Gating kinetics of GIRK channels are mediated by cytoplasmic residues adjacent to transmembrane domains

G-protein-coupled inwardly rectifying potassium channels (GIRK / Kir3.x) are involved in neurotransmission-mediated reduction of excitability. The gating mechanism following G protein activation of these channels likely proceeds from movement of inner transmembrane helices to allow K⁺ ions movement through the pore of the channel. There is limited understanding of how the binding of G-protein βγ subunits to cytoplasmic regions of the channel transduces the signal to the transmembrane regions. In this study, we examined the molecular basis that governs the activation kinetics of these channels, using a chimeric approach. We identified two regions as being important in determining the kinetics of activation. One region is the bottom of the outer transmembrane helix (TM1) and the cytoplasmic domain immediately adjacent (the slide helix); and the second region is the bottom of the inner transmembrane helix (TM2) and the cytoplasmic domain immediately adjacent. Interestingly, both of these regions are sufficient in mediating the kinetics of fast gating. This result suggests that there is a cooperative movement of both of these domains to allow fast and efficient gating of GIRK channels.     

Tectonics of a K+ channel: The importance of the N-terminus for channel gating

The small K⁺ channel Kcv represents the pore module of complex potassium channels. It was found that its gating can be modified by sensor domains, which are N-terminally coupled to the pore. This implies that the short N-terminus of the channel can transmit conformational changes from upstream sensors to the channel gates. To understand the functional role of the N-terminus in the context of the entire channel protein, we apply combinatorial screening of the mechanical coupling and long-range interactions in the Kcv potassium channel by reduced molecular models. The dynamics and mechanical connections in the channel complex show that the N-terminus is indeed mechanically connected to the pore domain. This includes a long rang coupling to the pore and the inner and outer transmembrane domains. Since the latter domains host the two gates of the channel, the data support the hypothesis that mechanical perturbation of the N-terminus can be transmitted to the channel gates. This effect is solely determined by the topology of the channel; sequence details only have an implicit effect on the coarse-grained dynamics via the fold and not through biochemical details at a smaller scale. This observation has important implications for engineering of synthetic channels on the basis of a K⁺ channel pore.     

Fast and slow gating are inherent properties of the pore module of the K+ channel Kcv

Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K⁺ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K⁺ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K⁺, single-channel conductance is 114 ± 11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. “Fast” gating, analyzed by β distributions, is responsible for the negative slope conductance in the single-channel current–voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a “slow” gating is revealed by the very low (in the order of 1–4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current.     

The voltage-sensing domain of a phosphatase gates the pore of a potassium channel

The modular architecture of voltage-gated K⁺ (Kv) channels suggests that they resulted from the fusion of a voltage-sensing domain (VSD) to a pore module. Here, we show that the VSD of Ciona intestinalis phosphatase (Ci-VSP) fused to the viral channel Kcv creates Kv_Synth1, a functional voltage-gated, outwardly rectifying K⁺ channel. Kv_Synth1 displays the summed features of its individual components: pore properties of Kcv (selectivity and filter gating) and voltage dependence of Ci-VSP (V_1/2 = +56 mV; z of ∼1), including the depolarization-induced mode shift. The degree of outward rectification of the channel is critically dependent on the length of the linker more than on its amino acid composition. This highlights a mechanistic role of the linker in transmitting the movement of the sensor to the pore and shows that electromechanical coupling can occur without coevolution of the two domains.     

Distinct lipid bilayer compositions have general and protein-specific effects on K+ channel function

It has become increasingly apparent that the lipid composition of cell membranes affects the function of transmembrane proteins such as ion channels. Here, we leverage the structural and functional diversity of small viral K⁺ channels to systematically examine the impact of bilayer composition on the pore module of single K⁺ channels. In vitro–synthesized channels were reconstituted into phosphatidylcholine bilayers ± cholesterol or anionic phospholipids (aPLs). Single-channel recordings revealed that a saturating concentration of 30% cholesterol had only minor and protein-specific effects on unitary conductance and gating. This indicates that channels have effective strategies for avoiding structural impacts of hydrophobic mismatches between proteins and the surrounding bilayer. In all seven channels tested, aPLs augmented the unitary conductance, suggesting that this is a general effect of negatively charged phospholipids on channel function. For one channel, we determined an effective half-maximal concentration of 15% phosphatidylserine, a value within the physiological range of aPL concentrations. The different sensitivity of two channel proteins to aPLs could be explained by the presence/absence of cationic amino acids at the interface between the lipid headgroups and the transmembrane domains. aPLs also affected gating in some channels, indicating that conductance and gating are uncoupled phenomena and that the impact of aPLs on gating is protein specific. In two channels, the latter can be explained by the altered orientation of the pore-lining transmembrane helix that prevents flipping of a phenylalanine side chain into the ion permeation pathway for long channel closings. Experiments with asymmetrical bilayers showed that this effect is leaflet specific and most effective in the inner leaflet, in which aPLs are normally present in plasma membranes. The data underscore a general positive effect of aPLs on the conductance of K⁺ channels and a potential interaction of their negative headgroup with cationic amino acids in their vicinity.     

Homology Models of the Tetramerization Domain of Six Eukaryotic Voltage-gated Potassium Channels Kv1.1-Kv1.6

Abstract

The homology models of the tetramerization (T1) domain of six eukaryotic potassium channels, Kv1.1-Kv1.6, were constructed based on the crystal structure of the Shaker T1 domain. The results of amino acid sequence alignment indicate that the T1 domains of these K⁺ channels are highly conserved, with the similarities varying from 77% between Shaker and Kv1.6 to 93% between Kv1.2 and Kv1.3. The homology models reveal that the T1 domains of these Kv channels exhibit similar folds as those of Shaker K⁺ channel. These models also show that each T1 monomer consists of three distinct layers, with N-terminal layer 1 and C- terminal layer 3 facing the cytoplasm and the membrane, respectively. Layer 2 exhibits the highest structural conservation because it is located around the central hydrophobic core. For each Kv channel, four identical subunits assemble into the homotetramer architecture around a four-fold axis through the hydrogen bonds and salt bridges formed by 15 highly conserved polar residues. The narrowest opening of the pore is formed by the four conserved residues corresponding to R115 of the Shaker T1 domain. The homology models of these Kv T1 domains provide particularly attractive targets for further structure-based studies.     

Divalent Cations Slow Activation of EAG Family K Channels through Direct Binding to S4

Voltage-gated K⁺ channels share a common voltage sensor domain (VSD) consisting of four transmembrane helices, including a highly mobile S4 helix that contains the major gating charges. Activation of ether-a-go-go (EAG) family K⁺ channels is sensitive to external divalent cations. We show here that divalent cations slow the activation rate of two EAG family channels (Kv12.1 and Kv10.2) by forming a bridge between a residue in the S4 helix and acidic residues in S2. Histidine 328 in the S4 of Kv12.1 favors binding of Zn²⁺ and Cd²⁺, whereas the homologous residue Serine 321 in Kv10.2 contributes to effects of Mg²⁺ and Ni²⁺. This novel finding provides structural constraints for the position of transmembrane VSD helices in closed, ion-bound EAG family channels. Homology models of Kv12.1 and Kv10.2 VSD structures based on a closed-state model of the Shaker family K⁺ channel Kv1.2 match these constraints. Our results suggest close conformational conservation between closed EAG and Shaker family channels, despite large differences in voltage sensitivity, activation rates, and activation thresholds.     

Possible function for virus encoded K+ channel Kcv in the replication of chlorella virus PBCV-1

The K⁺ channel Kcv is encoded by the chlorella virus PBCV-1. There is evidence that this channel plays an essential role in the replication of the virus, because both PBCV-1 plaque formation and Kcv channel activity in Xenopus oocytes have similar sensitivities to inhibitors. Here we report circumstantial evidence that the Kcv channel is important during virus infection. Recordings of membrane voltage in the host cells Chlorella NC64A reveal a membrane depolarization within the first few minutes of infection. This depolarization displays the same sensitivity to cations as Kcv conductance; depolarization also requires the intact membrane of the virion. Together these data are consistent with the idea that the virus carries functional K⁺ channels in the virion and inserts them into the host cell plasma membrane during infection.     

Gating pore currents,a new pathological mechanism underlying cardiac arrhythmias associated with dilated cardiomyopathy

The ability of cells to reliably fire action potentials is critically dependent upon the maintenance of a hyperpolarized resting potential, which allows voltage-gated Na⁺ and Ca²⁺ channels to recover from inactivation and open in response to a subsequent stimulus. Hodgkin and Huxley first recognized the functional importance a small, steady outward leak of K⁺ ions to the resting potential, action potential generation and cellular excitability, and we now appreciate the contribution of inward rectifier-type K⁺ channels (Kir or KCNJ channels) to this process. More recently, however, it has become evident that two-pore domain K⁺ (K2P) channels also contribute to the steady outward leak of K⁺ ions, and thus, maintenance of the resting potential. Molecular cloning efforts have demonstrated that K2P channel exist in yeast to humans, and represent a major branch in the K⁺ channel superfamily. Humans express 15 types of K2P channels, which are grouped into six subfamilies, based on similarities in amino acid sequence and functional properties. Although K2P channels are not voltage-gated, due to the absence of a canonical voltage sensor domain, their activity can be regulated by a variety of stimuli, including mechanical force, polyunsaturated fatty acids (PUFAs) (e.g., arachidonic acid), volatile anesthetics, acidity/pH, pharmacologic agents, heat and signaling events, such as phosphorylation and protein-protein interactions. K2P channels thus represent important regulators of cellular excitability by virtue of their impact on the resting potential, and as such, have garnered considerable attention in recent years.     

Pore-forming transmembrane domains control ion selectivity and selectivity filter conformation in the KirBac1.1 potassium channel

Potassium (K⁺) channels are membrane proteins with the remarkable ability to very selectively conduct K⁺ ions across the membrane. High-resolution structures have revealed that dehydrated K⁺ ions permeate through the narrowest region of the pore, formed by the backbone carbonyls of the signature selectivity filter (SF) sequence TxGYG. However, the existence of nonselective channels with similar SF sequences, as well as effects of mutations in other regions on selectivity, suggest that the SF is not the sole determinant of selectivity. We changed the selectivity of the KirBac1.1 channel by introducing mutations at residue I131 in transmembrane helix 2 (TM2). These mutations increase Na⁺ flux in the absence of K⁺ and introduce significant proton conductance. Consistent with K⁺ channel crystal structures, single-molecule FRET experiments show that the SF is conformationally constrained and stable in high-K⁺ conditions but undergoes transitions to dilated low-FRET states in high-Na⁺/low-K⁺ conditions. Relative to wild-type channels, I131M mutants exhibit marked shifts in the K⁺ and Na⁺ dependence of SF dynamics to higher K⁺ and lower Na⁺ concentrations. These results illuminate the role of I131, and potentially other structural elements outside the SF, in controlling ion selectivity, by suggesting that the physical interaction of these elements with the SF contributes to the relative stability of the constrained K⁺-induced SF configuration versus nonselective dilated conformations.     

Membrane anchoring and interaction between transmembrane domains are crucial for K+ channel function

The small viral channel Kcv is a Kir-like K(+) channel of only 94 amino acids. With this simple structure, the tetramer of Kcv represents the pore module of all complex K(+) channels. To examine the structural contribution of the transmembrane domains (TMDs) to channel function, we performed Ala scanning mutagenesis of the two domains and tested the functionality of the mutants in a yeast complementation assay. The data reveal, in combination with computational models, that the upper halves of both TMDs, which face toward the external medium, are rather rigid, whereas the inner parts are more flexible. The rigidity of the outer TMD is conferred by a number of essential aromatic amino acids that face the membrane and probably anchor this domain in the bilayer. The inner TMD is intimately connected with the rigid part of the outer TMD via π···π interactions between a pair of aromatic amino acids. This structural principle is conserved within the viral K(+) channels and also present in Kir2.2, implying a general importance of this architecture for K(+) channel function.     

C-Linker of Cyclic Nucleotide–gated Channels Controls Coupling of Ligand Binding to Channel Gating

Cyclic nucleotide–gated channels are composed of a core transmembrane domain, structurally homologous to the voltage-gated K⁺ channels, and a cytoplasmic ligand-binding domain. These two modules are joined by ∼90 conserved amino acids, the C-linker, whose precise role in the mechanism of channel activation by cyclic nucleotides is poorly understood. We examined cyclic nucleotide–gated channels from bovine photoreceptors and Caenorhabditis elegans sensory neurons that show marked differences in cyclic nucleotide efficacy and sensitivity. By constructing chimeras from these two channels, we identified a region of 30 amino acids in the C-linker (the L2 region) as an important determinant of activation properties. An increase in both the efficacy of gating and apparent affinity for cGMP and cAMP can be conferred onto the photoreceptor channel by the replacement of its L2 region with that of the C. elegans channel. Three residues within this region largely account for this effect. Despite the profound effect of the C-linker region on ligand gating, the identity of the C-linker does not affect the spontaneous, ligand-independent open probability. Based on a cyclic allosteric model of activation, we propose that the C-linker couples the opening reaction in the transmembrane core region to the enhancement of the affinity of the open channel for agonist, which underlies ligand gating.     

An ion gating mechanism of gastric H,K-ATPase based on molecular dynamics simulations

Gastric H,K-ATPase is an electroneutral transmembrane pump that moves protons from the cytoplasm of the parietal cell into the gastric lumen in exchange for potassium ions. The mechanism of transport against the established electrochemical gradients includes intermediate conformations in which the transferred ions are trapped (occluded) within the membrane domain of the pump. The pump cycle involves switching between the E1 and E2P states. Molecular dynamics simulations on homology models of the E2P and E1 states were performed to investigate the mechanism of K⁺ movement in this enzyme. We performed separate E2P simulations with one K⁺ in the luminal channel, one K⁺ ion in the occlusion site, two K⁺ ions in the occlusion site, and targeted molecular dynamics from E2P to E1 with two K⁺ ions in the occlusion site. The models were inserted into a lipid bilayer system and were stable over the time course of the simulations, and K⁺ ions in the channel moved to a consistent location near the center of the membrane domain, thus defining the occlusion site. The backbone carbonyl oxygen from residues 337 through 342 on the nonhelical turn of M4, as well as side-chain oxygen from E343, E795, and E820, participated in the ion occlusion. A single water molecule was stably bound between the two K⁺ ions in the occlusion site, providing an additional ligand and partial shielding the positive charges from one another. Targeted molecular dynamics was used to transform the protein from the E2P to the E1 state (two K⁺ ions to the cytoplasm). This simulation identified the separation of the water column in the entry channel as the likely gating mechanism on the luminal side. A hydrated exit channel also formed on the cytoplasmic side of the occlusion site during this simulation. Hence, water molecules became available to hydrate the ions. The movement of the M1M2 transmembrane segments, and the displacement of residues Q159, E160, Q110, and T152 during the conformational change, as well as the motions of E343 and L346, acted as the cytoplasmic-side gate.     

The mechano-gated K<Subscript>2P</Subscript> channel TREK-1

The versatility of neuronal electrical activity is largely conditioned by the expression of different structural and functional classes of K⁺ channels. More than 80 genes encoding the main K⁺ channel alpha subunits have been identified in the human genome. Alternative splicing, heteromultimeric assembly, post-translational modification and interaction with auxiliary regulatory subunits further increase the molecular and functional diversity of K⁺ channels. Mammalian two-pore domain K⁺ channels (K_2P) make up one class of K⁺ channels along with the inward rectifiers and the voltage- and/or calcium-dependent K⁺ channels. Each K_2P channel subunit is made up of four transmembrane segments and two pore-forming (P) domains, which are arranged in tandem and function as either homo- or heterodimeric channels. This novel structural arrangement is associated with unusual gating properties including “background” or “leak” K⁺ channel activity, in which the channels show constitutive activity at rest. In this review article, we will focus on the lipid-sensitive mechano-gated K_2P channel TREK-1 and will emphasize on the polymodal function of this “unconventional” K⁺ channel. EBSA Satellite meeting: Ion channels, Leeds, July 2007.     

Investigations of the Contribution of a Putative Glycine Hinge to Ryanodine Receptor Channel Gating

Ryanodine receptor channels (RyR) are key components of striated muscle excitation-contraction coupling, and alterations in their function underlie both inherited and acquired disease. A full understanding of the disease process will require a detailed knowledge of the mechanisms and structures involved in RyR function. Unfortunately, high-resolution structural data, such as exist for K⁺-selective channels, are not available for RyR. In the absence of these data, we have used modeling to identify similarities in the structural elements of K⁺ channel pore-forming regions and postulated equivalent regions of RyR. This has identified a sequence of residues in the cytosolic cavity-lining transmembrane helix of RyR (G⁴⁸⁶⁴LIIDA⁴⁸⁶⁹ in RyR2) analogous to the glycine hinge motif present in many K⁺ channels. Gating in these K⁺ channels can be disrupted by substitution of residues for the hinge glycine. We investigated the involvement of glycine 4864 in RyR2 gating by monitoring properties of recombinant human RyR2 channels in which this glycine is replaced by residues that alter gating in K⁺ channels. Our data demonstrate that introducing alanine at position 4864 produces no significant change in RyR2 function. In contrast, function is altered when glycine 4864 is replaced by either valine or proline, the former preventing channel opening and the latter modifying both ion translocation and gating. Our studies reveal novel information on the structural basis of RyR gating, identifying both similarities with, and differences from, K⁺ channels. Glycine 4864 is not absolutely required for channel gating, but some flexibility at this point in the cavity-lining transmembrane helix is necessary for normal RyR function.     

DPP6 Domains Responsible for Its Localization and Function

Dipeptidyl peptidase-like protein 6 (DPP6) is an auxiliary subunit of the Kv4 family of voltage-gated K⁺ channels known to enhance channel surface expression and potently accelerate their kinetics. DPP6 is a single transmembrane protein, which is structurally remarkable for its large extracellular domain. Included in this domain is a cysteine-rich motif, the function of which is unknown. Here we show that this cysteine-rich domain of DPP6 is required for its export from the ER and expression on the cell surface. Disulfide bridges formed at C349/C356 and C465/C468 of the cysteine-rich domain are necessary for the enhancement of Kv4.2 channel surface expression but not its interaction with Kv4.2 subunits. The short intracellular N-terminal and transmembrane domains of DPP6 associates with and accelerates the recovery from inactivation of Kv4.2, but the entire extracellular domain is necessary to enhance Kv4.2 surface expression and stabilization. Our findings show that the cysteine-rich domain of DPP6 plays an important role in protein folding of DPP6 that is required for transport of DPP6/Kv4.2 complexes out of the ER.     

A novel potassium channel encoded by Ectocarpus siliculosus virus

Kcv, the first identified viral potassium channel encoded by the green algae Paramecium bursaria chlorella virus (PBCV-1), conducted K(+) selective currents when expressed in heterologous systems. This K(+) channel was proposed to be important for PBCV-1 infection and replication. In the present study, we identified and functionally characterized a novel K(+) channel Kesv, encoded by Ectocarpus siliculosus virus that infects filamentous marine brown algae. Kesv encodes a protein of 124 amino acids and is 21.8% identical and 37.1% homologous to Kcv. Membrane topology programs predicted that Kesv consists of three transmembrane domains. When expressed in Xenopus oocytes, Kesv induced largely instantaneous, K(+) selective currents that were sensitive to block by Ba(2+) and amantadine. Thus, Kesv along with Kcv, constitutes an emerging family of viral potassium channels, which may play important roles in the life cycle of viruses.     

Overlapping Binding Sites of Structurally Different Antiarrhythmics Flecainide and Propafenone in the Subunit Interface of Potassium Channel Kv2.1

Kv2.1 channels, which are expressed in brain, heart, pancreas, and other organs and tissues, are important targets for drug design. Flecainide and propafenone are known to block Kv2.1 channels more potently than other Kv channels. Here, we sought to explore structural determinants of this selectivity. We demonstrated that flecainide reduced the K⁺ currents through Kv2.1 channels expressed in Xenopus laevis oocytes in a voltage- and time-dependent manner. By systematically exchanging various segments of Kv2.1 with those from Kv1.2, we determined flecainide-sensing residues in the P-helix and inner helix S6. These residues are not exposed to the inner pore, a conventional binding region of open channel blockers. The flecainide-sensing residues also contribute to propafenone binding, suggesting overlapping receptors for the drugs. Indeed, propafenone and flecainide compete for binding in Kv2.1. We further used Monte Carlo-energy minimizations to map the receptors of the drugs. Flecainide docking in the Kv1.2-based homology model of Kv2.1 predicts the ligand ammonium group in the central cavity and the benzamide moiety in a niche between S6 and the P-helix. Propafenone also binds in the niche. Its carbonyl group accepts an H-bond from the P-helix, the amino group donates an H-bond to the P-loop turn, whereas the propyl group protrudes in the pore and blocks the access to the selectivity filter. Thus, besides the binding region in the central cavity, certain K⁺ channel ligands can expand in the subunit interface whose residues are less conserved between K⁺ channels and hence may be targets for design of highly desirable subtype-specific K⁺ channel drugs.     

External Cd2+ and protons activate the hyperpolarization-gated K+ channel KAT1 at the voltage sensor

The functionally diverse cyclic nucleotide binding domain (CNBD) superfamily of cation channels contains both depolarization-gated (e.g., metazoan EAG family K⁺ channels) and hyperpolarization-gated channels (e.g., metazoan HCN pacemaker cation channels and the plant K⁺ channel KAT1). In both types of CNBD channels, the S4 transmembrane helix of the voltage sensor domain (VSD) moves outward in response to depolarization. This movement opens depolarization-gated channels and closes hyperpolarization-gated channels. External divalent cations and protons prevent or slow movement of S4 by binding to a cluster of acidic charges on the S2 and S3 transmembrane domains of the VSD and therefore inhibit activation of EAG family channels. However, a similar divalent ion/proton binding pocket has not been described for hyperpolarization-gated CNBD family channels. We examined the effects of external Cd²⁺ and protons on Arabidopsis thaliana KAT1 expressed in Xenopus oocytes and found that these ions strongly potentiate voltage activation. Cd²⁺ at 300 µM depolarizes the V₅₀ of KAT1 by 150 mV, while acidification from pH 7.0 to 4.0 depolarizes the V₅₀ by 49 mV. Regulation of KAT1 by Cd²⁺ is state dependent and consistent with Cd²⁺ binding to an S4-down state of the VSD. Neutralization of a conserved acidic charge in the S2 helix in KAT1 (D95N) eliminates Cd²⁺ and pH sensitivity. Conversely, introduction of acidic residues into KAT1 at additional S2 and S3 cluster positions that are charged in EAG family channels (N99D and Q149E in KAT1) decreases Cd²⁺ sensitivity and increases proton potentiation. These results suggest that KAT1, and presumably other hyperpolarization-gated plant CNBD channels, can open from an S4-down VSD conformation homologous to the divalent/proton-inhibited conformation of EAG family K⁺ channels.     

Isoflurane alters the structure and dynamics of GLIC

Pentameric ligand-gated ion channels are targets of general anesthetics. Although the search for discrete anesthetic binding sites has achieved some degree of success, little is known regarding how anesthetics work after the events of binding. Using the crystal structures of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), which is sensitive to a variety of general anesthetics, we performed multiple molecular dynamics simulations in the presence and absence of the general anesthetic isoflurane. Isoflurane bound to several locations within GLIC, including the transmembrane pocket identified crystallographically, the extracellular (EC) domain, and the interface of the EC and transmembrane domains. Isoflurane also entered the channel after the pore was dehydrated in one of the simulations. Isoflurane disrupted the quaternary structure of GLIC, as evidenced in a striking association between the binding and breakage of intersubunit salt bridges in the EC domain. The pore-lining helix experienced lateral and inward radial tilting motion that contributed to the channel closure. Isoflurane binding introduced strong anticorrelated motions between different subunits of GLIC. The demonstrated structural and dynamical modulations by isoflurane aid in the understanding of the underlying mechanism of anesthetic inhibition of GLIC and possibly other homologous pentameric ligand-gated ion channels.