Structural Basis of Inward Rectifying Potassium Channel Gating G. Loussouarn, T. Rose, and C.G. Nichols*

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1 Structural Basis of Inward Rectifying Potassium Channel Gating G. Loussouarn, T. Rose, and C.G. Nichols* the channels, and finally by the solving of high-resolution structures. The goal of this article is to review recent experiments that have begun to pin down the structural basis of the inward rectifier channel permeation pathway, and the gating processes that open and close the permeation pathway. The last 10 years have seen rapid advances in the understanding of potassium channel function. Since the first inward rectifying (Kir) channels were cloned in 1994, the structural basis of channel function has been significantly elucidated, and determination of the crystal structure of a bacterial K channel (KcsA) in 1998 provided an atomic resolution of the permeation pathway. This review considers recent experimental studies aimed at uncovering the structural basis of Kir channel activity, and the applicability of comparative models based on KcsA to illuminate Kir channel pore structure and opening and closing processes. (Trends Cardiovasc Med 2002; 12: ). 2002, Elsevier Science Inc. The cardiac action potential is an essentially uniform electrical signal that repeats every second for 70 years or more in humans. This reproducibility requires a carefully orchestrated turning on and off of ion channel conductances. Between 1960 and 1980, the major conductances that underlie the different phases of the cardiac action potential (Figure 1A) were delineated. In diastole, cardiac myocytes are held at a negative membrane potential (typically 60 to 80 mv) by the high resting K conductance of the so-called inward rectifier (i K1 ) channels. Depolarizing currents from neighboring cells move the membrane potential to the threshold for voltage-gated Na currents, which generate the upstroke of the action potential. Voltage-gated Ca 2 currents are a major driving force for G. Loussouarn and C.G. Nichols are at the Department of Cell Biology and Physiology, and T. Rose is at the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA. * Address correspondence to: C.G. Nichols, Department of Cell Biology and Physiology Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA. Tel.: ( 1) ; fax: ( 1) , cnichols@cellbio.wustl.edu. 2002, Elsevier Science Inc. All rights reserved /02/$-see front matter the maintenance of a depolarized plateau potential, and then the slow activation of additional voltage-gated K conductances and reactivation of the inward rectifier bring the membrane potential back to the resting state. Many other conductances carried by transporters, exchangers, and additional background nonselective or chlorideconducting channels contribute to and refine this signal. Of particular relevance in the current context, several additional inward rectifying K (Kir) channels, including adenosine triphosphate (ATP)- inhibited (K ATP ) and muscarinic acetylcholine (ACH) receptor-activated (K ACh ) channels, contribute to alterations of excitability in response to altered metabolic demands and hormonal influences (Snyders 1999). Over the last 15 years, advances in gene cloning have permitted the molecular identification of the channel proteins that underlie each of the currents. The Kir channels consist of about 15 members in six subfamilies. Of these, Kir2.x subunits encode classical inward rectifier (I K1 ) currents, Kir3.x subunits encode G-protein-gated K ACh currents, and Kir6.x subunits encode K ATP channels (Nichols and Lopatin 1997). Cloning has been followed by structure function studies based on the effect of mutations or chimeras on functional properties of Structural Similarities between Kir Channels and KcsA Compared with thousands of soluble protein structures, only tens of membrane protein structures are known; and among hundreds of K channels, only one bacterial channel, KcsA, has thus far been crystallized (Doyle et al. 1998). Both KcsA and Kir channel subunits consist of two transmembrane helices (TMs) bridged by an extracellular pore loop (P loop) that contains the signature K selectivity sequence (Gly-Tyr-Gly) (Figure 1B). Although KcsA aligns more closely with the last two TMs and P loop of the six TM voltage-gated (Kv) channels, the overall structure seems conserved in all K channels, and indeed in all cation channels. Biochemical (Raab-Graham and Vandenberg 1998) and electrophysiological (Shyng and Nichols 1997, Yang et al. 1995) studies on channels expressed in heterologous systems demonstrate that four Kir subunits coassemble to generate Kir channels, as in KcsA. Structural homology in and around the selectivity filter of KcsA and Kir channels is likely, because comparative models of Kir channels based on the KcsA structure (e.g., Figure 1D) are consistent with at least three different experimental observations. 1. Differences in unitary conductance between Kir6.1 and Kir6.2 are explained in the model by steric hindrance at the extracellular side of the selectivity filter (Repunte et al. 1999). 2. A disulfide bond between the extremities of the P loop (Cys 122 and Cys 154 in Kir2.1; Cho et al. 2000, Leyland et al. 1999) seems to be required for the proper folding of the protein. Although this bridge does not exist in KcsA, comparative models place these cysteines in close proximity in Kir2.1 (Cho et al. 2000, Leyland et al. 1999) and Kir6.2 (Loussouarn et al. 2000). Comparative modeling is based on relatively low sequence identity between the Kir channel and KcsA, and the two Kir2.1 models TCM Vol. 12, No. 6,

2 mentioned above utilize slightly different alignments. Nevertheless, both bring the two cysteines close enough for a disulfide bridge. 3. Mutations that alter pore block by external Rb and Cs in Kir2.1 are close to the selectivity filter and line the pore according to these models (Thompson et al. 2000). There are conflicting experimental studies that bring this proposed structural similarity into question. One of the main features of the KcsA selectivity filter is the interaction of K ions with the main chain carbonyl oxygens of the signature sequence. The interaction requires the Y78 side chain to face away from the pore, but in Kir2.1 mutant 254 TCM Vol. 12, No. 6, 2002

3 Figure 1. The molecular basis of Kir channel gating. (A) Depolarization is generated and maintained by Na and Ca currents (ina, ica). Voltage-gated K currents (Kv) and Kir channels contribute to repolarization and maintenance of a negative resting potential. (B) Kir-channel subunits consist of two transmembrane domains (M1, M2), separated by a pore loop (P loop) that contains the signature K -selectivity sequence, as well as extended cytoplasmic N and C termini. Major roles of the P loop, M2, and the C terminus have been implicated in gating (red). (C) Potential structural basis of different gating modes, and corresponding current voltage relationships. The P loop, M2, and C termini of two subunits are represented in each of the four diagrams, which are based on the KcsA structure. Nonconducting or closed conformations of the channel may arise from changes in the selectivity filter ( purple, top left), polyamine (spermidine) block in the pore (yellow, top right), or rearrangements of M2 (stabilized by, e.g., ATP; red, bottom left). The open channel, stabilized by C-terminal interactions with phosphoinositol biophosphate (PIP 2), requires an open conformation (stabilized by PIP 2 ) of both the selectivity filter and M2 (green, bottom right). (D) Comparative modeling of closed (left) and open (right) states of the Kir6.2 channel. The Kir6.2 tetramer, displayed in red ribbons, was built with the KcsA crystal structure used as a template. The three-dimensional model was embedded in a bilayer of lipids, displayed in gray. The space accessible to ions and water molecules within the channel is displayed as a cyan solid and opens to the extracellular (top) and intracellular (bottom) compartments. The model in an open state (right) was built from the closed-state model by holding a large pore-accessible sulfhydryl reagent (qbbr) within the bundle crossing (Loussouarn et al. 2001). Notice the discontinuity of the water-accessible space on the left and the continuity on the right, and the very mild variations of the red (closed) and green (open) protein ribbons. Most of the gating can occur by side-chain rearrangements. Permeation of K ions, indicated by a diagram representation (purple CPK in motion) is possible in the open state, but not the closed state. Y145C (the equivalent tyrosine being mutated to a cysteine) the cysteine side chain is accessible to Ag, hinting that it faces the pore (Dart et al. 1998). However, Ag accessibility was observed with the equivalent mutation in the voltagegated K (Kv) channel Shaker (Lu and Miller 1995), requiring that the two main mammalian K channel families have a different P-loop structure than KcsA, despite the strong homology in this region. More likely, the substituted cysteine side chain is accessible to Ag, even when the equivalent tyrosine side chain is facing away from the central axis. Finally, the accessibility of substituted cysteines in M2 helices of Kir2.1 and Kir6.2 are both consistent with the predictions of three-dimensional models built on KcsA as a template: the pattern of accessibility of the introduced cysteines to sulfhydryl reagents (Lu et al. 1999) or Cd 2 (Loussouarn et al. 2000) are consistent with M2 being helical in both cases, and with one side facing the pore. Apparently at odds with this parsimony is the finding that the Kir2.1 pore can accomodate large sulfhydryl groups at a section where KcsA s pore is very narrow (Lu et al. 1999). The fact that a single cadmium ion can block the pore at the equivalent section in Kir6.2 (Loussouarn et al. 2000) would counterargue that the pore should be as narrow as KcsA at this level. These seemingly mutually exclusive conclusions are resolved by the observation that the Kir6.2 pore can also accommodate the same large sulfhydryl reagents as Kir2.1 (Loussouarn et al. 2001). Modeling Kir6.2 on the KcsA structure indicates that the same conformation of the channel may allow coordination of a single Cd 2 by all four cysteines, or accommodation of four ethylamine derivatives, one at each cysteine, with the side chains angling up through the central cavity toward the selectivity filter (Loussouarn et al. 2001). Using a yeast genetic screen to identify the environment of residues by their tolerance to mutations, Minor et al. (1999) concluded that pore-lining residues in Kir2.1 correspond to non-pore-lining residues in the KcsA crystal. Furthermore, in the KcsA crystal, M1 interacts only with M2 of the same subunit, but in Kir2.1, second site suppressor mutations indicate that M1 interacts with M2 of the same subunit and with M2 of the neighboring subunit (Minor et al. 1999). The first discrepancy may be resolved if we use the alignment proposed by Doyle et al. (1998) for KcsA with Kir1.1 (ROMK), which is highly homologous to Kir2.1. This alignment is consistent with the disulfide bridge discussed above, and aligns the pore-lining residues determined by Minor et al. (1999) with the porefacing residues in the KcsA crystal (Capener et al. 2000). The second discrepancy may originate from the fact that different methods do not obviously report the same channel conformation. The KcsA crystal structure may represent the closed channel (Perozo et al. 1999), whereas the yeast selection experiments require open channels and it is more likely that the inferred structure represents the open channel. Conceivably, M1 moves from a position in which it interacts with M2 of another subunit (in the open conformation) to a position in which it does not (closed conformation), as suggested for Kir3.1 channels (Yi et al. 2001). In conclusion, none of the structural constraints inferred from mutagenesis studies are completely inconsistent with comparative models of Kir channels. Bearing in mind the fact that KcsA represents only one channel state and this is likely to be closed to permeant ions such models may be used as a starting point for considering molecular dynamics (Capener et al. 2000). Finally, it has recently been demonstrated that the pore of KcsA can functionally substitute for that of Kv or Kir channel pores, and maintain many regulatory processes (Lu et al. 2001). This work goes a long way toward strengthening the idea that there is no fundamental structural difference between the various K channels within the pore-forming regions. Where and What Are the Gates that Open K Channels? All potassium channels exist in at least one fully open and one fully closed state. Gating that is, the transitions between these states is controlled by a wide range of physiological variables. Those channels that predominantly control repolarization of the action potential are gated by voltage: membrane depolarization promotes opening and repolarization promotes closure. These channels are encoded by Kv subunits, each of which typically consist of six TMs (S1 S6), one of which (S4) contains repeated positive charges. Voltage is sensed by the movement of these charges within the membrane field, and systematic mutagenesis combined with fluorescent tagging experiments have provided significant insight into the nature of the S4 movements (reviewed in Bezanilla 2000). Other potassium channels that lack S4, including the two TM inward rectifiers, are generally gated by TCM Vol. 12, No. 6,

4 cytoplasmic ligands such as G proteins and intracellular nucleotides, and, again, systematic mutagenesis has begun to reveal the binding sites for these ligands. Moreover, for all the insights that have been gained with regard to channel structures that sense the gating ligands or voltage, a question that is only now being addressed is the nature of the gates themselves. In some cases, gating actually involves a block of the pore by the gating particle. Perhaps the best-delineated example is the fast, so-called N-type inactivation (closure after voltage-dependent opening) that occurs in some Kv and Na channels, and the inward rectification of Kir channels. N-type inactivation involves a blocking of the inner entrance of the channel by the N terminus of the channel (Hoshi et al. 1990). Recent studies that combined crystallization and mutagenesis experiments provide compelling evidence that a few amino acids near the N-terminal enter into the inner vestibule to cause this voltage-dependent block (Zhou et al. 2001). Inward rectification involves a pore block by intracellular Mg 2 and linear polyamines (putrescine, spermidine, and spermine) (Nichols and Lopatin 1997) (Figure 1C). Again, accumulating evidence indicates that the binding site for the polyamines is either in the inner vestibule, or the entrance to the selectivity filter itself (Oliver et al. 1998; Pearson and Nichols, 1998; Spassova and Lu, 1998), but confirmation awaits cocrystallization studies. In other cases, gating seems to involve a genuine closure of the permeation pathway, rather than an extrinsic block. What is the nature of such closing? Electron Paramagnetic Resonance experiments, which probe the environment and position of introduced cysteines in purified KcsA in solution, provide a picture of the probable structural basis of ph-dependent gating of this bacterial channel (Perozo et al. 1999). Transition from the open to the closed conformation is accompanied by significant motion of the transmembrane domains and by subtle motions of the lower part of the selectivity filter, and these two moving parts present as obvious candidates for the gates: (1) a small perturbation in the structure of the selectivity filter could be enough to disrupt potassium ion permeation; (2) movement of the M2 domain (and M1) could narrow the pore at the bundle crossing and prevent ion flow across this section. A type of inactivation that follows voltage-dependent activation (so-called C-type inactivation) in Kv1 channels seems to occur by a collapse of the selectivity filter, but activation involves residues at the cytoplasmic end of S6. Comparative modeling of Kv1 channels based on KcsA suggests that the activation gate may be due to a constriction at the bundle crossing (del Camino and Yellen 2001), consistent with the observed decrease in the accessibility of introduced cysteines above the bundle crossing in the closed state (for a review, see Yellen 1998). In cyclic nucleotide-gated (CNG) channels, there is also evidence that the selectivity filter, as well as conformational change of S6, is associated with cyclic nucleotide activation of the channel (Flynn et al. 2001). Although the data on Kv1 channels seems to provide compelling support for the argument that closure occurs by block of permeation at the bundle crossing, the rate of Ag modification of a cysteine introduced in the inner vestibule of CNG channels is not reduced in the closed state. This suggests that at least in the case of this K channel cousin, if a conformational change of the inner pore does occur when the channel closes, it does not constitute a gate to an ion as small as K (Flynn and Zagotta 2001). How Many Gates Are in Kir Channels? Both selectivity filter rearrangements and M2 motion have been proposed to occur, and may actually coexist, in Kir channels (Figure 1C). Analysis of single K ATP (Kir6.2) channels shows bursts of activity with fast transitions between open and closed states, separated by long closures. The long closures, but not the short intraburst closures, are dependent on intracellular ATP (ATPi), becoming longer as ATPi increases (Enkvetchakul et al. 2000). Long closures are also evident in other Kir channels. In Kir1.1, for example, intracellular acidification leads to the appearance of a long-lived closed state ( 1 s) (Choe et al. 1997). In both of these examples, decreased membrane phosphoinositol bisphosphate (PIP 2 ) favors the long closed state (Leung et al. 2000, Shyng et al. 2000, Shyng and Nichols 1998), increasing proton sensitivity of Kir1.1 (Leung et al. 2000), and increasing ATP sensitivity in Kir6.2 (Shyng et al. 2000), thus suggesting a similar gating mechanism, in which PIP 2 stabilizes the open state and ATP or H stabilizes the closed state. Mutations in M2 affect the slow gating in at least two different Kir channel families (Kir6.x and Kir3.x). In Kir6.2, both application of PIP 2 and mutations in M2 stabilize the open state, reducing the long-lived closed time (Figure 1C) (Enkvetchakul et al. 2000). Comparative modeling suggests that Cd 2 coordination by introduced cysteines in M2 requires some movement of the helices (Loussouarn et al. 2001). For all residues, PIP 2 reduces the Cd 2 on rate, possibly by stabilizing the open state and reducing the necessary motion (Loussouarn et al. 2000). In Kir3.x channels, mutations in M2 also stabilize the channel open state by increasing the burst duration (Sadja et al. 2001). Near the bundle crossing in M2, V188 tolerates only substitution by hydrophobic residues in the closed channel but can be replaced with other residues in a constitutively open channel, suggesting a change in the environment of this residue during gating, again consistent with M2 motion (Yi et al. 2001). This proposed flexibility of the helices is reminiscent of the gating mechanism suggested for KcsA (Perozo et al. 1999) and it is possible that the slow gate is itself generated by a constriction of the bundle crossing due to movement of the TMs, as discussed above for Kv1 channels (del Camino and Yellen 2001). In both the KcsA crystal and in Kir6.2 models based on KcsA, the bundle crossing is narrow enough to prevent the passage of potassium ions (Capener et al. 2000, Loussouarn et al. 2000, Shrivastava and Sansom 2000). The reductions of single-channel conductance that result from MTSEA modification of residues near the bundle crossing (Loussouarn et al. 2001, Lu et al. 1999) clearly indicate that this region can act as a significant barrier in the permeation process, yet the accessibility of sulfhydryl reagents much larger than K ions through this region to the inner vestibule also indicates that a certain minimum diameter must be achieved in the open state. Comparative models (Figure 1D) indicate that minimal backbone M2 movement, accompanied by side-chain rearrangement, may actually increase the diameter of the pore through this region con- 256 TCM Vol. 12, No. 6, 2002

5 siderably (Figure 1D). Because Kir6.2 channels are closed by ATP, it is experimentally feasible to examine substituted cysteine accessibility in open and closed states. Although accessibility of MTS reagents to some M2 cysteine substitutions is reduced in the ATP closed state, closure appears to accelerate MTS modification of other cysteines (L.R. Phillips, C.G. Nichols unpublished data). Thus the picture emerging from these studies may be more similar to that in CNG channels, in which M2 motions occur during gating, but the bundle crossing may not normally constitute a gate to permeant ions. The burst characteristics of Kir6.2 and other Kir channels are typically independent of gating ligands, suggesting the presence of an additional fast gate. Mutations in the selectivity filter have recently been demonstrated to modify the burst kinetics (Proks et al. 2001), consistent with the fast gate being in this part of the protein (Figure 1C). The kinetics are slightly different between channels, but analysis of Kir1.1/Kir2.1 chimeras again suggests that this difference is conferred by the region of the P loop that bears the selectivity filter (Choe et al. 1999). Singleresidue mutations (So et al. 2001) and even more subtle changes in the selectivity filter of Kir2.1 utilizing unnatural amino acids to alter the backbone chemistry (changing an amide carbonyl to an ester carbonyl) (Lu et al. 2001) lead to dramatic changes in the fast gating, again consistent with a gate in the selectivity filter. Perspective The goal of current research on the structure function relationships of ion channels is to define at the atomic level the fundamental processes and structures that govern the movement of ions through the channel pore, and the gating of the channel pores. It is becoming increasingly clear that all cation channels share the same fundamental pore architecture, and may share similar mechanisms of gating. Nevertheless, there are subtle yet important differences between channels that are dependent on the specific residues that contribute to the relevant segments and give rise to the significantly different physiologically relevant gating. Note added in proof: Recent crystallization of the MthK K channel (Jiang et al Nature 417: ) in a presumable open state has dramatically illustrated one mechanism of opening. This structure differs from that of KcsA in that the M2 helices are splayed open at 40, sufficient to generate a large opening at the equivalent position to the narrow bundle crossing in KcsA. It remains to be seen whether this mechanism holds for inward rectifier K channels. References Bezanilla F: The voltage sensor in voltage-dependent ion channels. Physiol Rev 80: Capener CE, Shrivastava IH, Ranatunga KM, et al.: Homology modeling and molecular dynamics simulation studies of an inward rectifier potassium channel. 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6 Raab-Graham KF, Vandenberg CA: Tetrameric subunit structure of the native brain inwardly rectifying potassium channel Kir 2.2. J Biol Chem 273:19,699 19,707. Repunte VP, Nakamura H, Fujita A, et al.: Extracellular links in Kir subunits control the unitary conductance of SUR/ Kir6.0 ion channels. EMBO J 18: Sadja R, Smadja K, Alagem N, et al.: Coupling Gbetagamma-dependent activation to channel opening via pore elements in inwardly rectifying potassium channels. Neuron 29: Shrivastava IH, Sansom MSP: Simulations of ion permeation through a potassium channel: molecular dynamics of KcsA in a phospholipid bilayer. Biophys J 78: Shyng SL, Barbieri A, Gumusboga A, et al.: Modulation of nucleotide sensitivity of ATP-sensitive potassium channels by phosphatidylinositol-4-phosphate 5-kinase. Proc Natl Acad Sci USA 97: Shyng SL, Nichols CG: Octameric stoichiometry of the KATP channel complex. 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Neuron 29: Zhou M, Morais-Cabral JH, Mann S, et al.: Potassium channel receptor site for the inactivation gate and quaternary amine inhibitors. Nature 411: PII S (02) TCM Scavenger Receptors that Recognize Advanced Glycation End Products Akira Miyazaki,* Hitoshi Nakayama, and Seikoh Horiuchi Scavenger receptors recognize modified low-density lipoproteins (LDLs) such as acetylated LDL and oxidized LDL. Advanced glycation end products (AGE), which are generated through long-term exposure of proteins to glucose, also behave as active ligands for some scavenger receptors, including class A scavenger receptor (SR-A) and class B scavenger receptors such as CD36 and scavenger receptor, class B, type I (SR-BI). SR-BI, the first identified high-density lipoprotein (HDL) receptor, plays key roles in reverse cholesterol transport by promoting selective uptake of cholesteryl esters (CE) in HDL by hepatocytes, and cholesterol efflux of unesterified cholesterol from peripheral cells to HDL. Using Chinese hamster ovary cells overexpressing SR-BI (CHO- SR-BI cells), it was demonstrated that AGE-bovine serum albumin binds to SR-BI and inhibits selective uptake of HDL-CE by CHO-SR-BI cells as well as cholesterol efflux from CHO-SR-BI cells to HDL, suggesting potential roles of AGE in diabetic dyslipidemia and accelerated atherosclerosis in diabetes. (Trends Cardiovasc Med 2002;12: ). 2002, Elsevier Science Inc. Advanced Glycation End Products In the Maillard reaction, proteins react with glucose to form Schiff bases and Amadori products. After long incubation, these early products are converted to advanced glycation end products (AGE), which are characterized physicochemically by fluorescence, brown color, and intra- or intermolecular cross-linking; and biologically by specific recognition by AGE receptors (Baynes and Thorpe, 1999, Horiuchi 1996, Vlassara 1997). Immuno- Akira Miyazaki Seikoh Horiuchi are at the Department of Biochemistry, Kumamoto University School of Medicine, and Hitoshi Nakayama is at the Department of Biofunctional Chemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan. * Address correspondence to: Akira Miyazaki, Department of Biochemistry, Kumamoto University School of Medicine, Honjo, Kumamoto , Japan. Tel.: ( 81) ; fax: ( 81) ; akiramyz@kaiju.medic.kumamoto-u.ac.jp. 2002, Elsevier Science Inc. All rights reserved /02/$-see front matter histochemical studies have revealed the presence of AGE-modified proteins in human and animal tissues under various pathological conditions related to aging and age-related disorders such as diabetic macro- and microangiopathy (Mitsuhashi et al. 1993, Nakamura et al. 1994), atherosclerosis (Kume et al. 1995), Alzheimer s disease (Smith et al. 1994, Vitek et al. 1994), and various types of amyloidosis characterized by deposition of abnormal amyloid fibril proteins (Miyata et al. 1993). AGE proteins are known to induce a variety of cellular events in vascular wall cells and other cells, possibly through the functions of so-called AGE receptors that recognize AGE, thereby modulating the above disease processes. Receptor for AGE (RAGE) and Galectin-3 as AGE Receptors Receptor for AGE (RAGE), which was originally purified from bovine lung endothelial cells (Neeper et al. 1992) as a 35- kda protein that belongs to the immu- 258 TCM Vol. 12, No. 6, 2002