wave propagation. In this proposed model, mechanical stress or an increase of calcium concentration in the first cell opens channels; ATP molecules are released and move extracellularly to the second cell; ATP activates the purinergic receptors on the second cell and induces IP3 production that consequently increases intracellular calcium concentration. Intracellular calcium opens the pannexin1 hemichannels on the second cell and ATP is released. In this way, the calcium response can get amplified and propagated a long distance (Locovei et al. 2006a). A model was proposed to explain the function of pannexin1 membrane channels in calcium wave propagation (Figure 1.9). Initially, the pannexin1 channel is activated by mechanical stress or cytoplasmic calcium increase. ATP is released to the extracellular space through pannexin1 channels and it binds to purinergic receptors on neighboring cells and induces IP3 production. IP3 then activates calcium channels in the ER and releases calcium that activates pannexin1 membrane channels in the second cell. ATP is released from the second cell and binds to purinergic receptors in the third cell and keeps the calcium wave propagating. Open pannexin1 channels release ATP that may bind to

27 15 purinergic receptors on the same cell, which then activate the channel again and release more ATP. This is a positive feedback that may lead to cell death. There must be some negative control to avoid positive feedback, such as desensitization or inhibitory factors. Recently it has been discovered that ATP itself is a direct inhibitor for pannexin1 channels (Qiu and Dahl 2008). Figure 1.10 Connexin peptide Gap27 reduced calcium wave propagation induced by mechanical stimulation in airway epithelial cells reversibly. The cell marked by the white arrow was stimulated by mechanical stress. Fura-2 was used to indicate the cytoplasmic calcium concentration. A. absence of inhibitors. B. 60 min incubation in 190µM Gap 27 solution. C. 18 min washout of inhibitory Gap 27 peptide (Boitano and Evans 2000). 1.5 Connexin Mimetic Peptide Connexin mimetic peptides were originally designed to mimic the extracellular docking gate of the gap junction. Gap junctions are composed of two apposing hexameric connexons. Under physiological condition, connexons are closed. A gap junction forms and opens when two connexons dock to each other. The docking process involves the

28 16 interaction of two extracellular loops of the connexins (Dahl et al. 1994; Dahl et al. 1992; White et al. 1995). It was expected that these peptides would open connexons by mimicking homophilic extracellular loop interactions between two opposing connexons. The expectation, however, was not supported by experiments; application of the peptides did not open connexons. On the contrary, the peptides specifically inhibited gap junction formation (Dahl et al. 1992; Dahl et al. 1994; Warner et al. 1995). This observation gave rise to the development of a series of connexin mimetic peptides as inhibitors of gap junction formation (Evans and Boitano 2001; Kwak and Jongsma 1999). Later, connexin mimetic peptides were reported to inhibit calcium wave propagation and ATP release. 43 Gap26 and 43 Gap27, sequences from the first extracellular loop and the second extracellular loop of Cx43 respectively, were reported to inhibit calcium waves in airway epithelial cells (Boitano et al. 2000), brain endothelial cells (Braet et al. 2003), bovine corneal endothelial cells (Gomes et al. 2005; Gomes et al. 2006; D Hondt et al. 2007) and retinal pigment epithelial cells (Pearson et al. 2005). 32 Gap27, a peptide with a sequence derived from the second extracellular loop of Cx32, was also reported to inhibit ATP release in the bladder cancer epithelial cell line ECV304 (De Vuyst et al. 2006). Figure 1.10 gives one example that 43 Gap 27 inhibited calcium wave propagation in airway epithelial cells, which is reversible (Boitano and Evans 2000). Because these peptides are thought to specifically interact with host connexins, the inhibition effect was interpreted to mean that specific interactions closed the connexin membrane channels and inhibited ATP release and calcium wave propagation. However, no test for a direct effect of peptides on connexin membrane channels was performed. All readouts of the peptide effects were indirect, such as calcium wave spread, ATP release or dye uptake. Recently it was reported

29 17 that 32 Gap24, a peptide with the sequence of the cytoplasmic loop of Cx32, was also effective in attenuating the measured parameters (De Vuyst et al. 2006), which raises more doubt on the specific interaction. I, therefore, tested the effects of these mimetic peptides on identified connexin membrane channels and on pannexin1 channels. 1.6 Pore Structure Unlike sodium channels, potassium channels or calcium channels, gap junctions do not exhibit specific ion or molecule selectivity, allowing passage of ions and metabolites with masses up to 1000 Daltons. But this does not mean molecules can pass through gap junctions indiscriminately. Gap junctions formed by different connexins still display modest charge selectivity (up to 10-fold), different size restriction and even dramatic selectivity for natural metabolites (up to 100-fold) (Suchyna et al. 1999; Nicholson et al. 2000; Goldberg et al. 1999). The residues lining the pore interact with permeates and should define the selectivity. Identifying pore structure is important to understand the role of different gap junction proteins. The substituted cysteine accessibility method (SCAM) has been successfully used to identify the pore lining residues in many membrane channels, such as ACh receptors (Akabas et al. 1991), GABA receptors (Xu and Akabas 1993), and connexons (Zhou et al. 1997, Skerrett et al. 2002, Kronengold et al. 2003). When a residue in the pore of a channel is replaced by cysteine that can be bound covalently by thiol reagents, application of thiol reagents will partially block the channel that can be detected by electrophysiological recording or other means. Our lab first used this method in chimeric Cx32E 1 43 and Cx46 connexons (Zhou et al. 1997). Strong inhibition was identified in two residues I33 and M34 in the middle of transmembrane segment 1 (TM1) of Cx32E 1 43,

30 18 while moderate reactivity was identified in several positions at TM3, suggesting that TM1 and TM3 cooperate to form the pore. The structure of the carboxyl terminus truncated Cx43 gap junction constructed from cryo electron microscopy data confirmed this conclusion. The SCAM was extended from TM1 to extracellular loop 1 (E1) and identified several more pore lining residues in TM1 and E1 (Kronengold et al. 2004). In the present study, I tested the pore lining residues in pannexin1, which is justified for two important reasons. First, pore lining residues determine the selectivity and permeability that is critical to understand the role of pannexin membrane channels. Second, the pore lining residues of connexons have been studied in detail and the result in pannexin1 can be compared and contrasted with connexins, which may give some clues to the questions whether two different families of proteins have similar pore structure and whether the difference in pore structure can explain the difference in physiological functions.

31 Chapter 2 Materials and Methods 2.1 Plasmid Mouse pannexin1 was kindly provided by Dr. Rolf Dermietzel (University of Bochum), and Cx46 was obtained from Dr. D. L. Paul (Harvard University). Cx32E 1 43 was constructed in our lab by replacing the first extracellular loop of Cx32 with that of Cx43 and the generation procedure was described previously (Pfahnl et al. 1997). 2.2 Mimetic Peptide The use of the peptides 32 Gap24, 43 Gap26, 43 Gap 27, and 10 Panx1 has been published (Evans and Boitano 2001, Pelegrin and Surprenant 2006, De Vuyst et al. 2006). The sequences of 32 Gap24 is GHGDPLHLEEVKC; the sequence of 43 Gap26 is VCYDKSFPISHVR; the sequence of 43 Gap 27 is SRPTEKTIFII; Pannexin1 peptides used here have the following sequences: 10 Panx1, WRQAAFVDSY; E1a, AQEISIGTQIS; E1b, SSFSWRQAAFVDS; E1c, SESGNLPLWLHK; E2a, SSLSDEFVCSIKS; and E2b, KSGILRNDSTVPDQ. Connexin peptides 43 Gap26 and 43 Gap 27 were obtained from Evans lab; pannexin peptides are synthesized by Sigma. 2.3 Mutagenesis Site directed mutations were generated according to the protocol of QuikChange II Site-Directed Mutagenesis Kit (#200524, Stratagene, CA). Template plasmid and two complementary mutation primers were needed for the PCR reaction that amplifies the mutant edition of the plasmid. For detailed procedure, see Appendix I. 19

32 20 Mutant plasmids then were transformed into competent E. coli cells by heat shock. Transformed cells grew in LB agar plate with selective antibiotic at 37 C for 16 hours. For detailed procedure, see Appendix II. One colony was picked from selective plates and grown in LB media with antibiotic for 16 hours. The mutation plasmid was purified from E. coli cells according to the protocol of QIAprep Spin Miniprep Kit (#27104, Qiagen). For detailed procedure, see Appendix III. Purified mutation plasmids were sequenced by Genewiz Co.. Pannexin1 encoding sequence is about 1300 base pairs. The reliable sequencing data cover about 800 base pairs. One primer with sequence just before the encoding area was used to sequence the first half of the encoding area and one primer with sequence in the middle of the encoding area was used to sequence the second half of the encoding area. 2.4 Synthesis of mrna The plasmid containing Cx32E 1 43 (pgem 3Z; Promega, Madison, WI) was linearized with Ssp1 and transcribed with SP6 polymerase. The plasmid containing Cx46 (rsp64t) was linearized with EcoR1 and transcribed with SP6 polymerase. Pannexin1, in pcs2, was linearized with Not I. In vitro transcription was performed with the polymerases T3 or SP6, using the Message Machine kit (Ambion, Austin, TX). mrnas were quantified by absorbance (260 nm), and the proportion of full-length transcripts was checked by agarose gel electrophoresis. In vitro transcribed mrnas (about 40 nl) were injected into Xenopus oocytes. For detailed procedures for in vitro mrna transcription, see Appendix IV.

33 Preparation of Oocytes All procedures were conducted in accordance with the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society. Preparation of oocytes was performed as described previously (Dahl 1992). Ovaries of Xenopus laevis were cut into small pieces and incubated in 2.5 mg/ml collagenase (Worthington, Lakewood, NJ) calcium free Oocyte Ringer 2 (OR2) solution, stirring at 1 turn/s at room temperature. Typically the incubation period was 3 hours for oocytes to be separated from the follicle cells. Extensive washing by regular OR2 followed and healthy looking oocytes were selected for mrna expression. 2.6 Electrophysiological techniques After injection of mrna, the oocytes were incubated at 18 C for h in oocyte Ringer solution (in mm): 82.5 NaCl, 2.5 KCl, 1 MgCl 2, 1 CaCl 2, 1 Na 2 HPO 4, and 5 HEPES, at ph 7.5. Whole cell membrane current of a single oocyte was measured using a two-microelectrode voltage clamp (Geneclamp 500B; Axon Instruments, Sunnyvale, CA) and recorded with a chart recorder (Soltec, San Fernando, CA). One electrode records membrane potential and the other is for current injection. The electrodes are connected to a voltage clamp circuit, which allows clamping at any arbitrary membrane potential by injecting current through the current electrode. Both voltage-measuring and current-passing microelectrodes were pulled with a P-97 Flaming/Brown micropipette puller (Sutter Instruments, CA) and filled with 3M KCl. The recording chamber was perfused continuously with solution. Membrane conductance was determined using voltage pulses. Oocytes expressing Cx32E 1 43 or Cx46 were held at - 20 mv, and depolarizing pulses of 5 seconds duration and of 5 or 10 mv amplitude were applied.

34 22 Oocytes expressing pannexin1 were held at - 60 mv, and pulses to +20 mv or +60 mv were applied to transiently open the channels. These pulse protocols were chosen for stable recording conditions for the various channels. To determine the junctional conductance, devitellinized oocytes were paired. Each oocyte of a pair was voltage clamped with two intracellular electrodes, and the membrane potential was held at -50 mv. 5 mv steps were applied to one oocyte. The transjunctional current was of the same magnitude but of opposite sign to the current required to keep the potential of the second oocyte constant. 2.7 Thiol Reagent The thiol reagent MBB (#442631, Calbiochem Co.) was dissolved in DMSO (Dimethyl Sulfoxide) to a stock concentration of 100mM. To get a working concentration of 100µM, 30µL DMSO stock solution was put at the bottom of a clean dry tube and 30mL OR 2 solution was poured into the tube along the wall immediately. (Notes: putting 30µL DMSO stock solution directly into 30mL OR2 with a pipette will not dissolve the DMSO solution.) The reaction between MBB and a thiol group is shown in Figure 2.1. MTSET (#T795901) and MTSES (#S672000) were purchased from Toronto Research Chemicals. MTSET and MTSES were dissolved in distilled water to a stock concentration of 100mM and aliquots were stored at -20 C. An aliquot was diluted in OR2 to the concentration of 1mM and used immediately due to the chemical instability of MTSET. The chemical formula of MTSET is and the chemical reaction

35 23 between MTS reagent and thiol group is. This reaction leads to a disulfide bond that can be broken by reducing agents. Figure 2.1 The chemical reaction between MBB and the thiol group of a cysteine. This reaction leads to a bond that cannot be reversed by reducing agents. 2.8 Dye uptake Cx32E143 or pannexin1-expressing oocytes were preincubated with the test substances in Ringer solution for 5 min. The cells were then transferred to a 100 mm potassium gluconate solution containing 10 mm 6-carboxyfluorescein and the test substances for 20 min. After extensive washing in Ringer solution, oocytes were frozen for cryosectioning. Data were analyzed with the software program NIH Image ( A straight line was drawn normal to the membrane

36 24 and a profile plot was generated. In the profile plot, the area above the autofluorescence level was measured. 2.9 Data Analysis To determine percent inhibition of membrane currents, lines were drawn on enlarged records through peak currents before and after peptide application and during peptide application. The same was done at the baseline to account for eventual baseline shifts. Percent inhibition was calculated, and two-tailed t-tests for correlated samples were performed for statistical analysis. In all figures, statistical significance is indicated as ** P < 0.01; * P < 0.05; not significant P > 0.05.

37 Chapter 3 Attenuation of Pannexin1 Channels by Mimetic Peptides Connexin mimetic peptides were first designed to mimic the extracellular docking gate of the gap junction. Gap junctions are composed of two opposing hexameric connexons. Under physiological condition, hexameric connexons are closed. A gap junction forms and opens when two connexons dock to each other. The docking process involves the interaction of two extracellular loops of the connexins (Dahl et al. 1992; White et al. 1995). The connexin peptides were expected to open connexons by mimicking the homophilic extracellular loop interaction of two opposed connexons. However, the peptides were not strong enough to open connexin channels. Instead, they attenuate gap junction formation (Dahl et al. 1992; Dahl et al. 1994; Warner et al. 1995). Later, connexin mimetic peptide 43 Gap26 and 43 Gap27, which are derived from the first and the second extracellular loops of Cx43, were observed to inhibit ATP release and calcium wave propagation (Boitano et al. 2000; Braet et al. 2003; Evans et al. 2006; D Hondt et al. 2007). Because open Cx43 connexons are detected under extreme conditions like holding membrane potential above +50 mv and exposing cells to zero extracellular calcium solution, it was expected that connexons could open with low probability in physiological conditions to release ATP. Thus, the specific binding between mimetic peptides and their host Cx43 was proposed to inhibit open Cx43 connexons, thus ATP release and calcium wave propagation. According to the same logic, Cx32 connexons are also inferred to be involved in ATP release and calcium wave propagation (De Vuyst et al. 2006). 25

38 Do Connexin Mimetic Peptides Inhibit Connexin Currents? In the literature, there is not a single report demonstrating the direct effects of connexin mimetic peptides on host connexons, most likely due to the lack of open Cx43 or Cx32 connexons in normal conditions. However, Cx32E 1 43 constructed by replacing the first extracellular loop of Cx32 with that of Cx43, forms open channels on the surface of oocytes that share gating and permeability properties with Cx32 gap junction (Pfahnl et al. 1997; Hu and Dahl 1999; Purnick et al. 2000). Unpaired Cx32E 1 43 connexons provide a good chance to test the direct effect of connexin mimetic peptides on host connexons, because the sequences of effective mimetic peptide 43 Gap26 and 32 Gap24 are derived from the first and the second extracellular loops of Cx32E We first tested the activity of mimetic peptide on gap junction channels as a positive control. Cx32E 1 43-expressing oocytes were devitellinized, treated with 43 Gap26 for 15 min, and paired in the continued presence of the peptide. Cx32E 1 43 junctional conductance was determined 3 h after pairing by dual-voltage clamp. The conductance was significantly lower than that of paired oocytes without the peptide, indicating that 43 Gap26 attenuated gap junction channel formation (figure 3.1). On the other hand, delayed application of the peptide to oocyte pairs with established gap junction channels did not acutely affect junctional conductance, suggesting that 43 Gap26 did not impact the existing Cx32E 1 43 gap junctions. These findings are in agreement with previous studies that have shown that the peptides interfere with the formation but not the function of gap junction channels (Dahl et al. 1992; Dahl et al. 1994; Warner et al. 1995; Braet et al. 2003; Gomes et al. 2005). Cx32E 1 43 channels expressed on the surface of single oocytes were opened by depolarization to -20 mv. Membrane conductance was measured by giving 5 mv voltage pulses. There was no acute effect of 43 Gap26 on Cx32E 1 43 conductance (Figure 3.2). The

39 27 effect of 32 Gap24 on Cx32E 1 43 current was also tested. There was no acute effect either (Figure 3.3). Application of CO 2 was used as positive control to support the existence of Cx32E 1 43 current, because cytoplasmic acidification closes Cx32E 1 43 connexons. These two observations suggested that connexin mimetic peptides do not inhibit their host connexin channels. Thus, these effective peptides must inhibit ATP release and calcium wave propagation by a different pathway. Figure Gap26 inhibited the formation of Cx32E 1 43 gap junction channels. Devitellinized oocytes expressing Cx32E 1 43 were paired, and junctional conductance was measured 3h after pairing. c, control without peptide; p, 200µM peptide was applied 15 min before pairing and remained in the bath solution without replenishment; pd, 200µM peptide was applied 2.5 h after pairing and remained in the solution. Values are expressed as means+se. The number of oocyte pairs analyzed is indicated above the bars. ns, not significant; **P < This experiment was performed by Dr. Meiyun Ma. Figure Gap26 did not acutely inhibit Cx32E 1 43 currents. In oocytes expressing Cx32E 1 43, the membrane potential was clamped at -20 mv. Membrane currents induced by 5 mv voltage steps were not acutely affected by 200 µm 43 Gap26. This experiment was performed by Dr. Meiyun Ma.

40 28 Figure Gap24 did not acutely inhibit Cx32E 1 43 currents. Oocytes expressing Cx32E 1 43 were held at -20 mv and 10 mv pulses were applied. Application of 32 Gap24 did not acutely inhibit the membrane currents while cytoplasmic acidification with CO 2 attenuated the currents. 3.2 Do Connexin Mimetic Peptides Inhibit Pannexin1 Currents? Previous observations suggest that pannexin1 forms ATP release channels (Bao et al. 2004, Locovei et al. 2006a and b). Therefore, the question arises: do connexin peptides inhibit ATP release and calcium wave by affecting pannexin1 channels? Pannexin1 was expressed on the surface of oocytes within 36 hours after injection. Oocytes expressing pannexin1 were held at -60 mv and opened by depolarization to +60 mv. Although pannexin 1 channels are unlikely to be opened by voltage under physiological conditions, this pulse protocol was chosen for its simplicity to analyze effects on the pannexin1 channel. In previous studies the mimetic peptides were used at concentrations of 200 to 500 M. Application of 200µM of the peptides 32 Gap24 and 43 Gap27 inhibited pannexin1 channels immediately and the inhibition subsided following washout (Figure 3.4, Figure 3.5). These observations were consistent with the effects of peptides on calcium wave propagation, supporting the function of pannexin1 channels as ATP release channels. The inhibition was also concentration dependent. 32 Gap24

41 29 inhibited pannexin1 current about 10% at 200µM, 25% at 500µM and 60% at 2mM (Figure 3.4). Application of 100µM Cbx was used as control to indicate the existence of pannexin1 current. Figure Gap24 acutely inhibited pannexin1 currents. Oocytes expressing mouse pannexin1 were held at -60 mv and pulses to +60 mv to activate pannexin channels were applied. Gap24 attenuated the currents in a dose dependent manner. Carbenoxolone also inhibited the currents. Figure Gap27 acutely inhibited pannexin1 currents. Oocytes expressing pannexin1 were held at -60 mv and pulses to +60 mv were applied. The current pulse was attenuated acutely by 200 µm 43 Gap27 peptide (Dr. Locovei did this part). 3.3 Do Pannexin Mimetic Peptides Inhibit Pannexon Currents? A pannexin1 mimetic peptide 10 panx1, derived from the first extracellular loop of pannexin1, was reported to inhibit dye uptake in macrophages implying the involvement of pannexin1 in macrophage cells (Pelegrin and Surprenant 2006). To test if pannexin mimetic peptides directly inhibit pannexin1 current, we synthesized another five small genuine pannexin peptides from the extracellular loops of pannexin1, E1a, Elb, Elc, E2a

42 30 and E2b. The first three are from the first extracellular loop; the last two are from the second extracellular loop. 10 panx1 is a 3 amino acid shorter version of peptide panxe1b. The inhibition level of these peptides at 200 µm concentration varied. Application of pannexin mimetic peptides E1b and E1c inhibited pannexon currents rapidly and reversibly (Figure 3.6A). The inhibition of Elb and E1c on pannexon currents were about 18 percent and 12 percent respectively, while the inhibition of other three peptides E1a, E2a and E2b, were relatively small, about 5 percent (Figure 3.6B). 10 panx1, a 3 amino acid shorter version of peptide panxe1b, inhibited pannexin currents to a similar extent as panx1e1b, about 18 percent (Figure 3.6B). 10 panx1 was also shown to inhibit Cx46 channels about 10%, indicating that 10 panx1 is not specific to the sequence of pannexin channels (Figure 3.7). Figure 3.6 Pannexin mimetic peptides acutely inhibited pannexin1 currents. A) Peptides Elb and E1c attenuate pannexin1 currents. Oocytes expressing pannexin1 were held at -60 mv and pulses to +60 mv were applied. B) Quantitative analysis of inhibition of membrane currents carried by pannexin1 channels, by various pannexin mimetic peptides at the concentration of 200 µm. E1a, E1b and E1c are from the first extracellular loop of pannexin1. E2a and E2b are from the second extracellular loop of pannexin1. 10 panx1 is similar to E1b, but 3 amino acid shorter. 10 panx1 was reported to inhibit dye uptake in macrophage cells (Pelegrin and Surprenant 2006). Means±SE are plotted. The number of oocytes analyzed is indicated above the bar. * indicates 95% confidence; ** indicates 99% confidence.

43 31 Figure µm pannexin peptide 10 panx1 inhibited Cx46 current about 10%. Oocytes expressing Cx46 were held at -60 mv, and pulses to 20 mv were applied to open Cx46 channels. 3.4 Do Mimetic Peptides Inhibit Pannexon Currents by Steric Block? Both pannexin and connexin mimetic peptides inhibit pannexin1 currents, suggesting that the inhibition is not specific. All these effective peptides share one common characteristic: the size of about 1400 Daltons, corresponding to the cut-off size of innexin gap junctions (Table 3.1). Therefore, the question was: Do these effective peptides attenuate pannexin1 channel current by steric block? Table 3.1 The sizes of effective mimetic peptides Peptides Amino Acid Sequence Molecular Weight 32 Gap24 GHGDPLHLEEVKC Gap26 PSFDSRHCIVKYV Gap27 SRPTEKTIFII panx1 WRQAAFVDSY 1242

44 32 In order to investigate the pure block effect of big molecules, we tested the inhibition effect of inert molecules like polyethylene glycols (PEG). Oocytes expressing pannexin1 were held at -60 mv and pulses to +60 mv were applied. The results showed that PEGs attenuated pannexin1 currents in a size dependent manner, with maximal inhibition at molecular weight 1500 (Figure 3.8), which is consistent with the size of effective mimetic peptides. PEG 1500 also attenuated the pannexon current in a dose dependent manner (Figure 3.9). PEG 1500 attenuated the pannexon current with 5 percent at 200 µm and 12 percent at 1 mm concentration. The inhibition level of 1 mm PEG 1500 is similar to that of 200 µm effective peptides (Figure 3.10). The discrepancy could be due to higher peptide concentrations at the channel mouth as a consequence of peptide-protein interactions. PEGs, based on their chemical composition, would be expected to have less affinity with proteins. The extracellular loops from opposing connexons interact with each other to form gap junction channels, indicating the interaction of pannexin mimetic peptides with pannexons that may increase the concentration of peptides at the mouth of pannexon channels and thus inhibit more by steric block. The inhibitory effect of 10 panx1 of 18 percent on pannexin currents is higher than that of 32 Gap24 of 10 percent and that of the scrambled version of 10 panx1 of 5 percent (Figure 3.11). The dose-response curves of pannexin1 currents inhibition by peptides 10 panx1, 32 Gap24, 10 px1scr, 32 Gap24scr and PEG1500 are summarized in Figure 3.12.

45 33 Figure 3.8 Size dependent inhibitions of pannexin1 currents by PEGs. PEG (polyethylene glycol) at the indicated molecular sizes was applied to pannexon currents. Oocytes expressing pannexin1 were held at -60 mv and pulses to +60 mv were applied. The current pulses were attenuated acutely by PEG molecules at the concentration of 1 mm. Means±SE are plotted. The number of oocytes analyzed is indicated above the symbols. Figure 3.9 PEG1500 acutely inhibited pannexin1 currents. PEG1500 attenuates pannexon currents in a dose dependent fashion. Oocytes expressing pannexin1 were held at -60 mv and pulses to +60 mv were applied.

46 Inhibition (%) 34 Figure 3.10 The inhibition level of 1mM PEG1500 is similar to that of 200µM 32 Gap24. Quantitative analysis of inhibition of pannexin1 currents by 32 Gap24, PEG1500 at the concentration of 200 µm and PEG1500 at 1 mm. Means±SE are plotted. The number of oocytes analyzed is indicated above the bar. * indicates 95% confidence; ** indicates 99% confidence ** ** * 3 ** 0 10panx1 10panx1scr Gap24 Gap24scr Figure 3.11 The inhibition effects of scrambled versions of 32 Gap24 and 10 panx1 on pannexin1 currents. 32 Gap24scr, with scrambled sequence of 32 Gap24, inhibit the pannexon currents; 10 panx1scr, with scrambled sequence of 10 panx1, slightly inhibit the pannexon currents. All these peptides were applied at the concentration of 200 µm. Means±SE are plotted. The number of oocytes analyzed is indicated above the bars. * indicates 95% confidence; ** indicates 99% confidence.

47 35 Figure 3.12 Dose response curves of pannexon inhibition by peptides and PEG1500. Peptides and PEG1500 at various concentrations were applied as indicated by the color codes. Oocytes expressing pannexin1 were held at -60 mv and pulses to +60 mv were applied. Means±SE are plotted. The number of oocytes analyzed is indicated beside the symbols. 3.5 Do Mimetic Peptides Inhibit Dye Uptake Through Pannexons? The inhibition level of 200 µm effective peptides on pannexon currents is about 15%, whereas the inhibition level of peptides on ATP release and calcium wave propagation is about 50%. One possible reason is that current is carried mainly by small ions that are much smaller than ATP. Effective peptides may have bigger blocking effects to larger molecules, such as ATP and dye molecules, than small ions. In order to test this hypothesis, we measured the effect of peptides on dye uptake through pannexin1 channels. Pannexin1-expressing oocytes were preincubated with 200µM peptide in Ringer solution for 5 min. The cells were then transferred to a 100 mm potassium gluconate solution containing 10 mm 6-carboxyfluorescein with MW 376 and 200 µm peptides for 20 min. Peptides 10 Panx1 and 32 Gap24 at 200 µm inhibited dye

48 36 uptake through pannexin1 channels by about 50%. Similar inhibition effects occurred for 1mM PEG 1500 (Figure 3.13A). As a negative control, no inhibition effects of these peptides and PEG 1500 were observed for dye uptake through Cx32E 1 43 channels (Figure 3.13B). In summary, these data suggested that the connexin or pannexin mimetic peptides may inhibit the pannexon by steric block and thereby inhibit the calcium wave propagation. We may still use the peptides as nonspecific pannexon blockers, but these peptides may have little to do with gap junction protein characteristics and the misleading terms connexin or pannexin mimetic peptides should be abandoned.

49 Figure 3.13 Effect of peptides and PEG molecules on dye uptake in oocytes expressing pannexin1 (A) or Cx32E 1 43 (B). Top: fluorescence micrographs of sections of oocytes exposed to 200µM peptides or 1mM PEG1500 as indicated. Note that dye uptake is more pronounced in Cx32E 1 43-expressing oocytes, probably because of much higher expression levels. Bottom: quantitative analysis of dye uptake. Data were analyzed with the software program NIH Image ( A straight line was drawn normal to the membrane and a profile plot was generated. Data are normalized to oocytes not exposed to peptide or PEG1500. Values are means + SE. The number of oocytes analyzed is indicated above the symbols. Two-tailed T tests were performed. ** P <0.01. Uninjected oocytes did not show any dye uptake (not shown). 37

50 Chapter 4 The Pannexin1 Pore: Pore Lining Segments and Residues In contrast to connexins, the innexin homolog, pannexin1, does not appear to form gap junction channels but instead forms unpaired membrane channels to allow ATP release and to be involved in calcium wave propagation. Although pannexins and connexins have no sequence homology, they function similarly and they share similar membrane topology, gating effects and pore size. Like connexins, pannexin1 has four transmembrane domains and two extracellular loops (Locovei et al. 2006b) and six subunits compose pannexin1 channels (Boassa et al. 2007). Pannexin1 channels are permeable to big molecules, such as ATP and carboxyfluorescein (Bao et al. 2004). Pannexin1 channels can be blocked by general gap junction channel blockers, such as carbenoxolone and β-glycyrrhetinic acid (Bruzzone et al. 2005). Now the question is whether the similarity extends to the pore structure of the channels. Substituted Cysteine Accessibility Method (SCAM) has been successfully used to identify the pore lining residues of many kinds of channels. When a residue in the pore of a channel is replaced by cysteine that can be bound covalently by thiol reagents, application of thiol reagents will cause a partial block the channel that can be detected by electrophysiological recording or other means. It is also possible that thiol reagents may work on a cysteine outside of the pore by changing the conformation of the pore allosterically. Experiments can be designed to discriminate these situations. A block is size dependent, while gating does not need a strict correlation. Apparently introduction of a reactive amino acid cysteine in theory could change the channel configuration. However, in practice this method has been successfully used in many channels because replacement 38

51 39 of amino acids by cysteine is tolerated almost as well as alanine replacement. It was first tried in ACh receptors (Akabas et al. 1991); then it was extended to GABA receptors (Xu and Akabas 1993), connexons (Zhou et al. 1997, Skerrett et al. 2002, Kronengold et al. 2003) and many other channel proteins. Our lab first used the Substituted Cysteine Availability Method to identify the pore lining residues in Cx32E 1 43 and Cx46 connexons (Zhou et al. 1997). Two residues, I33 and M34 in the middle of TM1 of Cx32E 1 43, were identified to react with extracellularly applied thiol reagents, while several moderate reactive residues were identified in TM3, suggesting that TM1 and TM3 cooperate to form the pore. 4.1 Cysteine at Position 426 in Pannexin1 are Reactive to MBB Softwares TMpred ( and TMHMM ( were used to predict the transmembrane domains of mouse pannexin1. The possible TM1 is from M38 to I60, TM2 from F108 to W127, TM3 from L217 to L236 and TM4 from L275 to I296. Pannexin1 has amino and carboxyl termini in the cytoplasm with two extracellular loops (Locovei et al. 2006b; Boassa et al. 2007). There are four signature cysteines in the extracellular loops, which are thought to form disulfide bonds. Mutations in any one of the four positions led to loss of channel function. There are another 6 cysteines in transmembrane and cytoplasmic parts (Figure 4.1). The reactivity of endogenous cysteines of pannexin1 with thiol reagents had to be tested before systematic scanning could be performed. Mouse pannexin1 was expressed in Xenopus oocytes; pannexons were opened by applying positive voltage pulses and the modification of the thiol reagents on endogenous cysteines could be tested by extracellular

52 40 application. Because of the large pore size of a pannexon, the large thiol reagent MBB (maliemidobutyryl biocytin, MW 537) was used. 100µM MBB substantially reduced pannexon current. The effect was partially reversible after washout (Figure 4.2A). Thus, the inhibition could be divided into two parts, reversible inhibition and irreversible inhibition Figure 4.1 Predicted topology of mouse pannexin1. Like connexins, pannexin1 had 4 transmembrane spans with two extracellular loops and cytoplasmic NH2 and COOH termini (Locovei et al. 2006, Boassa et al. 2007). The possible TM1 is from M38 to I60, TM2 from F108 to W127, TM3 from L217 to L236 and TM4 from L275 to I296 by hydrophobicity plot. The colors indicate the basic characteristics of amino acids. Green, hydrophobic; blue, positively charged; Red, negatively charged; yellow, cysteine; and pink, proline.

53 41 Figure 4.2 MBB effects on WT or mutant pannexin1 currents. A: application of 100 µm MBB inhibited pannexon current. Oocytes expressing mouse pannexin1 were held at -60mV and pulses to +60mV were applied to activate pannexons. Application of 100µM MBB greatly reduced pannexon current that was partially reversible after washing of MBB; additional application of 100µM MBB reduced the current in a small degree that was completely reversible after washing of MBB. The two-end arrow between dotted lines indicates the irreversible inhibition used to calculate percent inhibition in panel D and all other plots in this report. B: 1mM PEG400 or 1mM PEG600 reduced pannexin1 current that was reversible after washing of PEG molecules. C: MBB effects on mutant pannexin1 currents including C40S, C136S, C215S, C227S and C426S. Only the inhibitory effect on C426S was reversible after washout. D: quantitative analysis of inhibition of membrane currents carried by channels formed by WT pannexin1, C426S, C40S by 100µM MBB. Values are means+ SE. The number of oocytes analyzed is indicated above the bars. Reversible inhibition may be due to steric block of free MBB that can be demonstrated by application of inert PEG (polyethylene glycol) molecules with comparable size (Wang et al. 2007). The current was reduced when PEG400 or PEG600 molecules were in the pore. The current recovered when PEG molecules were washed away (Figure 4.2B). Irreversible inhibition was attributed to covalent modification that cannot be washed away (Figure 4.2A). Subsequent repeated applications of MBB yielded

54 42 only reversible inhibition. In the following part of this report, the inhibition refers to the irreversible inhibition. The irreversible inhibition effect of MBB on pannexon current indicated that covalent modification of some endogenous cysteines occurred. In order to identify which cysteine bound MBB and reduced pannexon current, I systematically constructed mutants by replacing each endogenous cysteine with serine one at a time. They were C40S, C136S, C215S, C227S, C346S, and C426S. The effect of MBB on these mutants was tested. Irreversible inhibition still existed in mutants C40S, C136S, C215S and C227S, but disappeared completely in mutant C426S (Figure 4.2 C and D), indicating that endogenous cysteines except C426 did not have measurable impact on pannexon current with MBB modification. Mutant C426S apparently behaved in the same way as wild type pannexin1 in known ways, such as expression time course in the oocytes, voltage dependence and response to the blocker carbenoxolone. Therefore, mutant C426S was a suitable substitute for wild type pannexin1 for cysteine scanning. 4.2 Several Positions in TM1 and E1 are Reactive to MBB Each residue along TM1 was substituted by cysteine, one at a time, in C426S pannexin1. Because thiol modifications of endogenous cysteines in C426S did not have measurable effect on pannexon current, the modification effect of extraneous cysteine was singled out. Mutations at positions I50, S51, L52, A53 and E57 in the outside half of TM1 as well as Q63 and I64 at the border of TM1 and E1 failed to form functional channels as determined by depolarization. At the other positions, cysteine mutants formed functional channels. MBB was applied among functional mutants and caused substantial inhibition at positions F54, I60, G61 and T62. Modest inhibition was found on I58C current, but the

55 43 effect was not significantly different from control. The mutations in the inside half of TM1 from M37C to L49C did not exhibit measurable inhibition by MBB (Figure 4.3). A B Figure 4.3 Inhibitory effects of MBB on membrane currents carried by TM1 cysteine mutant channels. A. Electrophysiological recordings of membrane currents carried by some TM1 cysteine mutant channels. Oocytes expressing mutant pannexin1 were held at -60mV and pulses to 60mV were applied to activate pannexons. To test for cysteine accessibility, 10µM MBB was applied. B. Quantitative analysis of inhibition of MBB on membrane currents carried by channels formed by cysteine mutants in TM1. X axis is positioned at the interface between cytoplasm and membrane. Values are means+ SE. The number of oocytes analyzed is indicated next to the bars. NC: no functional channel. Paired T test was performed between currents before and after thiol reagent application. * P <0.05, ** P<0.01.

56 44 Figure 4.4 Quantitative analysis of inhibition of MBB on membrane currents carried by channels formed by cysteine mutants in TM2. X axis is positioned at the interface between cytoplasm and membrane. Values are means+ SE. The number of oocytes analyzed is indicated next to the bars. NC: no functional channel. 4.3 No Positions in TM2 are Reactive to MBB Residues in TM2 were systematically substituted by cysteine one at a time on the basis of C426S pannexin1. Cysteine substitution along TM2 except Y121 formed functional channels in the membrane of oocytes. MBB did not display noticeable inhibition at any position in TM2 (Figure 4.4). 4.4 No Positions in TM3 are Reactive to MBB Residues in TM3 from L217 to L236 were systematically substituted by cysteine one at a time on the basis of C426S pannexin1. Mutants at positions Y229, Y232, Y233 and S235 in the outside half of TM3 and I223 in the inside half of TM3 failed to form

57 45 functional channels as determined by depolarization. MBB was tested in all functional mutants and exhibited no inhibition at any of these positions (Figure 4.5). 4.5 No Positions in TM4 are Reactive to MBB Residues in TM4 were systematically substituted by cysteine one at a time on the basis of C426S pannexin1. Cysteine substitutions along TM4 except V291 formed functional channels. MBB did not display noticeable inhibition at all positions in TM 4 (Figure 4.6). Figure 4.5 Quantitative analysis of inhibition effect of MBB on TM3 mutant currents. X axis is positioned at the interface between cytoplasm and membrane. Values are means+ SE. The number of oocytes analyzed is indicated near the bars. NC: no functional channel.

58 46 Figure 4.6 Quantitative analysis of inhibition of MBB on TM4 mutant currents. X axis is positioned at the interface between cytoplasm and membrane. Values are means+ SE. The number of oocytes analyzed is indicated near the bars. NC: no functional channel. 4.6 Several Positions in TM1 and E1 are also Reactive to MTSET Cysteine scanning in all four transmembrane domains by using the thiol reagent MBB identified several positions in the outside portion of TM1, corresponding to the outside half of the pore. In order to check whether some reactive residues that lined the cytoplasmic half of the pore were modified by MBB but did not attenuate pannexon currents significantly, a small positive charged thiol reagent MTSET (2-trimethylammonioethyl-methane thiosulfonate) with MW 278 was used. Small MTSET may have larger block effect than big MBB if the pore could allow for several bindings for MTSET while allow only one binding for MBB (Figure 4.7).

59 47 Figure 4.7 Schematic demonstration of binding between cysteine and thiol reagents. If there is only one binding site, a large molecule will inhibit the pore more than a small molecule. However, pannexons have six subunits (Boassa et al. 2007). The pore may accommodate six MTSET while only accommodate one MBB. In this situation, MTSET may have more inhibition effect than MBB. Like MBB, MTSET exhibited significant inhibition at positions T62, G61 and I60 and no measurable inhibition in the inside half of TM1 from V38 to L47 except that V43C and G44C were not determined. MTSET exhibited large inhibition at position I58 and relatively small but significant inhibition at position L49 while MBB did not display inhibition in these two positions (Figure 4.8). To our surprise, MTSET caused enhancement at position F54 rather than inhibition as seen with MBB. Because MBB is negatively charged and MTSET is positively charged, we first hypothesized that positive charge may change the inner environment of the channel significantly and enhance the movement of ions. We tested the effect of a similar but negatively charged MTS molecule, MTSES, and a neutral maleimide, pyrenyl maleimide, on F54C channel currents. Both negative MTSES and neutral pyrenyl maleimide displayed similar enhancement as positive MTSET (Figure 4.9), indicating that the enhancement of currents is not related to a charge effect in the pore. We also tested the effects of a neutral MTS molecule, MTSBn, on the F54C current. Application of MTSBn induced a large current and destabilized the channels so that it could not be clamped. The second possible cause of enhancement is that MTSET may work as a reducing agent to affect the disulfide bonds in the channels so that

60 48 the conductance increases, but application of the reducing agent DTT reduced the current a little rather than induce further enhancement (Figure 4.9). Another possible cause of the enhancement is that introduction of a cysteine in position F54 may change the structure moderately, including the increase of the crevice in the channel, so that small MTS molecules MTSET (MW 278), MTSES (MW 233), MTSBn (MW 202) or small neutral pyrenyl maleimide (MW 297) could enter the crevice, bind to C227 in the protein and then increase the channel current by freezing the channels at high conductance conformation while big MBB (MW 537) cannot. It had been reported that the reactivity of MTSET with the thiol group of a cysteine is very high and the reagent may react with a minor channel conformation and modify cysteines partially buried in the crevice of the protein rather than the freely accessible surface of the protein (Liu et al. 1996, Holmgren et al. 1996, Lu and Miller 1995). The two thiol reagents MBB and MTSET contribute to identification of residues that may line the outside half of the pore. They are L49, F54, I58, I60, G61 and T62. The first 3 residues L49, F54 and I58 are separated by 3 or 4 residues, consistent with alpha helix structure and the following three amino acids I60, G61 and T62 do not show helix characteristics, consistent with their predicted position at TM1 and E1 border. Examples of thiol reagent modification of mutant pannexon currents are displayed in Figure 4.8A. The inside half of TM1 was not susceptible to thiol reaction by MBB and MTSET.

61 49 A B Figure 4.8 Inhibitory effects of MTSET on membrane currents carried by channels formed by TM1 cysteine mutants. A. Electrophysiological recordings of membrane currents carried by some TM1 cysteine mutant channels. Oocytes expressing mutant pannexin1 were held at -60mV and pulses to 20mV were applied to activate pannexons. To test for cysteine accessibility, 1mM MTSET was applied. B. Quantitative analysis of inhibition of MTSET on membrane currents carried by channels formed by cysteine mutants in TM1. X axis is positioned at the interface between cytoplasm and membrane. Values are means+ SE. The number of oocytes analyzed is indicated next to the bars. NC: no functional channel; ND not determined. Paired T test was performed between currents before and after thiol reagent application. * P <0.05, ** P<0.01.

62 50 Figure 4.9 the effects of various thiol reagents on F54C currents. Oocytes expressing F54C pannexin1 were held at -60mV and pulses to 60mV or 20mV were applied to activate pannexons. To test for cysteine accessibility, 10 M MBB, 100 M pyrenyl maleimide, 1mM MTSET, 1mM MTSES or 1mM MTSBn were applied. Pulses to 60 are only applied to MBB application. 4.7 No Positions in the Inner Portion of TM2 are Reactive to MTSET MTSET was tested in the inside half of TM2 and no inhibitory effects were exhibited. At position L125, MTSET enhanced the mutant conductance; at position Y121, the mutant did not form functional channels; at other positions from L119 to W127, MTSET did not have significant effect on the mutant conductance (Figure 4.10). 4.8 Two Positions in the Inside Portion of TM3 are Reactive to MTSET MTSET was tested in mutants at positions in the inside half of TM3 and exhibited inhibition at positions L217 and V221. The cysteine scanning was then extended to the cytoplasmic side from R216 to I213 and no measurable inhibition at these nearby residues

63 51 was identified (Figure 4.11). Examples of MTSET inhibition at position L217 and V221 are displayed in Figure 4.11 A and B. 4.9 No Positions in the Inner Portion of TM4 are Reactive to MTSET MTSET was tested in the inside half of TM4 and no inhibitory effects were exhibited. At position V291, the mutant does not form functional channels; at other positions from P288 to I296, MTSET did not have significant effect on the mutant conductance (Figure 4.12). Figure 4.10 Quantitative analysis of inhibition of membrane currents carried by channels formed by cysteine mutants in the inside portion of TM2 by MTSET. X axis is positioned at the interface between cytoplasm and membrane. Values are means+ SE. The number of oocytes analyzed is indicated next to the bars. NC: no functional channel.

64 52 TM3 Figure 4.11 Inhibitory effects of MTSET on membrane currents carried by channels formed by cysteine mutants at the inside portion of TM3. A: electrophysiological recording of membrane currents carried by V221C channels. B. Electrophysiological recording of membrane currents carried by L217C channels. Oocytes expressing mutant pannexin1 were held at -60mV and pulses to 20mV were applied to activate pannexons. To test for cysteine accessibility, 1mM MTSET was applied. C. Quantitative analysis of inhibition of MTSET on membrane currents carried by channels formed by cysteine mutants in the inside portion of TM3. X axis is positioned at the interface between cytoplasm and membrane. Values are means+ SE. The number of oocytes analyzed is indicated next to the bars. NC: no functional channel. Paired T test was performed between currents before and after thiol reagent application. * P < Several Positions at the Carboxyl Terminus are Reactive to MBB MBB inhibition at position C426 at the end of the carboxyl terminus suggested that the carboxyl terminus might bend back to the pore area or even form part of the pore. To test whether additional amino acids are involved, cysteine mutagenesis was extended from S425 to S406 on the basis of C426S pannexin1. Mutants were expressed in the oocytes and MBB was tested. Except for S417C, which failed to form functional channels, cysteine substitutions at positions from S425 through N416 displayed inhibition by MBB (Figure 4.13), suggesting the possibility that the end part of carboxyl terminus is near the pore if not part of it.

65 53 Figure 4.12 Quantitative analysis of inhibition of membrane currents carried by channels formed by cysteine mutants in the inside portion of TM4 by MTSET. X axis is positioned at the interface between cytoplasm and membrane. Values are means+ SE. The number of oocytes analyzed is indicated next to the bars. NC: no functional channel Summary The inhibitory effects of thiol reagents MBB and MTSET on the conductance of cysteine mutants are summarized in Figure Excluding the C terminus, we identified 8 possible residues lining the pore: L49, F54, I58, I60, G61 and T62 in the outside half of TM1 and extension to E1, L217 and V221 in the inside half of TM3 (Figure 4.15A). The 2 residues L217 and V221 in TM3 and 3 residues L49, F54 and I58 in TM1 are separated by 3 or 4 residues, exhibiting helix characteristics, consistent with their predicted positions in the membrane; the reactivity of successive three amino acids (I60, G61 and T62) that may

66 54 lie in E1 did not display helix characteristics. Our data suggested that the outside portion of TM1 and E1, and inside half of TM3 form the pore of pannexons. In addition to providing information about pore structure, our data also shed light on the conformation of pannexons. Mutations at positions I50, S51, L52, A53, E57 and Q56 in outside half of TM1, Q63 and I64 at the border of TM1 and E1, Y121 in the inside half of TM2, Y229, Y232, Y233 and S235 in the outside half of TM3, I223 and R216 in the inside half of TM3, V291 in the inside half of TM4 failed to form functional channels on the membrane of oocytes (Figure 4.15B), indicating their importance in maintaining the structural integrity of pannexons. Figure 4.13 Quantitative analysis of inhibition of membrane currents carried by channels formed by cysteine mutants in the carboxyl terminus by MBB. X axis is positioned at the end of carboxyl terminus. Values are means+ SE. The number of oocytes analyzed is indicated next to the bars. NC: no functional channel. Paired T test was performed between currents before and after thiol reagent application. * P <0.05, ** P<0.01.

67 MBB MTSET 55 TM1 TM2 TM3 TM4 Figure 4.14 Summary of quantitative analysis of inhibition of membrane currents carried by channels formed by cysteine mutants in all four transmembrane domains by MBB and MTSET. X axis is positioned at the edge of cytoplasm and membrane. Values are means+ SE. The number of oocytes analyzed is indicated next to the bars. NC: no functional channel; ND not determined. Paired T test was performed between currents before and after thiol reagent application. * P <0.05, ** P<0.01.

68 Figure 4.15 Summary of the positions with thiol reagent reactivity or loss of function. A: The positions of amino acids were marked where cysteine substitutions displayed inhibition by thiol reagents, indicating that they might line the pore. B: The positions of amino acids were marked where cysteine substitutions failed to form functional channel as determined by depolarization, indicating they might play an important role in maintaining the structural integrity of pannexons. 56

69 Chapter 5 Conclusion and Discussion In vertebrates, connexons typically are closed. When two apposing connexons meet each other, they will form open gap junction channels between two neighboring cells. Pannexins, which were originally identified as gap junction proteins, are very different. Pannexin1 does not form gap junction channels under normal conditions (Dahl and Locovei 2006; Huang et al. 2007a; Ransford et al. 2007; Boassa et al. 2007; Penuela et al. 2007). Instead, pannexin1 forms functional membrane channels (Bruzzone et al. 2003; Bao et al. 2004), which can open under physiological conditions. Pannexin1 channels have been suggested to be ATP release channels involved in calcium wave propagation (Bao et al. 2004; Locovei et al. 2006a, b; Romanov et al. 2007; Huang et al. 2007b). Here I provide additional evidence for an ATP release function of pannexin1 channels. In addition, I report about attempts to elucidate the pore structure of pannexin1 channels. 5.1 The Inhibitory Effect of Mimetic Peptides does not Indicate the Involvement of Connexons But Instead Implicates Pannexin1 Channels in ATP Release ATP release plays an important role in calcium wave propagation to coordinate neighboring cells, but how ATP is released from the cytoplasm to the extracellular space is unknown. Connexons were proposed to be the channels to release ATP due to the large size of the pore, although connexons typically do not open in physiological conditions. Low level connexon channel activity was observed in extreme conditions, such as zero extracellular calcium or high positive membrane potential above 50 mv (Li et al. 1996; Contreras et al. 2003). It was argued that the brief, infrequent connexon channel openings 57

70 58 might be sufficient for dye or ATP to flux from cytoplasm to extracellular space (Contreras et al. 2003). Unspecific gap junction blockers, including α-glycyrrhetinic acid, carbenoxolone and flufenamic acid, and gap junction mimetic peptides, are widely used for identification of connexons. Gap junction mimetic peptides are based on a presumed specific interaction of peptides with connexons. Gap junction protein mimetic peptides were originally created with the assumption that they might mimic the docking interaction in gap junction channel formation and thus open unpaired connexons. If that were true, the techniques commonly used in membrane channels, including whole cell voltage clamp and patch clamp, could also be easily performed in gap junction research. However, these small peptides did not open connexons. Instead, they inhibited gap junction formation (Dahl 1992; Dahl et al. 1994; Warner et al. 1995). The working explanation is that the specific interaction between mimetic peptides and connexons is not sufficient to activate the docking gate but is strong enough to hinder the docking interaction between two connexons. As possible specific gap junction blockers, a series of connexin mimetic peptides from various host connexins were developed (Chaytor et al. 1997; Chaytor et al. 1998; Kwak and Jongsma 1999). Because the peptides only inhibit the formation of new gap junctions rather than inhibiting existing gap junctions, the inhibitory action on gap junctions is slow and depends on the turnover rate of gap junction proteins. Recently, in a series of papers (Table 5.1), faster actions of connexin mimetic peptides were observed to inhibit ATP release and calcium wave propagation. Because connexin peptides are presumed to specifically affect connexons, these phenomena were interpreted as proofs of open connexons and their involvement in ATP release. However, in these studies, no direct effect of peptides on functional

71 59 connexons is tested. Moreover, the rationale for a specific action of gap junction protein mimetic peptides as inhibitors of unpaired connexons is ambiguous. If the mechanism of action of the peptides involved a specific gating process, one might expect these peptides to open connexons, assuming the peptides were to mimic the action of the docking process in gap junction channel formation, rather than close connexons. Whether connexin mimetic peptides are specific and whether these peptides inhibit connexons are not unequivocally proved. The observation that peptide Gap24 derived from cytoplasmic loops of Cx32 was more effective to inhibit ATP release than extracellular peptides further blurred the argument that gap junction mimetic peptides are specific inhibitor of unpaired connexons (De Vuyst et al. 2006; De Vuyst et al. 2007). Furthermore, the high peptide concentrations required for significant effects on ATP release and calcium wave propagation exclude high-affinity peptide protein interaction. Table 5.1 List of publications where gap junction mimetic peptides were used in channel identification Am J Physiol Lung cell Mol Physiol 279: L623-L (Boitano and Evans 2000) Cell Calcium 33: 37 48, 2003 (Braet et al. 2003b) Cell Commun Adhes 10: , 2003 (Leybaert et al. 2003) J Cell Physiol 197, , 2003 (Braet et al. 2003a) J Neurochem 88, , 2004 (Vandamme et al. 2004) Neuron 46: , 2005 (Pearson et al. 2005) Invest Ophthalmol Vis Sci 46: , 2005 (Gomes et al. 2005) Embo J 25: 34 44, 2006 (De Vuyst et al. 2006) Biochem J 397: 1 14, 2006 (Evans et al. 2006) Exp Eye Res 83: , 2006 (Gomes et al. 2006) J Biol Chem 281, , 2006 (Takeuchi et al. 2006) Circ Res 99: , 2006 (Eltzschig et al. 2006) Invest Ophthalmol Vis Sci 48: , 2007 (D Hondt et al. 2007a) Mol Biol Cell 18: 34 46, 2007 (De Vuyst et al. 2007) Invest Ophthalmol Vis Sci 48, , 2007 (D Hondt et al. 2007b) Am J Physiol Heart Circ Physiol (Shintani-Ishida et al. 2007) Proc Natl Acad Sci U S A 104, , 2007 (Retamal et al. 2007) Embo J. 26, , 2007 (Romanov et al. 2007) The major obstacle to measure the direct effect of mimetic peptide on connexons is that connexons typically do not form unpaired open channels. Even under extreme

72 60 conditions, such as holding membrane potential above +50mV, the connexon channel activity is so low that single channel events can be resolved in whole cell recordings (Contreras et al. 2003). The chimera Cx32E 1 43 happens to form open connexons on the cell membrane in response to moderate depolarization (Pfahnl et al. 1997). Expression of the chimera results in cell death unless measures are taken to keep the connexons closed. Fortuitously, this can be simply achieved by incubating the cells in Ringer solution with the calcium concentration increased to 5 mm. For acute experiments on the chimera connexons, the calcium concentration is then reduced to the normal level of 1 mm. The gating and permeability of this mutant channel are consistent with that of Cx32 gap junction channels and the unitary conductance, about 50 ps, is twice that of a Cx32 gap junction channel (Pfahnl et al. 1997; Purnick et al. 2000). Cx32E 1 43 connexons keep most properties of Cx32 connexons except the open probability on the cell membrane. In addition, this chimera connexon contains the sequence of two most widely used mimetic peptides 32 Gap24 and 43 Gap26. Therefore, Cx32E 1 43 connexons are suitable to test the effects of gap junction mimetic peptides. Our data show that 43 Gap26, derived from the first extracellular loop of Cx32E 1 43, did inhibit the formation of Cx32E 1 43 gap junction channels between two oocytes, supporting the proposal that the peptide protein binding forms steric hindrance to reduce the interaction between two connexons. However, 43 Gap26 did not inhibit Cx32E 1 43 connexons at a noticeable level, which is contrary to the assertion that specific peptide connexon interaction inhibits open connexons and thus ATP release. Another effective peptide 32 Gap24, derived from the cytoplasmic loop of Cx32E 1 43, did not have any effect on Cx32E 1 43 connexon current, either. It is formally possible that the chimera mutation

73 61 changes the docking interaction between extracellular loops of apposing connexons as compared to wild-type Cx32 or Cx43. However, the general properties of the Cx32E 1 43 channel, such as the ability to form gap junction channels, its single channel conductance and its formation of gap junction channels that can be inhibited by mimetic peptide 43 Gap26, indicate that the peptide connexon interaction is preserved. Therefore, these effective peptides probably inhibit some other channels rather than connexons to attenuate ATP release. Pannexin1 channels recently have been strongly supported to be the ATP release channels. Our data show that inhibitory peptides, such as 32 Gap24 and 43 Gap27, did inhibit pannexin1 channel currents. In addition, pannexin mimetic peptide 10 panx1, which had been reported to inhibit dye uptake in macrophage cells (Pelegrin and Surprenant 2006), also attenuate pannexin1 currents. These peptide effects on Cx32E 1 43 and pannexin1 channels consistently support the model that pannexin1 channels rather than connexin channels act as ATP release channels. Therefore, the inhibitory effect of connexin mimetic peptides on ATP release and calcium wave propagation is likely indicative of the channel activity of pannexin1 channels rather than that of connexin channels. 5.2 Mimetic Peptides Work on Pannexin1 Channels Through Steric Block Our data show that a scrambled version of 10 panx1 was less effective in inhibiting pannexin1 currents than the original peptides, consistent with the weak inhibition of scrambled peptides used as control in ATP release and calcium wave propagation (Pearson et al. 2005). This apparently implies that pannexin1 exhibits a sequence specific binding site for the peptides. However, many inhibitory peptides with diverse sequences and origins, such as 32 Gap24, 43 Gap26, 43 Gap27, 10 panx1 and several other pannexin mimetic

74 62 peptides, all inhibited pannexin1 currents, suggesting that a high affinity of specific peptide-pannexon binding is unlikely. In addition, 10 panx1 also inhibits Cx46 currents, supporting the nonspecific peptide protein interaction. Scrambled versions of the peptides are generally accepted as a control for sequence specificity. However, this control method has flaws. For a 10mer, the possible sequences and ways of folding are about 3.6 million, or ten factorial. One scrambled version may change the way it folds significantly, while another may change its folding only mildly. Our experiment using the scrambled versions of 32 Gap24 and 10 panx1 indicated these two different possibilities. The scrambled version of 32 Gap24 showed slightly lower inhibitory effect than 32 Gap24, while the scrambled version of 10 panx1 had much less inhibitory effect than 10 panx1. Connexin and pannexin mimetic peptides are all of similar molecular size of 1400 Daltons. The pannexin channels could accommodate the entry of molecules in this size range, and their presence would effectively block channel currents. Therefore, a mechanism involving steric block of the channel was considered. Consistent with such a mechanism, PEG 1500 attenuated pannexin1 currents in a similar way as the peptides. The accessibility limit for PEG falls into as the same size range as the cut-off limit for the permeation of tracer molecules through innexin gap junction channels in invertebrates. However, higher concentrations of PEG 1500 than peptide were required to achieve equivalent degrees of inhibition of channel currents. This discrepancy could be due to slightly higher peptide concentrations at the channel mouth as a consequence of unspecific peptide-protein interactions. PEGs, based on their chemical composition, would be expected to interact less frequently with proteins. This argument could also explain the

75 63 difference between mimetic peptides and their scrambled versions. Mimetic peptides seem to have better chance to interact with the extracellular loops of the proteins and thus increasing their concentration at the mouth of the channels. Moreover, despite the same amino acid content, the scrambled peptides do not need to fold the same way as the authentic sequence. They could be either larger and be partially excluded from the channel or they may be smaller and occlude less of the channel. At the concentrations used in ATP-release studies, none of the peptides exceeded 20% inhibition of channel currents, indicating that the mechanism of channel closure is inefficient in terms of interference with the permeation of small ions. However, inhibition of dye uptake was much more substantial. This suggests that the transit of larger molecules, including ATP and dyes, is more severely affected by the peptides because of steric constraints on the channel. This could explain the high levels of inhibition of downstream events reported in other studies (Boitano et al. 2000, Gomes et al. 2005, Gomes et al. 2006, D Hondt et al. 2007, Pearson et al. 2005, De Vuyst et al. 2006). Consistent with the steric block mechanism, the effective peptides and PEG1500, because of its size, are excluded from the Cx32E 1 43 channels with small conductance (50 ps) and thus have no effect on the currents or dye uptake. In summary, our data suggest that the connexin mimetic peptides do not inhibit their host connexons but instead inhibit pannexin1 channels. Our data also raise doubt about using mimetic peptides for channel identification. Neither connexin nor pannexin mimetic peptides exhibit the specificity and efficacy required for channel identification.

76 Pannexin1 Channels Share Similar Pore Structure with Connexons Although pannexin1 shares no sequence similarity with connexins, it shares similarity with connexins in higher level of protein structure. For example, pannexin1 has similar membrane topology as connexins. Like connexins, pannexin1 channels have six subunits and respond to gap junction blockers including carbenoxolone and β-glycyrrhetinic acid (Locovei et al. 2006b; Boassa et al 2007; Bruzzone et al. 2005). Our question is whether they extend their similarities to the pore structure. Although gap junction channels composed of different connexins demonstrate variety in pore size, voltage dependence and selectivity, they probably share a conserved structural conformation according to the three dimensional cryo electron microscopy density maps of gap junction channels composed of Cx43L263Δ and Cx26M34A (Unger et al. 1999; Oshima et al. 2007). Cx43L263Δ is a Cx43 mutation where the large carboxyl terminus is deleted at L263. Cx26M34A is a Cx26 point mutation at M34. Both structures consistently display similar 24 alpha-helical packing and the general shape of the pore, even though the two proteins use totally different mutagenesis method (Figure 5.1). The only big difference is that a density core exists in the map of Cx26M34A gap junction channels, probably because the large lump of carboxyl terminus was kept in Cx26M34A but deleted in Cx43L263Δ mutant. Given that the sequence conservation of M1 to M4 in connexins and the similar structure demonstrated in Cx43 and Cx26 mutants, it is likely that all gap junction channels composed of connexins share similar structural characteristics, including pore structure. In both cryoem density maps, transmembrane span B and transmembrane span C in each connexin subunit are observed to line the pore. Span B is the major span that lines the pore in the narrow part while Span C lines the wide cytoplasmic mouth of the pore (Figure

77 65 5.1), but it is difficult to assign transmembrane segments M1 to M4 to the spans in the density maps, due to a resolution limit of about 7 Å. Fortunately, other methods can be used to solve this problem, for example, the SCAM method. Figure 5.1 The structural views of gap junction channels composed by Cx43L263Δ (A) and Cx26M34A (B). The left side shows the view looking toward the extracellular loop. The right side shows the side view of one Cx43L263Δ subunit or two opposing Cx26M34A subunits and the above space is the cytoplasm (Unger et al. 1999; Oshima et al. 2007). The scale bar is 20Å. Three different labs performed cysteine scanning in Cx46 connexons, Cx32E 1 43 connexons and Cx32 gap junction channels (Zhou et al. 1997; Kronengold et al. 2003; Skerrett et al. 2002). However, the data are inconsistent and result in two different models. Based on the data from Cx46 connexons and Cx32E 1 43 connexons, the outside half of TM1 and the initial part of E1 is the major span to line the pore (Zhou et al. 1997; Kronengold et al. 2003). In addition, data from Cx32E 1 43 suggested TM3 also lined the pore at the cytoplasmic mouth two years before the first connexon structure was published (Zhou et al. 1997). Based on the data from Cx32 gap junction channels, reactive residues

78 66 are found in all four spans and TM3 and TM2 are suggested to be the most accessible spans in the pore. The inconsistencies may arise from the thiol reagents used, the functional states of the channel, the measuring techniques, the sources of artifact and the constraints inherent in the mutagenesis analysis. It is premature to have a conclusion about the general atomic model for connexons. The identification of pore structure of a far-related pannexin1 channel by using SCAM will give independent insight into the structure of gap junction channels. Provided that the inhibitory effects of thiol reagents are due to blocking, our data suggest that the external portion of pore is lined by E1 and TM1 while the internal portion of the pore is lined by TM3, consistent with the SCAM data from Cx46 connexons and Cx32E 1 43 connexons. The SCAM data from Cx46 connexons, Cx32E 1 43 connexons and pannexin1 channels do not agree with the data from Cx32 gap junction channels. One simple reason could be that unpaired connexons or pannexons are different from gap junction channels and the pore structure may change during the docking interaction. However, the channel properties, including unit conductance, pharmacological study, selectivity and permeability instead indicate that a connexon is half of one gap junction channel and the pore structure does not change significantly during the docking interaction. In addition, the technique used for measuring the effect of thiol reagents on the Cx32 gap junction channel is problematic. In order to apply the thiol reagent to the gap junction area, one oocyte was cut and the thiol reagent had to pass the long way through the yolk, a procedure prone to possible artifacts (Skerrett et al. 2002). Therefore, it is highly possible that the thiol reagent reactivity in Cx32 gap junction channels is artifactual and should not be used with strong confidence.

79 67 Figure 5.2 Mapping the thiol reagent reactivity into pore structure. A. A map of reactive residues in pannexin1. The green spots indicate reactive positions T62, G61, I60, I58, F54 in TM1 and V221, L217 in TM3 from outside to inside. B. Side view of a half of a gap junction channel composed by Cx43L263Δ. The top is the extracellular space and the bottom is the cytoplasm. The red asterisk marks the narrowest part of the channel where the aqueous pore is about 15 Å in diameter (Adapt from Unger et al. 1999). The inhibition of thiol reagents MBB or MTSET at the initial part of E1 (I60, G61 and T62) and outside portion of M1 (I58) is substantial, while at inner positions, including the portions of M1 (F54, L49) and TM3 (L217, V221), the inhibition is weak. Because the inhibition level is significantly different along the pore, the pore is unlikely to be a cylinder. Given that the initial portion of E1, the outside half of M1 and inside half of M3 are predicted to line the pore from extracellular space to the cytoplasm and the thiol reagents will have bigger blocking effect in the narrow area than that in wide area, the extracellular mouth of the pore is probably narrower than the cytoplasmic mouth of the pore. This conclusion is consistent with the structure of the gap junction channels constructed from cryoem data (Unger et al. 1999; Oshima et al. 2007), where the pore is narrowest just outside of the plane of plasma membrane and has a wide opening to cytoplasm (Figure 5.2).