Dynamic PIP₂ interactions with voltage sensor elements contribute to KCNQ2 channel gating

Significance

Many membrane protein functions require phosphatidylinositol-4,5-bisphosphate (PIP₂), a minor phospholipid in the inner leaflet of plasma membranes; however the underlying mechanisms have not been well elucidated. Here, we show that PIP₂ might differentially regulate the activities of highly homologous proteins. The voltage-gated potassium (Kv) channels Shaker, Kv1.2 and KCNQ2 have similar structural arrangements and voltage sensing mechanisms. Previous studies showed that PIP₂ decreases the voltage sensitivity of the Shaker and Kv1.2 channels; we observed that PIP₂ up-regulates the voltage sensitivity of the KCNQ2 channel. The differential interactions of PIP₂ with these channels contribute to the diversity of PIP₂ regulations. Our data suggest that the effects of PIP₂ and its interactions with membrane proteins should be studied at a finer scale.

Abstract

The S4 segment and the S4–S5 linker of voltage-gated potassium (Kv) channels are crucial for voltage sensing. Previous studies on the Shaker and Kv1.2 channels have shown that phosphatidylinositol-4,5-bisphosphate (PIP₂) exerts opposing effects on Kv channels, up-regulating the current amplitude, while decreasing the voltage sensitivity. Interactions between PIP₂ and the S4 segment or the S4–S5 linker in the closed state have been highlighted to explain the effects of PIP₂ on voltage sensitivity. Here, we show that PIP₂ preferentially interacts with the S4–S5 linker in the open-state KCNQ2 (Kv7.2) channel, whereas it contacts the S2–S3 loop in the closed state. These interactions are different from the PIP₂–Shaker and PIP₂–Kv1.2 interactions. Consistently, PIP₂ exerts different effects on KCNQ2 relative to the Shaker and Kv1.2 channels; PIP₂ up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel. Disruption of the interaction of PIP₂ with the S4–S5 linker by a single mutation decreases the voltage sensitivity and current amplitude, whereas disruption of the interaction with the S2–S3 loop does not alter voltage sensitivity. These results provide insight into the mechanism of PIP₂ action on KCNQ channels. In the closed state, PIP₂ is anchored at the S2–S3 loop; upon channel activation, PIP₂ interacts with the S4–S5 linker and is involved in channel gating.

A series of ion channels, such as inward rectifier K⁺ (Kir) channels, transient receptor potential channels, and voltage-gated channels, are sensitive to the presence of phosphatidylinositol-4,5-bisphosphate (PIP₂) in membranes (1–4). Structural studies on Kir channels (1, 2, 5) demonstrated that PIP₂ directly interacts with the channels. Subsequent studies supported that PIP₂ also interacts directly with voltage-gated potassium (Kv) channels (6–19). Several positive residues that may be critical for PIP₂ activity have been identified (7, 11, 18, 20–24). Previous studies on Kv1.2 and Shaker channels showed that PIP₂ exerts opposing effects on Kv channels, up-regulating the current amplitude, while leading to a decrease in voltage sensitivity (7, 18). The S4 segment and the S4–S5 linker of Kv channels are crucial for voltage sensing. The interactions of PIP₂ with the S4 segments and the S4–S5 linkers of the closed-state Shaker and Kv1.2 channels underlie the loss-of-function effect of PIP₂ on voltage sensitivity (7, 18).

The KCNQ (Kv7) family of slowly activated outwardly rectifying potassium channels is one of the Kv channel families that are sensitive to the presence of PIP₂ in the membrane. KCNQ channels have been widely studied because of their important biological and pharmacological functions. Retigabine, a first-in-class K⁺ channel opener used for the treatment of epilepsy, adopts a unique mechanism to enhance the activity of KCNQ channels (25). PIP₂ is important for the functions of KCNQ channels. Reduction of PIP₂ affinity caused by congenic mutations of KCNQ channels is associated with long QT syndrome, suggesting critical physiological implications of PIP₂ on KCNQ channels (23, 26). We reported that PIP₂ also alters the pharmacological selectivity of KCNQ potassium channels (6). Zaydman et al. (27) showed that the coupling of voltage sensing and pore opening in the KCNQ1 channel requires PIP₂ and suggested there is a PIP₂ interaction site at the interface between the voltage-sensing domain (VSD) and the central pore domain (PD). However, the effects and interactions of PIP₂ on KCNQ channels are not well understood.

Here, by combining molecular dynamics (MD) simulations, mutagenesis, and electrophysiological determinations, we observed that the effects and interactions of PIP₂ on KCNQ2 are different relative to the Shaker and Kv1.2 channels. PIP₂ up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel. PIP₂ preferentially interacts with the S4–S5 linker of the open-state KCNQ2 channel and does not interact with the S4 segment or S4-S5 linker of the closed state. In the closed state, PIP₂ only interacts with the S2–S3 loop. Furthermore, our electrophysiological experiments suggest that disruption of the interaction of PIP₂ with the S4–S5 linker may decrease the voltage sensitivity and current amplitude, whereas disruption of the interaction with the S2–S3 loop only alters the current amplitude of the channel. These results provide insights into the mechanism of PIP₂ action on Kv channels.

Results

Agonistic Effects of PIP₂ on the Voltage Sensitivity of the KCNQ2 Channel.

Depletion of PIP₂ suppresses the current of the KCNQ2 channel (6, 11, 19). However, the effects of PIP₂ on the voltage sensitivity of the KCNQ2 channel remain unclear. Here, the essential roles of PIP₂ in KCNQ2 channel activity were examined and validated in CHO–K1 cells. We first cotransfected KCNQ2 and Musicrinic receptor 1 (M1) in the cells, where PIP₂ is reduced through the phospholipase C (PLC)-mediated lipid hydrolysis (3, 14). Consistent with previous reports (6, 11, 19), we observed that activation of the M1 receptor by Oxo-M (5 μM) induces a dramatic suppression of the current amplitude (Fig. 1A). Next, we decreased the PIP₂ concentration using a voltage-sensitive phosphatase from Danio rerio (Dr–VSP), which hydrolyzes PIP₂ at highly depolarized voltages (e.g., +120 mV) and transiently reduces the PIP₂ level (28). In the cells cotransfected with KCNQ2 and Dr–VSP, the current is significantly reduced upon +120 mV depolarization (Fig. 1B). The closing of the KCNQ2 channel in response to decreased PIP₂ concentration is consistent with previous observations (6). The M1 and Dr–VSP experiments support the positive effects of PIP₂ on current amplitude. However, because the depletion of PIP₂ by both methods completely abolishes the function of the KCNQ2 channels, the influences of PIP₂ on voltage sensitivity could not be analyzed. Therefore, we evaluated the effects of increased PIP₂ levels on the KCNQ2 channel. A phosphatidylinositol-5-kinase (PI5-K) was coexpressed with the KCNQ2 channel. Overexpression of PI5-K can elevate the PIP₂ membrane concentration to millimolar levels (17). When coexpressed with PI5-K, the current amplitude of the KCNQ2 channel at +50 mV (saturated voltage potential) is significantly higher than the control (Fig. 1 C and E). Noticeably, the conductance-voltage (G-V) curve is significantly left-shifted (Fig. 1D). Therefore, unlike the PIP₂-mediated decrease in the voltage sensitivity of the Shaker and Kv1.2 channels, we observed that PIP₂ up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel.

Fig. 1.

Effects of manipulating PIP₂ concentrations by M1 receptors, Dr–VSP, and PI5-K on KCNQ2 channels. (A) Representative traces and time course of KCNQ channel currents in CHO–K1 cells transfected with KCNQ2 and the M1 receptors. Oxo-M at 5 μM suppresses the KCNQ2 channel current rapidly; representative traces are shown as indicated. (B) Representative traces and time course of KCNQ2 channel current in the cells cotransfected with KCNQ2 and Dr–VSP. Protocol shown was used to elicit KCNQ2 channel currents and activate Dr–VSP. Depolarization to +120 mV caused a dramatic decrease of KCNQ2 channel currents. (C) Representative traces of KCNQ2 channels in cells without (Left) and with (Right) cotransfection with PI5-K. The holding potential is −80 mV, followed by a series of depolarization steps from −70 to +50 mV in 10-mV increments, followed by a 1000-ms hyperpolarization step to −120 mV to record the tail current. (D) Voltage activation curves of the KCNQ2 channels in cells without and with cotransfection with PI5-K as indicated. (E) Histogram summarizing the current densities of KCNQ2 channels when PIP₂ concentrations were manipulated as indicated.

Interactions of PIP₂ with the Open and Closed States of KCNQ2.

Elucidation of the interactions between PIP₂ and KCNQ2 channels would facilitate the understanding of PIP₂ regulation of these channels. Previous studies of the Shaker and Kv1.2 channels have identified the interactions of PIP₂ with the S4–S5 linker of the closed-state channels; the role of PIP₂ in controlling the stability of the voltage sensor in the closed state could explain the loss-of-function effects of PIP₂ on voltage sensitivity of the channels (7, 18). To predict the interactions of PIP₂ with the KCNQ2 channel, we performed all-atom MD simulations on both the open and closed conformations of the KCNQ2 channel in the presence of PIP₂ molecules in the palmitoyloleoyl phosphatidylcholine (POPC) bilayer. Simulations were performed using the program GROMACS 4.6.1 (29). Simulations with the CHARMM36 force field (30) and the CHARMM27 force field (31) yielded similar PIP₂ interactions (Figs. 2 and 3 and Fig. S1), indicating the reproducibility of the MD simulation results. For simplicity, only the simulations with the CHARMM36 force field are discussed in detail below.

Fig. 2.

Trajectories of PIP₂ molecules in the simulation of the open-state KCNQ2 channel. Three PIP₂ molecules move to the S4–S5 linker. The channel is shown in gray ribbon, viewed from the intracellular side.

Fig. 3.

Trajectories of PIP₂ molecules in the simulation of the closed-state KCNQ2 channel. All PIP₂ molecules move to the S2–S3 loop. The channel is shown in gray cartoon, viewed from the intracellular side.

Similar to other Kv channels, the transmembrane (TM) domain of the KCNQ channel can be divided into three parts: the S1–S4 segments forming the VSD, a canonical PD (S5–P–S6), and the S4–S5 linker, which couples the movement of the VSD to the opening or closing of the PD. In addition to the TM domain, positive residues in the cytoplasmic C terminus just after the end of S6 (referred to as the “CCT region”) are believed to contribute to PIP₂ sensitivity (3, 11, 20, 21, 24, 27). We modeled the TM domain (residues 95–312) and CCT region (residues 313–337) for the open-state KCNQ2 channel based on the crystal structures of Kv1.2 (PDB code 2A79) (32) and an activated KcsA K⁺ channel (PDB code 3PJS) (33). The TM domain of the closed-state KCNQ2 was modeled based on the closed-state conformation of the Kv1.2/Kv2.1 chimera channel reported by Jensen et al. (34), and the CCT region was modeled based on the crystal structure of the closed KcsA channel (PDB code 3EFF) (35). We used various methods to test the reliability of the modeled structures (Figs. S2–S4) (SI Materials and Methods provides details). The structure of each state was simulated in a POPC bilayer in the presence of four PIP₂ molecules (Fig. S5). In the initial simulation systems, the PIP₂ molecules were placed in the inner leaflet of the bilayer, far from the channel. The closest distance between PIP₂ and the channel was more than 15 Å. Each system was subjected to a 200-ns MD simulation.

Fig. 2 shows the diffusion trajectories of the four PIP₂ molecules in the simulation of the open-state KCNQ2 channel. Three PIP₂ molecules diffused to the S4–S5 linker, interacting with K230 at the linker. Statistically, the S4–S5 linker may be a putative PIP₂ interaction site with relatively higher potency. Fig. 3 shows the diffusion trajectories of the four PIP₂ molecules in the simulation of the closed state. Although the initial positions of the PIP₂ molecules in this system are similar to those in the open-state system, PIP₂ shows significantly different activity. All PIP₂ molecules moved to the S2–S3 loop and interact with K162 and other positive residues in the loop. None of the PIP₂ molecules interact with the S4 segment or the S4–S5 linker in this simulation. These results indicate that the S2–S3 loop might be a potential PIP₂ interaction region on the closed-state channel.

Mutagenesis and Electrophysiological Experiments.

The unbiased MD simulations identified the potential interaction sites for PIP₂ in the open- and closed-state KCNQ2 channels. Based on the simulation data, we performed mutagenesis and electrophysiological experiments to examine the roles of K230 in the S4–S5 linker and K162 in the S2–S3 loop in determining the PIP₂ regulation of the KCNQ2 channel. Mutation of K230 to alanine (A) does not exhibit detectable current at all; therefore, K230 was mutated to leucine (L). Whole-cell recordings of WT KCNQ2 and the KCNQ2^K230L and KCNQ2^K162A mutants are shown in Fig. 4. The K230L mutation causes significantly decreased current density, and the mutant channel has a right-shifted voltage-dependent activation curve in whole-cell recordings relative to the WT channel (Fig. 4 A–C). To determine the responses of the WT and mutants channels to PIP₂, we expressed WT KCNQ2, KCNQ2^K230L, and KCNQ2^K162A in Xenopus oocytes and recorded the channels using excised inside-out patches (Fig. 5). Fig. 5 A and B shows the responses of the WT KCNQ2 and KCNQ2^K230L channels to fast application of incremental concentrations of diC8–PIP₂ and 60 μg/mL poly-lysine to the inner face of the patch. Compared with WT KCNQ2, the KCNQ2^K230L mutant has significantly reduced sensitivity to dic8–PIP₂. These data indicate that the interactions between K230 and PIP₂ affect the voltage sensitivity and current amplitude of the channel.

Fig. 4.

Distinct roles of K230 and K162 in KCNQ2 channel function. (A) Representative traces of WT and mutant KCNQ2 channels. (B) Histogram summarizing the current densities of WT and mutant channels. (C and D) G-V curves of the WT and K230L and K162A mutant channels (*P < 0.05).

Fig. 5.

PIP₂ sensitivities of the WT and mutant channels. (A–C) Representative traces of inside-out patch recordings showing the PIP₂ sensitivities of the WT and mutant channels as indicated. Application of poly-lysine (P–L) 60 μg/mL inhibits the current of all of the tested channels. (D) Histogram summarizing the dic8–PIP₂ (100 μM) responses of the indicated channels (*P < 0.05).

The MD simulations suggested that K162 interacts with PIP₂ in the closed-state KCNQ2 channel (Fig. 3). Consistently, we observed decreased current amplitude in the K162A mutant channel in whole-cell recordings and significantly decreased sensitivity to diC8–PIP₂ in inside-out patch experiments (Figs. 4 and 5). Interestingly, the K162A mutation does not alter the voltage sensitivity of the channel (Fig. 4D), indicating that the PIP₂–K162 and PIP₂–K230 interactions affects channel gating differently.

Discussion

PIP₂ up-regulates the current amplitude and decreases the voltage sensitivity of the Shaker and Kv1.2 channels (7, 18). PIP₂ also up-regulates the current amplitude of the KCNQ2 channel. However, we observed that the effects of PIP₂ on the voltage sensitivity of the KCNQ2 channel are different from the loss-of-function effects on the voltage sensitivity of the Shaker and Kv1.2 channels. At elevated PIP₂ concentrations, the G-V curve of the KCNQ2 channel is significantly left-shifted, suggesting the increased voltage sensitivity of the channel (Fig. 1D). These results indicate the diversity of PIP₂ effects on Kv channels.

The different functional effects of PIP₂ can be attributed to differences in the interaction of PIP₂ with the channels. The conformational transitions of the S4 segment and the S4–S5 linker upon membrane potential changes are critical for voltage sensing of Kv channels. Previous studies on the Shaker and Kv1.2 channels suggested that PIP₂ could interact with the S4 segment and the S4–S5 linker in the closed state (7, 18). Three residues (K312, K322, and R326) in the S4–S5 linker and three residues (R303, K306, and R309) in the bottom of the S4 segment have been described as PIP₂ interaction sites in the closed state. Interaction of PIP₂ with the S4 segment and S4–S5 linker in the closed state might underlie the loss-of-function effects of PIP₂ on voltage sensitivity of the channels (7, 18). The MD simulations on the closed-state KCNQ2 channel suggested that PIP₂ interacts with the S2–S3 loop rather than the voltage-sensing-related S4 segment and S4–S5 linker. Consistent with the simulation results, a mutation that decreased the PIP₂ interaction with the S2–S3 loop did not alter the voltage sensitivity of the channel.

The MD simulations on the open-state KCNQ2 channel indicated that PIP₂ might interact with K230 of the S4–S5 linker. The position of K230 in the KCNQ2 channel is equivalent to the position of R326 in the Kv1.2 channel (Fig. S2A). Therefore, our results are consistent with previous observations; i.e., the residue equivalent to R326 of Kv1.2 is a PIP₂ interaction site. On the other hand, we observed that PIP₂ interacts with the S4–S5 linker only in the open state of the KCNQ2 channel, controlling the stability of the voltage sensor in the open state rather than the closed state. Therefore, PIP₂ increases the voltage sensitivity of the KCNQ2 channel. Consistently, disruption of the interaction of PIP₂ with the S4–S5 linker by a single mutation decreases voltage sensitivity of the channel.

Based on the MD simulations, mutagenesis, and electrophysiological experiments, we propose the following mechanism for the action of PIP₂ on the KCNQ2 channel (Fig. 6). Upon membrane hyperpolarization, the open state transfers to the closed state, and PIP₂ loses contact with the S4–S5 linker; however, PIP₂ is still retained in the vicinity due to interactions with the S2–S3 loop. The S2–S3 loop of the KCNQ2 channel is long and contains several positive residues. As indicated by multiple sequence alignments with well-known Kv channels (Fig. S6), the HERG (Kv11.1) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels also have long S2–S3 loops with several positive residues, whereas the S2–S3 loops of other channels are short; this is a potential basis for the sensitivity of the KCNQ, HERG, and HCN channels to PIP₂ (10, 11, 36).

Fig. 6.

Mechanism of PIP₂ action on the KCNQ2 channel. In the closed state, PIP₂ is anchored at the S2–S3 loop (Bottom). Upon channel activation, PIP₂ interacts with the S4–S5 linker and is involved in channel gating (Top). PIP₂ is shown in magenta.

K230 is conserved in KCNQ2-5 channels but not in KCNQ1. The corresponding residue in the KCNQ1 channel is glutamine (Q260). The S4–S5 linker of the KCNQ1 channel contains a positive residue (R259) in a forward position. To test whether the open-state KCNQ1 channel and other isoforms interact differently with PIP₂, we performed out an additional 200-ns simulation on the open-state KCNQ1 channel. As shown in Fig. S7, the PIP₂ molecules interact with R259 of the KCNQ1 channel. This result suggests that the S4–S5 linker of the open-state KCNQ1 channel is also a PIP₂ interaction site. However, in the simulation of the KCNQ1 channel, the PIP₂ molecules simultaneously interact with the S2–S3 loop. Structurally, PIP₂ diffuses to the VSD–PD interface in the simulations of both channels, but the detailed interactions are different. This difference indicates that PIP₂ may exert specific effects on the KCNQ1 channel.

Positive residues after the S6 segment are proposed to be critical for PIP₂ interaction (3, 11, 20, 21, 24, 27). However, our simulations do not provide any evidence that the CCT region is a PIP₂ interaction site. We performed inside-out patch experiments to test the roles of two positively charged residues in the CCT region, K327 and K331, in PIP₂ regulation. The KCNQ2^K327A and KCNQ2^K331A mutant channels do not show significantly reduced sensitivity to dic8–PIP₂ (Fig. S8). These results indicate that the role of the CCT region in the KCNQ channel might be not related to PIP₂ interaction.

MD simulation is of increasing importance to analyzing protein–lipid interactions. For example, MD simulations have been successfully applied to identify cholesterol binding or interaction sites for a variety of G protein-coupled receptors (37–40). Here, we show that MD simulation is a powerful method to predict PIP₂ interactions with membrane proteins. However, the 200-ns time scale of the current simulations is not sufficient to resolve all uncertainties. Comparison of the strengths of PIP₂ interaction with distinct sites requires long-time MD simulations. Furthermore, long-time MD simulations in combination with other experiments are needed to investigate PIP₂ interactions during the conformational transitions between open and closed states. These interactions are crucial for the effects of PIP₂ on the activation kinetics of the channels, such as the effect on the VSD–PD coupling. Our future studies will focus on such simulations and related experiments.

Materials and Methods

Homology Modeling.

The crystal structure of Kv1.2 (PDB code 2A79) (32) provides information on the open-state conformation. Using very long-time all-atom MD simulations, Jensen et al. (34) reported the closed-state conformation of the Kv1.2/Kv2.1 “paddle chimera” channel. Based on these structures, we modeled the structures of the transmembrane domain in open- and closed-state KCNQ2 channels. The structures of the cytoplasmic C terminus of the open- and closed-state KCNQ2 channels were modeled based on the open- and closed-state KcsA structures (PDB codes 3PJS and 3EFF) (33, 35), respectively. We used various methods to test the open- and closed-state homology models of the KCNQ2 channel.

Simulation Systems.

The open- and closed-state KCNQ2 channel models were embedded separately in a 130 × 130 Å POPC bilayer by aligning the protein’s axis of symmetry with the bilayer normal. In each system, lipids located within 1 Å of the KCNQ2 channel were removed, and four PIP₂ molecules were added manually to the inner leaflet of the bilayer. The initial position of each PIP₂ molecule was more than 15 Å away from any atom of the channel (Fig. S5). Subsequently, each system was solvated by TIP3P waters with 0.15 M KCl. Each simulation system included ∼172,000 atoms (130 × 130 × 110 Å).

MD Simulation.

MD simulations were performed using the GROMACS 4.6.1 package (29) with the Isobaric-Isothermal (NPT) ensemble and periodic boundary condition. The CHARMM36–CAMP force field (30) was applied for the protein and the POPC phospholipids, and Lupyan et al.’s (41) PIP₂ model was used. Energy minimizations were first performed to relieve unfavorable contacts, followed by equilibration steps of 27 ns in total to equilibrate the lipid bilayer and the solvent, with restraints on PIP₂ and the main chain of the transmembrane domain. We simultaneously relaxed all of the loops during the equilibration steps to obtain more reasonable loop conformations. After the equilibration steps, the PIP₂ molecules were still distant from the KCNQ2 channel; the minimum distances between PIP₂ molecules and the KCNQ2 channel were still more than 15 Å. Subsequently, a 200-ns production run was performed for each system. The KCNQ2 channel contains many cytoplasmic domains (536 residues) after residue 337. The motions of the C-terminal residues 313–337 should be restrained by these cytoplasmic domains. However, we could not build the structures of these cytoplasmic domains. Therefore, to reflect the effects of the missing cytoplasmic domains on the motion of residues 313–337, conformational restraints were applied on the Cα atoms of these residues. The temperature of each system was maintained at 300 K using the v-rescale method (42) with a coupling time of 0.1 ps. The pressure was kept at 1 bar using the Berendsen barostat (43) with τ_p = 1.0 ps and a compressibility of 4.5 × 10⁻⁵ bar⁻¹. SETTLE (44) constraints and LINCS (45) constraints were applied on the hydrogen-involved covalent bonds in water molecules and in other molecules, respectively, and the time step was set to 2 fs. Electrostatic interactions were calculated with the Particle-Mesh Ewald (PME) algorithm (46) with a real-space cutoff of 1.4 nm.

cDNA and Mutagenesis.

The voltage-gated potassium channel KCNQ2 cDNA was a gift from M. Sanguinetti (University of Utah, Salt Lake City, UT). The muscarinic receptor 1 (M1) cDNA was a gift from Hailin Zhang (University of Hebei, Shijiazhuang, China). The Danio rerio voltage-sensitive phosphatase (Dr–VSP) cDNA was a gift from Yasushi Okamura (Okazaki Institute for Integrative Bioscience, Okazaki, Japan). The PI(4)P-5 kinase cDNA was a gift from Dominik Oliver (Institute for Physiology and Pathophysiology, Marburg, Germany). Point mutations were introduced using the Quick Change II site-directed mutagenesis kit (Stratagene) and verified by DNA sequencing.

Electrophysiological Recording in CHO–K1 Cells.

The ruptured whole-cell patch voltage clamp recordings were performed using cultured CHO–K1 cells at room temperature with an Axopatch-200B amplifier (Molecular Devices). The electrodes were pulled from borosilicate glass capillaries (TW150-4, World Precision Instruments). When filled with the intracellular solution, the electrodes had an average resistance of 3–5 Mohms. The pipette solution contained 145 mM KCl, 1 mM MgCl₂, 5 mM EGTA, 10 mM Hepes (pH set to 7.3 using KOH), and 5 mM MgATP. During the recording, constant perfusion of extracellular solution was maintained using a BPS perfusion system (ALA Scientific Instruments). The extracellular solution contained 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1.5 mM MgCl₂, 10 mM Hepes (pH set to 7.4 using NaOH), and 10 mM glucose. The signals were filtered at 2 kHz, digitized using a DigiData 1440A, and analyzed with pClamp 9.2 software (Molecular Devices). The series resistance was compensated by 60–80%.

Oocyte Preparation and Macropatch Recording.

The Xenopus laevis oocytes were prepared and injected according to previously reported protocols (47). The mRNA for each channel or mutant was generated using the Ambion’s mMESSAGE mMACHINE T7 Kit. The mRNA was injected at 10–30 ng/oocyte, and recordings were performed 3–7 d later. For macropatch recordings, electrodes with resistances 1.5–2.0 Mohms were filled with filter ND96 solution containing 2 mM KCl, 91 mM NaCl, 1 mM MgCl₂, 5 mM NaOH, and 5 mM Hepes (pH set to 7.4 using NaOH). The internal (bath solution) was a high-potassium solution containing 96 mM KCl, 5 mM EDTA, and 10 mM Hepes (pH set to 7.4 using KOH). Before establishing an inside-out patch, the bath solution was switched to the recording solution, which contained 60 mM KCl, 5 mM EGTAK₂, 5 mM KF, 0.1 mM Na₃VO₄, 10 mM K₄P₂O₇, and 10 mM Hepes (pH 7.4). The recordings were performed using an EPC10 amplifier and the “Pulse” software (Heka) at room temperature. The solutions were applied using the DAD12 (ALA Instruments) fast perfusion system.

Details of the homology modeling, cell culture, and transient transfection experiments are provided in the SI Materials and Methods.