Patch-Clamp Fluorometry: Electrophysiology meets Fluorescence

1250 Biophysical Journal Volume 106 March 2014 1250 1257 Patch-Clamp Fluorometry: Electrophysiology meets Fluorescence Jana Kusch * and Giovanni Zifarelli * Universitätsklinikum Jena, Institut für Physiologie II, Jena, Germany; and Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Genova, Italy ABSTRACT Ion channels and transporters are membrane proteins whose functions are driven by conformational changes. Classical biophysical techniques provide insight into either the structure or the function of these proteins, but a full understanding of their behavior requires a correlation of both these aspects in time. Patch-clamp and voltage-clamp fluorometry combine spectroscopic and electrophysiological techniques to simultaneously detect conformational changes and ionic currents across the membrane. Since its introduction, patch-clamp fluorometry has been responsible for invaluable advances in our knowledge of ion channel biophysics. Over the years, the technique has been applied to many different ion channel families to address several biophysical questions with a variety of spectroscopic approaches and electrophysiological configurations. This review illustrates the strength and the flexibility of patch-clamp fluorometry, demonstrating its potential as a tool for future research. INTRODUCTION Ion channels are pore-forming membrane proteins, which allow ions to permeate across the membrane. They are involved in basic physiological processes such as the control of resting and action potentials, ionic homeostasis, exocytosis, and muscle contraction. Their activity is regulated by specific stimuli, including voltage, ligand binding, temperature, or mechanical stress. These stimuli cause conformational rearrangements that result in various gating transitions between different functional states: open, closed, inactivated, and desensitized. The function and regulation of ion channels have been extensively investigated over several decades using electrophysiological techniques, while detailed structural information has been provided by x-ray crystallography (1) and nuclear magnetic resonance spectroscopy (2). However, none of these approaches permit a direct correlation between ion channel conformations and the associated functional states. To overcome this limitation, different kinds of spectroscopic techniques have been combined with classical electrophysiological approaches to simultaneously monitor and thus directly correlate ion channel function with conformational changes. The first steps in this direction were made in the 1990s through the establishment of the so-called voltage-clamp fluorometry (VCF) that employed either the two-electrode voltage-clamp (3) or the cut-open technique (4) to investigate the movements of the Shaker potassium channel voltage sensor during the process of voltage gating. Soon after, the technique was applied to investigate other ion channels and transporters. VCF relies on the sensitivity of specific fluorophores to the hydrophobicity of their local environment (3). Site-directed fluorescence labeling is achieved by coupling the fluorophores to the sulfhydryl Submitted October 17, 2013, and accepted for publication February 6, 2014. *Correspondence: jana.kusch@med.uni-jena.de or zifarelli@ge.ibf.cnr.it Editor: Brian Salzberg. group of cysteine residues inserted at desired locations within the protein. Conformational changes in the region around the fluorophore lead to changes of its environment and thereby to changes of its emission spectra (3,4). Although VCF provides structural information of limited resolution, it does enable parallel recording of conformational changes and ion-transport activity in a near-native membrane environment and thus provides the possibility to directly correlate structure and function. Moreover, VCF allows the detection of conformational changes associated with electrically silent states (closed, inactivated, and desensitized states) (5,6) and to monitor the activity of transporters that are not electrogenic and cannot be investigated by standard electrophysiological techniques (7). In particular, VCF applying the cut-open technique (8) provides increased time resolution of electrophysiological measurements and access to the intracellular side of the proteins compared to standard voltage-clamp (9). Inspired by the principles of VCF, Zheng and Zagotta (10,11) established the patch-clamp fluorometry technique (PCF), an approach combining fluorescence recordings and patch-clamp recordings in the inside-out configuration. In general, PCF offers three major advantages over VCF: 1. Direct and fast access to the intracellular side of ion channels with the possibility to label intracellular amino acids and apply intracellular ligands and channel modulators to control channel gating; 2. Better signal/noise of fluorescence signals by excluding the intracellular milieu causing high autofluorescence; and 3. High time resolution of current recordings. Subsequently, PCF has been applied in a variety of electrophysiological and spectroscopic approaches to an enormous number of different ion channels. This review cannot cover this huge field in detail, but will focus on what is generally considered the classical PCF method based on the inside-out Ó 2014 by the Biophysical Society 0006-3495/14/03/1250/8 $2.00 http://dx.doi.org/10.1016/j.bpj.2014.02.006

Patch-Clamp Fluorometry: Electrophysiology meets Fluorescence 1251 configuration of the patch-clamp technique. We summarize the main technical developments in the context of the results obtained (for other excellent reviews, see Zheng (12) and Taraska and Zagotta (13)). Other PCF approaches based on different electrophysiological configurations will be discussed only briefly. CLASSICAL PCF FLUORESCENCE AND CURRENT RECORDINGS IN EXCISED INSIDE-OUT PATCHES In principle, PCF can be applied to study different biophysical properties of any type of ion channel, provided that they are expressed in the plasma membrane (Fig. 1). However, so far the technique has been used to investigate two major classes of biophysical properties: 1. Conformational changes associated with gating in cyclic nucleotide-gated (CNG) and K þ channels, and 2. The relation of ligand binding and gating in the closely related hyperpolarization-activated and cyclic-nucleotide modulated (HCN) and CNG channels. These applications are discussed in detail in the following sections. Relating conformational changes and ion channel gating Conformational rearrangements due to cyclic-nucleotide binding in CNG channels CNG channels play fundamental roles in both visual and olfactory signal transduction (14). While structurally similar to voltage-gated cation channels, CNG channels are only weakly voltage-dependent and are activated by binding of FIGURE 1 Technical overview of patch-clamp fluorometry. The enormous variability of PCF, given by the number of possible combinations of spectroscopic and electrophysiological tools, makes it a potent approach for studying ion channels and transporters, thus overcoming the limitations of the individual techniques. cyclic nucleotides to an intracellular C-terminal binding domain (CNBD) present in each of the four subunits. The CNBD is linked to the pore region by a complex a-helical structure, the so-called C-linker (15). PCF permitted detailed investigation of the spatial arrangements within the C-terminal channel portion and it provided important insights into how conformational changes due to cyclic nucleotide binding are transmitted from the cytoplasmic CNBD to the transmembrane domain (10,16). The first study dealing with this problem was the pioneering work of Zheng and Zagotta in 2000 (10). They investigated the conformational changes near the residue C481, located in the C-linker region of rod CNGA1 subunits, by fluorescence quenching. This residue was labeled by perfusing excised inside-out patches with the maleimideconjugated dye AlexaFluor 488, which is able to covalently bind to the sulfhydryl group of the cysteine residue. Quenching was assessed by the anionic quencher iodide and the cationic quencher thallium, respectively, in the absence and in the presence of cgmp, i.e., in closed and open channels. Iodide had a higher quenching efficiency in the channel s closed state whereas thallium had a higher quenching efficiency in the open state. This suggested that positively charged or dipolar residues present in the C-linker around C481 in the closed state rearrange upon channel activation, producing a movement of the positive charges away from the local environment of the dye. Although this structural insight was relatively limited, Zheng and Zagotta (10) showed very elegantly the potential of the technique in providing simultaneous information about structural changes and channel function. In particular, they demonstrated for the first time that rearrangements of the C-linker are associated with activation of CNG channels (10). A more detailed structural investigation of gating conformational changes produced by cyclic nucleotide binding in rod CNG channels was performed by applying PCF in combination with Förster resonance energy transfer (FRET), a technique that allows the monitoring of the relative distance between fluorophores (see Fig. 2 s legend for further description) (16). FRET was measured between either GFP or the dyes AlexaFluor 488 and AlexaFluor 568 labeling cysteine residues inserted at different sites in the C-terminus (used as donors) and dipicrylamine, a small, negatively charged nonfluorescent membrane probe (used as acceptor). Quantification of FRET signals were based on a spectral approach that monitors the whole spectrum of the dyes fluorescence emission rather than a selected wavelength with several advantages over other methods (12). Because dipicrylamine moves in a voltage-dependent manner either into the inner or outer leaflet of the membrane, FRET could provide information about the relative distances between the respective membrane leaflet and the different C-terminal regions labeled. Comparison of FRET data in the presence and absence of the agonist cgmp showed that, upon channel activation, none of the

1252 Kusch and Zifarelli FIGURE 2 FRET, one of the most often used fluorescence phenomena in PCF. (A) The Jablonski diagram shows the ground (S 0 ) and excited (S 1,S 2 ) states of two fluorophores composing a Förster resonance energy transfer (FRET) pair. FRET is the transfer of the excited state energy from an initially excited donor molecule D to an acceptor A that does not necessarily need to be a fluorescent molecule (67). This energy transfer is the result of long-range dipole-dipole interactions between D and A. The rate of energy transfer depends upon 1), the spectral overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor; 2), the quantum yield of the donor; 3), the relative orientation of D and A transition dipoles; and 4), the distance between D and A. In detail, the FRET efficiency E is inversely proportional to the sixth power of the distance r between D and A, when the distance is close to the Förster distance R 0. R 0 is the distance at which the energy transfer efficiency is 50% (see formula in Lakowicz (68)). (B) FRET typically occurs over distances comparable to the dimensions of macromolecules, and therefore has been intensively used as a type of spectroscopic ruler in studying conformational changes in ion channel proteins. FRET produces a decrease of D fluorescence (shown as blue fluorophore, e.g., CFP) and an increase in A fluorescence (shown as yellow fluorophore, e.g., YFP), and can therefore be quantified either by donor dequenching in which an increase in D fluorescence is measured after complete photobleaching of A, or by enhanced acceptor emission (also called sensitized emission ) in which the parameter measured is the increase in fluorescence intensity of A due to FRET with D. Alternatively, FRET quantification can be performed by fluorescence lifetime measurements of D and A (69). C-terminal subdomains moved perpendicular to the membrane. There was only a minor lateral movement, indicating that subtle conformational changes are sufficient to drive channel activation (16). Conformational rearrangements due to Ca 2þ -calmodulin binding in CNG channels PCF employing FRET was also applied to study the mechanism of Ca 2þ -calmodulin (Ca/CaM) modulation in olfactory (17) and rod CNG channels (18). CNG channel opening leads to an increased intracellular Ca 2þ level that causes a downregulation of the channels via Ca/CaM binding. This realizes a negative feedback circuit, which is essential for olfactory adaptation and light adaptation in retinal cones (14). CFP and YFP were fused either to the N- or C- terminus or both of CNG chimeric channels. FRET could be observed only between CFP and YFP present on different subunits, directly confirming (to our knowledge, for the first time) a cytoplasmic intersubunit interaction between N- and C- termini in full-length CNG channels. Moreover, upon application of Ca/CaM to the intracellular side of an excised patch, the FRET signal decreased with the same time course as the ionic current which demonstrated that Ca/CaM binding is directly responsible for the separation or reorientation of N- and C-termini, disturbing their interaction and thereby leading to channel inhibition (17). The mechanism of Ca/CaM binding was also directly investigated in rod CNG channels perfusing the intracellular side of excised patches with CaM molecules conjugated with AlexaFluor 488 (Ca/CaM-488) and monitoring the binding/unbinding time courses as well as FRET between Ca/CaM-488 and channel-attached CFP. The results showed a direct association between CaM and the N-terminus of CNGB1 subunits and a rotation or separation of the N- and C-termini in the presence of Ca/CaM (18). Conformational rearrangements due to ph changes in Kir1.1 channels PCF was applied to investigate the conformational changes related to ph-gating in Kir1.1 inwardly rectifying potassium channels (19). Kir1.1 channels are responsible for K þ secretion in the kidney (20) and are exquisitely sensitive to intracellular ph. Several hypotheses were proposed to explain the mechanism of ph regulation (21). Lee and Shieh (19) adopted PCF employing FRET between CFP and YFP to investigate ph-dependent conformational changes in the N- and C-termini. Because both fluorescent probes change their spectral properties in the ph range that modulates Kir1.1, a channel mutant was used instead of the wildtype, characterized by a shift of the ph dependence of gating to higher ph values, which do not affect the probe s spectra. Lee and Shieh (19) found that a change of the ph from 7.4 to 10, leading to channel opening in the mutant, was accompanied by a decrease of FRET efficiency, indicating an increased distance between N- and C-termini. Voltage- and Ca 2þ -dependent conformational changes in BK channels Large-conductance voltage- and calcium-dependent potassium (BK) channels are important for neuronal firing, neurosecretion, and smooth muscle tone (22). They show the typical structural features of voltage-gated potassium channels but, in addition, each subunit is characterized by a large cytoplasmic C-terminus. This channel portion comprises two regulatory domains of conductance (RCK domains), also referred to as the gating ring, where Ca 2þ binding promotes channel opening (23). This represents an important feedback system to control the intracellular Ca 2þ level. Crystal structures of the isolated gating ring

Patch-Clamp Fluorometry: Electrophysiology meets Fluorescence 1253 indicated Ca 2þ -induced conformational changes (24 26), but a full understanding of the coupling between conformational changes due to Ca 2þ binding to the cytoplasmic domain and channel activity is still missing. Miranda et al. (27) investigated this problem by applying PCF employing FRET in inside-out patches of Xenopus laevis oocytes. They studied the relative movements of subunits at three different locations within the gating ring. One site was located in the linker between RCK1 and RCK2, a second within the RCK2 domain, and a third was adjacent to the Ca 2þ binding site known as the calcium bowl. CFP or YFP was attached to either of these sites in one subunit and the labeled subunits were coexpressed. FRET was measured as emission spectra ratio. Ca 2þ -dependent FRET signals arising from fluorophores on different subunits indicated larger rearrangements of the gating ring than predicted by existing x-ray structures of the isolated gating ring. Surprisingly, these rotation-like rearrangements are not simply related to the open-closed transitions of the channel, which is in contrast to the currently accepted model of Ca 2þ - and voltage-dependent gating in BK channels (28). Relating ligand binding to ion channel gating Traditional ligand-binding assays, such as radioligand binding assays, isothermal titration calorimetry, or fluorescence polarization, do not relate conformational information to functional states, because they solely detect ligand binding to either isolated membrane preparations (often containing premature channel proteins) or to purified proteins that are not in their natural environment. Chang and Weiss (29) made a first step in the direction of directly relating binding and gating experimentally. They measured binding of tritium-labeled GABA and ionic current through GABA receptors in the same cell, but not simultaneously. Simultaneous recording of both processes were realized, to our knowledge for the first time, when employing fluorescently-labeled ligands in PCF approaches (30). Cyclic-nucleotide binding and gating in CNG channels To study ligand binding and gating in CNG channels using PCF, a fluorescent cgmp analog was used that activates homotetrameric CNGA2 channels in a similar manner as the physiological ligand cgmp (30). Simultaneously measured concentration-activation and concentration-binding curves reflecting channel behavior under steady-state conditions, together with multiple current-time courses induced by cgmp concentration jumps, were used to perform a global fit analysis to establish a kinetic 10-state Markov model i.e., the C4L model. The C4L model contains binding steps for each of the four closed states, among which the second is the most critical, switching the channel from a mostly closed to a maximally open channel. Interestingly, the established Monod-Wyman-Changeux model in Goulding et al. (31) and the coupled dimer model in Liu et al. (32) were unsatisfactory in describing the experimental data. To further analyse ligand binding and activation gating in CNGA2 channels in terms of Markovian models, PCF employing fcgmp was additionally performed at increased time resolution (33). Global fit analysis revealed two pathways for ligand unbinding: whereas the partially liganded channel unbinds its ligands slowly and only from closed states, the fully liganded channel reaches an open state from which it unbinds all four ligands very rapidly. The authors concluded that this concentration-dependent gating enables the channel to respond to changes in the cyclic nucleotide concentration with different kinetics. PCF was furthermore successfully applied to study ligand binding to different types of subunits in heterotetrameric olfactory CNG channels (34). It could be shown that the modulatory subunit CNGA4 but not CNGB1b is able to bind cgmp when forming a heterotetrameric channel together with the principle subunit CNGA2, whereas both modulatory subunits can bind camp. This suggests an important role of the CNGB1b subunit in ligand discrimination. Cyclic-nucleotide binding and gating in HCN channels A few years later, the same group successfully transferred this technical approach to homotetrameric HCN2 channels using a fluorescently-labeled camp analog (fcamp) (Fig. 3) (35). HCN channels play an important role in regulating heartbeat, and are involved in several neuronal processes (36). They are activated by hyperpolarizing voltages and modulated by the binding of camp. camp binding shifts the steady-state activation to less negative potentials, enhances the maximal open probability, and accelerates the activation kinetics. This duality of activating stimuli allows for changes of the true affinity of the ligand induced by channel activation to be monitored. Performing fluorescence and current recordings under steady-state and non-steadystate conditions provided the experimental proof that ligand binding and channel gating interdepend on each other: The affinity increased during channel activation, but decreased during channel deactivation (Fig. 3). Furthermore, the authors showed that ligand binding to only two of the four subunits is sufficient to maximally activate the channel, resembling the results on CNGA2 channels (30). To study ligand binding and unbinding behavior of these channels in more detail, Kusch et al. (37) performed fast fcamp-concentration jumps to record ligand-induced channel activation and deactivation kinetics in parallel to the respective kinetics of the increase and decrease in ligand binding. Using the whole set of kinetic data, they performed a global-fit analysis to estimate a full set of rate and equilibrium constants. This involved a 10-state Markov model, which was similar to the one used for describing CNG channel gating (30), but not identical because it also included

1254 Kusch and Zifarelli FIGURE 3 Measuring binding and gating in HCN2 channels using fluorescently-labeled camp. (A) A confocal image showing a pipette tip containing an inside-out patch excised from an X. laevis oocyte expressing HCN2 channels. The patch was perfused with the fluorescent camp analog fcamp (green, 0.75 mm) and a reference dye DY647 (red, 1mM). DY647 does not label the channel and is used for subtraction of the background fluorescence (reprinted from Neuron, 2010, Kusch et al., 67:75 85, with permission from Elsevier). The graph shows the distribution of the fluorescence intensities for both dyes along the pipette axis (indicated by the white line). The peak of the fcamp fluorescence (highlighted with white background) indicates the specific labeling of the channel, whereas the DY647 signal is purely determined by the geometry of the pipette and the patch membrane. Only the dome of the patch, assumed to be responsible for the main portion of the ionic current flow, is taken into account for quantifying ligand binding. (B) Representative simultaneous measurements of fluorescence (green) and current (black) traces, indicating binding and gating, respectively, upon application of an activating voltage step from 30 to 130 mv (black triangle) in the presence of 0.75 mm fcamp (reprinted from Neuron, 2010, Kusch et al., 67:75 85, with permission from Elsevier). The fluorescence signal was inverted and normalized with respect to the current trace. (Insets) Confocal images at the respective time points (gray lines). binding events to the open states. A complex cooperativity between the four subunits has been proposed, leading to the conclusion that channel states with zero, two, or four ligands bound are energetically preferred (37 39). Wu et al. (40) established a slightly different approach using the camp analog 8-NBD-cAMP, which is strongly fluorescent in a hydrophobic environment, such as in the cyclic-nucleotide binding pocket, but weakly fluorescent in an aqueous environment. They found the same increase in camp binding affinity during channel activation, as described earlier in Kusch et al. (35). However, whereas the kinetics of affinity decrease due to channel deactivation superimposed with the deactivation kinetics in Kusch et al. (35), Wu et al. (40) found it to be slower. Due to the similarities in the activation-related results of these two studies, this difference is quite surprising, but could be due to the different types of camp analogs used. Nevertheless, the fact that most of the findings are comparable suggests that PCF approaches employing fluorescently-labeled ligands are suitable to study ligand binding in ion channels. Wu et al. (41) used the same approach to investigate which discrete elements are responsible for this state-dependent interaction between camp and whole channel protein. They performed PCF to study ligand binding in the presence of the ionic blockers, Cs þ and Mg 2þ, respectively, both acting close to or within the selectivity filter, and the open channel blocker ZD7288, acting close to the activation gate at the intracellular end of the transmembrane S6 domain. Only the ZD7288 block reduced the activity-dependent increase in camp binding significantly. This led to the conclusion that movements in the channel activation gate, a region ~50 Å apart from the ligand-binding domain, directly affect ligand binding affinity. Wu et al. (41) consolidated this functional connection by an alanine scan of the intracellular end of S6. Additionally, using a combination of NMA (normal mode analysis)-based covariance analysis (a computational approach for studying protein dynamics) and PCF, it was found that residues distant from either the CNBD or the activation gate can significantly alter the channel response to camp (42). However, these residues only had minimal effects on the local interaction between camp and the isolated C-LinkerþCNBD fragment. These results emphasize the need to investigate camp-whole channel interactions rather than just interactions between camp and isolated CNBDs to learn about the channel s binding behavior (40 42). To have a closer look at the ligand entry-exit path of cyclic-nucleotide binding domains of HCN channels, Xu et al. (43) investigated a mutation of a conserved serine residue (44) in the core of the CNBD, which reduces the sensitivity to camp, but leaves the gating efficacy unchanged. A release of 8-NBD-cAMP from the mutant channel, which was almost twice as fast as from the wild-type, prompted the authors to conclude that the enormous reduction of camp sensitivity was due to a destabilization of

Patch-Clamp Fluorometry: Electrophysiology meets Fluorescence 1255 the camp molecule in the binding pocket. X-ray crystallography experiments included in this study indicated that the serine residue helps to keep an adjacent loop in an ordered structure, which is known to be important for the campdependent responses of the full-length channel (43 45). PCF EMPLOYING FLUORESCENTLY LABELED MEMBRANE LIPIDS Recently, Nomura et al. (46) reported the application of PCF to study bacterial mechanosensitive ion channels of small (MscS) and large (MscL) conductance, functioning as safety valves, which protect bacteria from rupturing upon a challenge by hypoosmotic shock (47). The dependence of channel activation on membrane tension was monitored after co-reconstituting both channel types into artificial liposomes containing azetolectin (99.9%) and rhodamine-conjugated phosphatidylethanolamine (0.1%). In contrast to classical-type PCF approaches, fluorescence was used to determine the radius of inside-out patches to calculate membrane tension. Thus, simultaneously measured current and tension values could be related directly. From a technical point of view, this study underlines nicely the high flexibility of PCF, being convertible from Xenopus oocyte membranes to other membrane systems (46). PCF-APPROACHES USING OTHER PATCH- CLAMP CONFIGURATIONS PCF has also been applied in combination with other patchclamp configurations (whole-cell and cell-attached) using mammalian cells as expression systems to investigate the link among gating rearrangements and activation in voltage-gated Ca 2þ channels (48 50), voltage-gated K þ channels (51), the Cl channel ClC-0 (52), TRPV1 channels (53), and P 2 X 2 receptors (54), and to elucidate the mechanism of PIP 2 regulation of K þ channels (55) and Ca 2þ channels (56). PCF employing advanced spectroscopic approaches like semiconfocal epifluorescence and total internal reflection fluorescence were used by Blunck et al. (57) to investigate gating-dependent conformational changes in the voltage-dependent prokaryotic sodium channel NaChBac. Furthermore, the cell-attached configuration was combined with confocal fluorescence microscopy to study ligand binding in nicotinic acetylcholine receptors at the single molecule level by Schmauder et al. (58). OUTLOOK PCF has provided a wealth of information on several aspects of ion channel function, using the power of simultaneous measurements of currents through electrophysiological techniques and of conformational changes through spectroscopic approaches such as fluorescence quenching and FRET (Fig. 1). Despite the results already achieved, the technique is still under continuous development, particularly in its spectroscopic component. For example, approaches like tryptophan quenching of bimane (59) and transition metal FRET (60) have already been applied to resolve conformational changes of smaller scale compared to classical GFP-based FRET. The use of small fluorophores and short linkers can minimize the effects of the labeling procedure on channel function and lead to more accurate FRET measurements (61). Another promising tool is the insertion of fluorescent unnatural amino acids, as shown for muscle nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes (62). Recently, unnatural amino acids were also used in VCF experiments on Shaker K þ channels (63). Further developments are expected from the broadening of the PCF approach to the single-channel level. First efforts in this direction have already been made in cell-attached patches (58) and artificial lipid bilayers containing reconstituted proteins (64 66). 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