Novel Kv7.1-Phosphatidylinositol 4,5-Bisphosphate Interaction Sites Uncovered by Charge Neutralization Scanning

K_v7.1 to K_v7.5 α-subunits belong to the family of voltage-gated potassium channels (K_v). Assembled with the β-subunit KCNE1, K_v7.1 conducts the slowly activating potassium current I_Ks, which is one of the major currents underlying repolarization of the cardiac action potential. A known regulator of K_v7 channels is the lipid phosphatidylinositol 4,5-bisphosphate (PIP₂). PIP₂ increases the macroscopic current amplitude by stabilizing the open conformation of 7.1/KCNE1 channels. However, knowledge about the exact nature of the interaction is incomplete. The aim of this study was the identification of the amino acids responsible for the interaction between K_v7.1 and PIP₂. We generated 13 charge neutralizing point mutations at the intracellular membrane border and characterized them electrophysiologically in complex with KCNE1 under the influence of diC₈-PIP₂. Electrophysiological analysis of corresponding long QT syndrome mutants suggested impaired PIP₂ regulation as the cause for channel dysfunction. To clarify the underlying structural mechanism of PIP₂ binding, molecular dynamics simulations of K_v7.1/KCNE1 complexes containing two PIP₂ molecules in each subunit at specific sites were performed. Here, we identified a subset of nine residues participating in the interaction of PIP₂ and K_v7.1/KCNE1. These residues may form at least two binding pockets per subunit, leading to the stabilization of channel conformations upon PIP₂ binding.     

Bimodal regulation of an Elk subfamily K+ channel by phosphatidylinositol 4,5-bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates Shaker K⁺ channels and voltage-gated Ca²⁺ channels in a bimodal fashion by inhibiting voltage activation while stabilizing open channels. Bimodal regulation is conserved in hyperpolarization-activated cyclic nucleotide–gated (HCN) channels, but voltage activation is enhanced while the open channel state is destabilized. The proposed sites of PIP₂ regulation in these channels include the voltage-sensor domain (VSD) and conserved regions of the proximal cytoplasmic C terminus. Relatively little is known about PIP₂ regulation of Ether-á-go-go (EAG) channels, a metazoan-specific family of K⁺ channels that includes three gene subfamilies, Eag (Kv10), Erg (Kv11), and Elk (Kv12). We examined PIP₂ regulation of the Elk subfamily potassium channel human Elk1 to determine whether bimodal regulation is conserved within the EAG K⁺ channel family. Open-state stabilization by PIP₂ has been observed in human Erg1, but the proposed site of regulation in the distal C terminus is not conserved among EAG family channels. We show that PIP₂ strongly inhibits voltage activation of Elk1 but also stabilizes the open state. This stabilization produces slow deactivation and a mode shift in voltage gating after activation. However, removal of PIP₂ has the net effect of enhancing Elk1 activation. R347 in the linker between the VSD and pore (S4–S5 linker) and R479 near the S6 activation gate are required for PIP₂ to inhibit voltage activation. The ability of PIP₂ to stabilize the open state also requires these residues, suggesting an overlap in sites central to the opposing effects of PIP₂ on channel gating. Open-state stabilization in Elk1 requires the N-terminal eag domain (PAS domain + Cap), and PIP₂-dependent stabilization is enhanced by a conserved basic residue (K5) in the Cap. Our data shows that PIP₂ can bimodally regulate voltage gating in EAG family channels, as has been proposed for Shaker and HCN channels. PIP₂ regulation appears fundamentally different for Elk and KCNQ channels, suggesting that, although both channel types can regulate action potential threshold in neurons, they are not functionally redundant.     

Phosphatidylinositol-4,5-bisphosphate, PIP2, controls KCNQ1/KCNE1 voltage-gated potassium channels: a functional homology between voltage-gated and inward rectifier K+ channels

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is a major signaling molecule implicated in the regulation of various ion transporters and channels. Here we show that PIP(2) and intracellular MgATP control the activity of the KCNQ1/KCNE1 potassium channel complex. In excised patch-clamp recordings, the KCNQ1/KCNE1 current decreased spontaneously with time. This rundown was markedly slowed by cytosolic application of PIP(2) and fully prevented by application of PIP(2) plus MgATP. PIP(2)-dependent rundown was accompanied by acceleration in the current deactivation kinetics, whereas the MgATP-dependent rundown was not. Cytosolic application of PIP(2) slowed deactivation kinetics and also shifted the voltage dependency of the channel activation toward negative potentials. Complex changes in the current characteristics induced by membrane PIP(2) was fully restituted by a model originally elaborated for ATP-regulated two transmembrane-domain potassium channels. The model is consistent with stabilization by PIP(2) of KCNQ1/KCNE1 channels in the open state. Our data suggest a striking functional homology between a six transmembrane-domain voltage-gated channel and a two transmembrane-domain ATP-gated channel.     

Distinct subdomains of the KCNQ1 S6 segment determine channel modulation by different KCNE subunits

Modulation of voltage-gated potassium (K_V) channels by the KCNE family of single transmembrane proteins has physiological and pathophysiological importance. All five KCNE proteins (KCNE1–KCNE5) have been demonstrated to modulate heterologously expressed KCNQ1 (K_V7.1) with diverse effects, making this channel a valuable experimental platform for elucidating structure–function relationships and mechanistic differences among members of this intriguing group of accessory subunits. Here, we specifically investigated the determinants of KCNQ1 inhibition by KCNE4, the least well-studied KCNE protein. In CHO-K1 cells, KCNQ1, but not KCNQ4, is strongly inhibited by coexpression with KCNE4. By studying KCNQ1-KCNQ4 chimeras, we identified two adjacent residues (K326 and T327) within the extracellular end of the KCNQ1 S6 segment that determine inhibition of KCNQ1 by KCNE4. This dipeptide motif is distinct from neighboring S6 sequences that enable modulation by KCNE1 and KCNE3. Conversely, S6 mutations (S338C and F340C) that alter KCNE1 and KCNE3 effects on KCNQ1 do not abrogate KCNE4 inhibition. Further, KCNQ1-KCNQ4 chimeras that exhibited resistance to the inhibitory effects of KCNE4 still interact biochemically with this protein, implying that accessory subunit binding alone is not sufficient for channel modulation. These observations indicate that the diverse functional effects observed for KCNE proteins depend, in part, on structures intrinsic to the pore-forming subunit, and that distinct S6 subdomains determine KCNQ1 responses to KCNE1, KCNE3, and KCNE4.     

Ca2+-Calmodulin and PIP2 interactions at the proximal C-terminus of Kv7 channels

In the heart, co-assembly of Kv7.1 with KCNE1 produces the slow I_KS potassium current, which repolarizes the cardiac action potential and mutations in human Kv7.1 and KCNE1 genes cause cardiac arrhythmias. The proximal Kv7.1 C-terminus binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP₂) and recently we revealed the competition of PIP₂ with the calcified CaM N-lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor a LQT mutation. Data indicated that PIP₂ and Ca²⁺-CaM perform the same function on I_KS channel gating to stabilize the channel open state. Here we show that similar features were observed for Kv7.1 currents expressed alone. We also find that conservation of homologous residues in helix B of other Kv7 subtypes confer similar competition of Ca²⁺-CaM with PIP2 binding to their proximal C-termini and suggest that PIP2-CaM interactions converge to Kv7 helix B to modulates channel activity in a Kv7 subtype-dependent manner.     

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Phosphatidylinositol (4,5)-bisphosphate (PIP₂) is a phospholipid of the plasma membrane that has been shown to be a key regulator of several ion channels. Functional studies and more recently structural studies of Kir channels have revealed the major impact of PIP₂ on the open state stabilization. A similar effect of PIP₂ on the delayed rectifiers Kv7.1 and Kv11.1, two voltage-gated K⁺ channels, has been suggested, but the molecular mechanism remains elusive and nothing is known on PIP₂ effect on other Kv such as those of the Shaker family. By combining giant-patch ionic and gating current recordings in COS-7 cells, and voltage-clamp fluorimetry in Xenopus oocytes, both heterologously expressing the voltage-dependent Shaker channel, we show that PIP₂ exerts 1) a gain-of-function effect on the maximal current amplitude, consistent with a stabilization of the open state and 2) a loss-of-function effect by positive-shifting the activation voltage dependence, most likely through a direct effect on the voltage sensor movement, as illustrated by molecular dynamics simulations.     

KCNE variants reveal a critical role of the beta subunit carboxyl terminus in PKA-dependent regulation of the IKs potassium channel

Co-assembly of KCNQ1 with different accessory, or beta, subunits that are members of the KCNE family results in potassium (K⁺) channels that conduct functionally distinct currents. The alpha subunit KCNQ1 conducts a slowly-activated delayed rectifier K⁺ current (I_Ks), a major contributor to cardiac repolarization, when co-assembled with KCNE1 and channels that favor the open state when co-assembled with either KCNE2 or KCNE3. In the heart, stimulation of the sympathetic nervous system enhances I_Ks. A macromolecular signaling complex of the I_Ks channel including the targeting protein Yotiao coordinates up- or down- regulation of channel activity by protein kinase A (PKA) phosphorylation and dephosphorylation of molecules in the complex. β-adrenergic receptor mediated I_Ks up-regulation, a functional consequence of PKA phosphorylation of the KCNQ1 amino terminus (N-T), requires co-expression of KCNQ1/Yotiao with KCNE1. Here, we report that co-expression of KCNE2, like KCNE1, confers a functional channel response to KCNQ1 phosphorylation, but co-expression of KCNE3 does not. Amino acid sequence comparison among the KCNE peptides, and KCNE1 truncation experiments, reveal a segment of the predicted intracellular KCNE1 carboxyl terminus (C-T) that is necessary for functional transduction of PKA phosphorylated KCNQ1. Moreover, chimera analysis reveals a region of KCNE1 sufficient to confer cAMP-dependent functional regulation upon the KCNQ1_KCNE3_Yotiao channel. The property of specific beta subunits to transduce post-translational regulation of alpha subunits of ion channels adds another dimension to our understanding molecular mechanisms underlying the diversity of regulation of native K⁺ channels.     

Functional interactions of voltage sensor charges with an S2 hydrophobic plug in hERG channels

Human ether-à-go-go–related gene (hERG, Kv11.1) potassium channels have unusually slow activation and deactivation kinetics. It has been suggested that, in fast-activating Shaker channels, a highly conserved Phe residue (F290) in the S2 segment forms a putative gating charge transfer center that interacts with S4 gating charges, i.e., R362 (R1) and K374 (K5), and catalyzes their movement across the focused electric field. F290 is conserved in hERG (F463), but the relevant residues in the hERG S4 are reversed, i.e., K525 (K1) and R537 (R5), and there is an extra positive charge adjacent to R537 (i.e., K538). We have examined whether hERG channels possess a transfer center similar to that described in Shaker and if these S4 charge differences contribute to slow gating in hERG channels. Of five hERG F463 hydrophobic substitutions tested, F463W and F463Y shifted the conductance–voltage (G-V) relationship to more depolarized potentials and dramatically slowed channel activation. With the S4 residue reversals (i.e., K525, R537) taken into account, the closed state stabilization by F463W is consistent with a role for F463 that is similar to that described for F290 in Shaker. As predicted from results with Shaker, the hERG K525R mutation destabilized the closed state. However, hERG R537K did not stabilize the open state as predicted. Instead, we found the neighboring K538 residue to be critical for open state stabilization, as K538R dramatically slowed and right-shifted the voltage dependence of activation. Finally, double mutant cycle analysis on the G-V curves of F463W/K525R and F463W/K538R double mutations suggests that F463 forms functional interactions with K525 and K538 in the S4 segment. Collectively, these data suggest a role for F463 in mediating closed–open equilibria, similar to that proposed for F290 in Shaker channels.     

Concerted All-or-none Subunit Interactions Mediate Slow Deactivation of Human ether-à-go-go-related Gene K+ Channels

During the repolarization phase of a cardiac action potential, hERG1 K⁺ channels rapidly recover from an inactivated state then slowly deactivate to a closed state. The resulting resurgence of outward current terminates the plateau phase and is thus a key regulator of action potential duration of cardiomyocytes. The intracellular N-terminal domain of the hERG1 subunit is required for slow deactivation of the channel as its removal accelerates deactivation 10-fold. Here we investigate the stoichiometry of hERG1 channel deactivation by characterizing the kinetic properties of concatenated tetramers containing a variable number of wild-type and mutant subunits. Three mutations known to accelerate deactivation were investigated, including R56Q and R4A/R5A in the N terminus and F656I in the S6 transmembrane segment. In all cases, a single mutant subunit induced the same rapid deactivation of a concatenated channel as that observed for homotetrameric mutant channels. We conclude that slow deactivation gating of hERG1 channels involves a concerted, fully cooperative interaction between all four wild-type channel subunits.     

Eag Domains Regulate LQT Mutant hERG Channels in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human Ether á go-go Related Gene potassium channels form the rapid component of the delayed-rectifier (I_Kr) current in the heart. The N-terminal ‘eag’ domain, which is composed of a Per-Arnt-Sim (PAS) domain and a short PAS-cap region, is a critical regulator of hERG channel function. In previous studies, we showed that isolated eag (i-eag) domains rescued the dysfunction of long QT type-2 associated mutant hERG R56Q channels, by substituting for defective eag domains, when the channels were expressed in Xenopus oocytes or HEK 293 cells.Here, our goal was to determine whether the rescue of hERG R56Q channels by i-eag domains could be translated into the environment of cardiac myocytes. We expressed hERG R56Q channels in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and measured electrical properties of the cells with whole-cell patch-clamp recordings. We found that, like in non-myocyte cells, hERG R56Q had defective, fast closing (deactivation) kinetics when expressed in hiPSC-CMs. We report here that i-eag domains slowed the deactivation kinetics of hERG R56Q channels in hiPSC-CMs. hERG R56Q channels prolonged the AP of hiPSCs, and the AP was shortened by co-expression of i-eag domains and hERG R56Q channels. We measured robust Förster Resonance Energy Transfer (FRET) between i-eag domains tagged with Cyan fluorescent protein (CFP) and hERG R56Q channels tagged with Citrine fluorescent proteins (Citrine), indicating their close proximity at the cell membrane in live iPSC-CMs. Together, functional regulation and FRET spectroscopy measurements indicated that i-eag domains interacted directly with hERG R56Q channels in hiPSC-CMs. These results mean that the regulatory role of i-eag domains is conserved in the cellular environment of human cardiomyocytes, indicating that i-eag domains may be useful as a biological therapeutic.     

KCNE4 can co-associate with the I(Ks) (KCNQ1-KCNE1) channel complex

Voltage-gated potassium (K(V)) channels can form heteromultimeric complexes with a variety of accessory subunits, including KCNE proteins. Heterologous expression studies have demonstrated diverse functional effects of KCNE subunits on several K(V) channels, including KCNQ1 (K(V)7.1) that, together with KCNE1, generates the slow-delayed rectifier current (I(Ks)) important for cardiac repolarization. In particular, KCNE4 exerts a strong inhibitory effect on KCNQ1 and other K(V) channels, raising the possibility that this accessory subunit is an important potassium current modulator. A polyclonal KCNE4 antibody was developed to determine the human tissue expression pattern and to investigate the biochemical associations of this protein with KCNQ1. We found that KCNE4 is widely and variably expressed in several human tissues, with greatest abundance in brain, liver and testis. In heterologous expression experiments, immunoprecipitation followed by immunoblotting was used to establish that KCNE4 directly associates with KCNQ1, and can co-associate together with KCNE1 in the same KCNQ1 complex to form a 'triple subunit' complex (KCNE1-KCNQ1-KCNE4). We also used cell surface biotinylation to demonstrate that KCNE4 does not impair plasma membrane expression of either KCNQ1 or the triple subunit complex, indicating that biophysical mechanisms probably underlie the inhibitory effects of KCNE4. The observation that multiple KCNE proteins can co-associate with and modulate KCNQ1 channels to produce biochemically diverse channel complexes has important implications for understanding K(V) channel regulation in human physiology.     

Transition between conformational states of the TREK-1 K2P channel promoted by interaction with PIP2

Members of the TREK family of two-pore domain potassium channels are highly sensitive to regulation by membrane lipids, including phosphatidylinositol-4,5-bisphosphate (PIP₂). Previous studies have demonstrated that PIP₂ increases TREK-1 channel activity; however, the mechanistic understanding of the conformational transitions induced by PIP₂ remain unclear. Here, we used coarse-grained molecular dynamics and atomistic molecular dynamics simulations to model the PIP₂-binding site on both the up and down state conformations of TREK-1. We also calculated the free energy of PIP₂ binding relative to other anionic phospholipids in both conformational states using potential of mean force and free-energy-perturbation calculations. Our results identify state-dependent binding of PIP₂ to sites involving the proximal C-terminus, and we show that PIP₂ promotes a conformational transition from a down state toward an intermediate that resembles the up state. These results are consistent with functional data for PIP₂ regulation, and together provide evidence for a structural mechanism of TREK-1 channel activation by phosphoinositides.     

Control of pH and PIP2 Gating in Heteromeric Kir4.1/Kir5.1 Channels by H-Bonding at the Helix-Bundle Crossing

Inhibition by intracellular H⁺ (pH gating) and activation by phosphoinositides such as PIP₂ (PIP₂ gating) are key regulatory mechanisms in the physiology of inwardly-rectifying potassium (Kir) channels. Our recent findings suggest that PIP₂ gating and pH gating are controlled by an intrasubunit H-bond at the helix-bundle crossing between a lysine in TM1 and a backbone carbonyl group in TM2. This interaction only occurs in the closed state and channel opening requires this H-bond to be broken, thereby influencing the kinetics of PIP₂- and pH-gating in Kir channels. In this addendum, we explore the role of H-bonding in heteromeric Kir4.1/Kir5.1 channels. Kir5.1 subunits do not possess a TM1 lysine. However, homology modelling and molecular dynamics simulations demonstrate that the TM1 lysine in Kir4.1 is capable of H-bonding at the helix-bundle crossing. Consistent with this, the rates of pH and PIP₂ gating in Kir4.1/Kir5.1 channels (two H-bonds) were intermediate between those of wild-type homomeric Kir4.1 (four H-bonds) and Kir4.1(K67M) channels (no H-bonds) suggesting that the number of H-bonds in the tetrameric channel complex determines the gating kinetics. Furthermore, in heteromeric Kir4.1(K67M)/Kir5.1 channels, where the two remaining H-bonds are disrupted, we found that the gating kinetics were similar to Kir4.1(K67M) homomeric channels despite the fact that these two channels differ considerably in their PIP₂ affinities. This indicates that Kir channel PIP₂ affinity has little impact on either the PIP₂- or pH-gating kinetics.     

Modulation of the Olfactory CNG Channel by Ptdlns(3,4,5)P<Subscript>3</Subscript>

Recent data suggest that the 3-phosphoinositides can modulate cyclic nucleotide signaling in rat olfactory receptor neurons (ORNs). Given the ability of diverse lipids to modulate ion channels, we asked whether phosphatidylinositol 3,4,5-trisphosphate (PIP₃) can regulate the olfactory cyclic nucleotide-gated (CNG) channel as a possible mechanism for this modulation. We show that applying PIP₃ to the intracellular side of inside-out patches from rat ORNs inhibits activation of the olfactory CNG channel by cAMP. The effect of PIP₃ is immediate and partially reversible, and reflects an increase in the EC₅₀ of cAMP, not a reduction in the single-channel current amplitude. The effect of PIP₃ is significantly stronger than that of PIP₂; other phospholipids tested have no appreciable effect on channel activity. PIP₃ similarly inhibits the recombinant heteromeric (A2/A4) and homomeric (A2) olfactory CNG channel expressed in HEK293 cells, suggesting that PIP₃ acts directly on the channel. These findings indicate that 3-phosphoinositides can be functionally important regulators of CNG channels.     

Sequence of Gating Charge Movement and Pore Gating in hERG Activation and Deactivation Pathways

K_V11.1 voltage-gated K⁺ channels are noted for unusually slow activation, fast inactivation, and slow deactivation kinetics, which tune channel activity to provide vital repolarizing current during later stages of the cardiac action potential. The bulk of charge movement in human ether-a-go-go-related gene (hERG) is slow, as is return of charge upon repolarization, suggesting that the rates of hERG channel opening and, critically, that of deactivation might be determined by slow voltage sensor movement, and also by a mode-shift after activation. To test these ideas, we compared the kinetics and voltage dependence of ionic activation and deactivation with gating charge movement. At 0 mV, gating charge moved ∼threefold faster than ionic current, which suggests the presence of additional slow transitions downstream of charge movement in the physiological activation pathway. A significant voltage sensor mode-shift was apparent by 24 ms at +60 mV in gating currents, and return of charge closely tracked pore closure after pulses of 100 and 300 ms duration. A deletion of the N-terminus PAS domain, mutation R4AR5A or the LQT2-causing mutation R56Q gave faster-deactivating channels that displayed an attenuated mode-shift of charge. This indicates that charge movement is perturbed by N- and C-terminus interactions, and that these domain interactions stabilize the open state and limit the rate of charge return. We conclude that slow on-gating charge movement can only partly account for slow hERG ionic activation, and that the rate of pore closure has a limiting role in the slow return of gating charges.     

Structure of KCNE1 and implications for how it modulates the KCNQ1 potassium channel

KCNE1 is a single-span membrane protein that modulates the voltage-gated potassium channel KCNQ1 (K V7.1) by slowing activation and enhancing channel conductance to generate the slow delayed rectifier current ( I Ks) that is critical for the repolarization phase of the cardiac action potential. Perturbation of channel function by inherited mutations in KCNE1 or KCNQ1 results in increased susceptibility to cardiac arrhythmias and sudden death with or without accompanying deafness. Here, we present the three-dimensional structure of KCNE1. The transmembrane domain (TMD) of KCNE1 is a curved alpha-helix and is flanked by intra- and extracellular domains comprised of alpha-helices joined by flexible linkers. Experimentally restrained docking of the KCNE1 TMD to a closed state model of KCNQ1 suggests that KCNE1 slows channel activation by sitting on and restricting the movement of the S4-S5 linker that connects the voltage sensor to the pore domain. We postulate that this is an adhesive interaction that must be disrupted before the channel can be opened in response to membrane depolarization. Docking to open KCNQ1 indicates that the extracellular end of the KCNE1 TMD forms an interface with an intersubunit cleft in the channel that is associated with most known gain-of-function disease mutations. Binding of KCNE1 to this "gain-of-function cleft" may explain how it increases conductance and stabilizes the open state. These working models for the KCNE1-KCNQ1 complexes may be used to formulate testable hypotheses for the molecular bases of disease phenotypes associated with the dozens of known inherited mutations in KCNE1 and KCNQ1.     

Discovery of a Novel Activator of KCNQ1-KCNE1 K+ Channel Complexes

KCNQ1 voltage-gated K⁺ channels (Kv7.1) associate with the family of five KCNE peptides to form complexes with diverse gating properties and pharmacological sensitivities. The varied gating properties of the different KCNQ1-KCNE complexes enables the same K⁺ channel to function in both excitable and non excitable tissues. Small molecule activators would be valuable tools for dissecting the gating mechanisms of KCNQ1-KCNE complexes; however, there are very few known activators of KCNQ1 channels and most are ineffective on the physiologically relevant KCNQ1-KCNE complexes. Here we show that a simple boronic acid, phenylboronic acid (PBA), activates KCNQ1/KCNE1 complexes co-expressed in Xenopus oocytes at millimolar concentrations. PBA shifts the voltage sensitivity of KCNQ1 channel complexes to favor the open state at negative potentials. Analysis of different-sized charge carriers revealed that PBA also targets the permeation pathway of KCNQ1 channels. Activation by the boronic acid moiety has some specificity for the Kv7 family members (KCNQ1, KCNQ2/3, and KCNQ4) since PBA does not activate Shaker or hERG channels. Furthermore, the commercial availability of numerous PBA derivatives provides a large class of compounds to investigate the gating mechanisms of KCNQ1-KCNE complexes.     

Interactions of H562 in the S5 Helix with T618 and S621 in the Pore Helix Are Important Determinants of hERG1 Potassium Channel Structure and Function

hERG1 is a member of the cyclic nucleotide binding domain family of K⁺ channels. Alignment of cyclic nucleotide binding domain channels revealed an evolutionary conserved sequence HwX(A/G)C in the S5 domain. We reasoned that histidine 562 in hERG1 could play an important structure-function role. To explore this role, we created in silica models of the hERG1 pore domain based on the KvAP crystal structure with Rosetta-membrane modeling and molecular-dynamics simulations. Simulations indicate that the H562 residue in the S5 helix spans the gap between the S5 helix and the pore helix, stabilizing the pore domain, and that mutation at the H562 residue leads to a disruption of the hydrogen bonding to T618 and S621, resulting in distortion of the selectivity filter. Analysis of the simulated point mutations at positions 562/618/621 showed that the reciprocal double mutations H562W/T618I would partially restore the orientation of the 562 residue. Matching hydrophobic interactions between mutated W562 residue and I618 partially compensate for the disrupted hydrogen bonding. Complementary in vitro electrophysiological studies confirmed the results of the molecular-dynamics simulations on single mutations at positions 562, 618, and 621. Experimentally, mutations of the H562 to tryptophan produced a functional channel, but with slowed deactivation and shifted V_1/2 of activation. Furthermore, the double mutation T618I/H562W rescued the defects seen in activation, deactivation, and potassium selectivity seen with the H562W mutation. In conclusion, interactions between H562 in the S5 helix and amino acids in the pore helix are important determinants of hERG1 potassium channel function, as confirmed by theory and experiment.     

Intracellular Long-Chain Acyl CoAs Activate TRPV1 Channels

TRPV1 channels are an important class of membrane proteins that play an integral role in the regulation of intracellular cations such as calcium in many different tissue types. The anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂) is a known positive modulator of TRPV1 channels and the negatively charged phosphate groups interact with several basic amino acid residues in the proximal C-terminal TRP domain of the TRPV1 channel. We and other groups have shown that physiological sub-micromolar levels of long-chain acyl CoAs (LC-CoAs), another ubiquitous anionic lipid, can also act as positive modulators of ion channels and exchangers. Therefore, we investigated whether TRPV1 channel activity is similarly regulated by LC-CoAs. Our results show that LC-CoAs are potent activators of the TRPV1 channel and interact with the same PIP₂-binding residues in TRPV1. In contrast to PIP₂, LC-CoA modulation of TRPV1 is independent of Ca²⁺ _i, acting in an acyl side-chain saturation and chain-length dependent manner. Elevation of LC-CoAs in intact Jurkat T-cells leads to significant increases in agonist-induced Ca²⁺ _i levels. Our novel findings indicate that LC-CoAs represent a new fundamental mechanism for regulation of TRPV1 channel activity that may play a role in diverse cell types under physiological and pathophysiological conditions that alter fatty acid transport and metabolism such as obesity and diabetes.     

Structural Determinants of Phosphatidylinositol 4,5-Bisphosphate (PIP2) Regulation of BK Channel Activity through the RCK1 Ca2+ Coordination Site

Big or high conductance potassium (BK) channels are activated by voltage and intracellular calcium (Ca²⁺). Phosphatidylinositol 4,5-bisphosphate (PIP₂), a ubiquitous modulator of ion channel activity, has been reported to enhance Ca²⁺-driven gating of BK channels, but a molecular understanding of this interplay or even of the PIP₂ regulation of this channel''s activity remains elusive. Here, we identify structural determinants in the KDRDD loop (which follows the αA helix in the RCK1 domain) to be responsible for the coupling between Ca²⁺ and PIP₂ in regulating BK channel activity. In the absence of Ca²⁺, RCK1 structural elements limit channel activation through a decrease in the channel''s PIP₂ apparent affinity. This inhibitory influence of BK channel activation can be relieved by mutation of residues that (a) connect either the RCK1 Ca²⁺ coordination site (Asp³⁶⁷ or its flanking basic residues in the KDRDD loop) to the PIP₂-interacting residues (Lys³⁹² and Arg³⁹³) found in the αB helix or (b) are involved in hydrophobic interactions between the αA and αB helix of the RCK1 domain. In the presence of Ca²⁺, the RCK1-inhibitory influence of channel-PIP₂ interactions and channel activity is relieved by Ca²⁺ engaging Asp³⁶⁷. Our results demonstrate that, along with Ca²⁺ and voltage, PIP₂ is a third factor critical to the integral control of BK channel activity.