Activation gating of hERG potassium channels: S6 glycines are not required as gating hinges

The opening of ion channels is proposed to arise from bending of the pore inner helices that enables them to pivot away from the central axis creating a cytosolic opening for ion diffusion. The flexibility of the inner helices is suggested to occur either at a conserved glycine located adjacent to the selectivity filter (glycine gating hinge) and/or at a second site occupied by glycine or proline containing motifs. Sequence alignment with other K+ channels shows that hERG possesses glycine residues (Gly648 and Gly657) at each of these putative hinge sites. In apparent contrast to the hinge hypotheses, substitution of both glycine residues for alanine causes little effect on either the voltage-dependence or kinetics of channel activation, and open state block by intracellular blockers. Substitution of the glycines with larger hydrophobic residues causes a greater propensity for the channel to open. We propose that in contrast to Shaker the pore of hERG is intrinsically more stable in the open than the closed conformation and that substitution at Gly648 or Gly657 further shifts the gating equilibrium to favor the open state. Molecular dynamics simulations indicate the S6 helices of hERG are inherently flexible, even in the absence of the glycine residues. Thus hERG activation gating exhibits important differences to other Kv channels. Our findings indicate that the hERG inner helix glycine residues are required for the tight packing of the channel helices and that the flexibility afforded by glycine or proline residues is not universally required for activation gating.     

Investigating the putative glycine hinge in Shaker potassium channel

The crystal structure of an open potassium channel reveals a kink in the inner helix that lines the pore (Jiang, Y.X., A. Lee, J.Y. Chen, M. Cadene, B.T. Chait, and R. MacKinnon. 2002. Nature 417:523-526). The putative hinge point is a highly conserved glycine residue. We examined the role of the homologous residue (Gly466) in the S6 transmembrane segment of Shaker potassium channels. The nonfunctional alanine mutant G466A will assemble, albeit poorly, with wild-type (WT) subunits, suppressing functional expression. To test if this glycine residue is critical for activation gating, we did a glycine scan along the S6 segment in the background of G466A. Although all of these double mutants lack the higher-level glycosylation that is characteristic of mature Shaker channels, one (G466A/V467G) is able to generate voltage-dependent potassium current. Surface biotinylation shows that functional and nonfunctional constructs containing G466A express at comparable levels in the plasma membrane. Compared with WT channels, the shifted-glycine mutant has impairments in voltage-dependent channel opening, including a right-shifted activation curve and a decreased rate of activation. The double mutant has relatively normal open-channel properties, except for a decreased affinity for intracellular blockers, a consequence of the loss of the side chain of Val467. Control experiments with the double mutants M440A/G466A and G466A/V467A suggest that the flexibility provided by Gly466 is more important for channel function than its small size. Our results support roles for Gly466 both in biogenesis of the channel and as a hinge in activation gating.     

Energetics of pore opening in a voltage-gated K(+) channel

Voltage-dependent gating in K(+) channels results from the mechanical coupling of voltage sensor movements to pore opening. We used single and double mutations in the pore of the Shaker K(+) channel to analyze a late concerted pore opening transition and interpreted the results in the context of known K(+) channel structures. Gating sensitive mutations are located at mechanistically informative regions of the pore and are coupled energetically across distances up to 15 A. We propose that the pore is intrinsically more stable when closed, and that to open the pore the voltage sensors must exert positive work by applying an outward lateral force near the inner helix bundle.     

A gating hinge in Na+ channels; a molecular switch for electrical signaling

Voltage-gated sodium channels are members of a large family with similar pore structures. The mechanism of opening and closing is unknown, but structural studies suggest gating via bending of the inner pore helix at a glycine hinge. Here we provide functional evidence for this gating model for the bacterial sodium channel NaChBac. Mutation of glycine 219 to proline, which would strongly favor bending of the alpha helix, greatly enhances activation by shifting its voltage dependence -51 mV and slowing deactivation by 2000-fold. The mutation also slows voltage-dependent inactivation by 1200-fold. The effects are specific because substitutions of proline at neighboring positions and substitutions of other amino acids at position 219 have much smaller functional effects. Our results fit a model in which concerted bending at glycine 219 in the S6 segments of NaChBac serves as a gating hinge. This gating motion may be conserved in most members of this large ion channel protein family.     

Selectivity filter gating in large-conductance Ca(2+)-activated K+ channels

Membrane voltage controls the passage of ions through voltage-gated K (K(v)) channels, and many studies have demonstrated that this is accomplished by a physical gate located at the cytoplasmic end of the pore. Critical to this determination were the findings that quaternary ammonium ions and certain peptides have access to their internal pore-blocking sites only when the channel gates are open, and that large blocking ions interfere with channel closing. Although an intracellular location for the physical gate of K(v) channels is well established, it is not clear if such a cytoplasmic gate exists in all K(+) channels. Some studies on large-conductance, voltage- and Ca(2+)-activated K(+) (BK) channels suggest a cytoplasmic location for the gate, but other findings question this conclusion and, instead, support the concept that BK channels are gated by the pore selectivity filter. If the BK channel is gated by the selectivity filter, the interactions between the blocking ions and channel gating should be influenced by the permeant ion. Thus, we tested tetrabutyl ammonium (TBA) and the Shaker "ball" peptide (BP) on BK channels with either K(+) or Rb(+) as the permeant ion. When tested in K(+) solutions, both TBA and the BP acted as open-channel blockers of BK channels, and the BP interfered with channel closing. In contrast, when Rb(+) replaced K(+) as the permeant ion, TBA and the BP blocked both closed and open BK channels, and the BP no longer interfered with channel closing. We also tested the cytoplasmically gated Shaker K channels and found the opposite behavior: the interactions of TBA and the BP with these K(v) channels were independent of the permeant ion. Our results add significantly to the evidence against a cytoplasmic gate in BK channels and represent a positive test for selectivity filter gating.     

Sequence-function analysis of the K+-selective family of ion channels using a comprehensive alignment and the KcsA channel structure

Sequence-function analysis of K(+)-selective channels was carried out in the context of the 3.2 A crystal structure of a K(+) channel (KcsA) from Streptomyces lividans (Doyle et al., 1998). The first step was the construction of an alignment of a comprehensive set of K(+)-selective channel sequences forming the putative permeation path. This pathway consists of two transmembrane segments plus an extracellular linker. Included in the alignment are channels from the eight major classes of K(+)-selective channels from a wide variety of species, displaying varied rectification, gating, and activation properties. Segments of the alignment were assigned to structural motifs based on the KcsA structure. The alignment's accuracy was verified by two observations on these motifs: 1), the most variability is shown in the turret region, which functionally is strongly implicated in susceptibility to toxin binding; and 2), the selectivity filter and pore helix are the most highly conserved regions. This alignment combined with the KcsA structure was used to assess whether clusters of contiguous residues linked by hydrophobic or electrostatic interactions in KcsA are conserved in the K(+)-selective channel family. Analysis of sequence conservation patterns in the alignment suggests that a cluster of conserved residues is critical for determining the degree of K(+) selectivity. The alignment also supports the near-universality of the "glycine hinge" mechanism at the center of the inner helix for opening K channels. This mechanism has been suggested by the recent crystallization of a K channel in the open state. Further, the alignment reveals a second highly conserved glycine near the extracellular end of the inner helix, which may be important in minimizing deformation of the extracellular vestibule as the channel opens. These and other sequence-function relationships found in this analysis suggest that much of the permeation path architecture in KcsA is present in most K(+)-selective channels. Because of this finding, the alignment provides a robust starting point for homology modeling of the permeation paths of other K(+)-selective channel classes and elucidation of sequence-function relationships therein. To assay these applications, a homology model of the Shaker A channel permeation path was constructed using the alignment and KcsA as the template, and its structure evaluated in light of established structural criteria.     

Collapse of conductance is prevented by a glutamate residue conserved in voltage-dependent K(+) channels

Voltage-dependent K(+) channel gating is influenced by the permeating ions. Extracellular K(+) determines the occupation of sites in the channels where the cation interferes with the motion of the gates. When external [K(+)] decreases, some K(+) channels open too briefly to allow the conduction of measurable current. Given that extracellular K(+) is normally low, we have studied if negatively charged amino acids in the extracellular loops of Shaker K(+) channels contribute to increase the local [K(+)]. Surprisingly, neutralization of the charge of most acidic residues has minor effects on gating. However, a glutamate residue (E418) located at the external end of the membrane spanning segment S5 is absolutely required for keeping channels active at the normal external [K(+)]. E418 is conserved in all families of voltage-dependent K(+) channels. Although the channel mutant E418Q has kinetic properties resembling those produced by removal of K(+) from the pore, it seems that E418 is not simply concentrating cations near the channel mouth, but has a direct and critical role in gating. Our data suggest that E418 contributes to stabilize the S4 voltage sensor in the depolarized position, thus permitting maintenance of the channel open conformation.     

A Conserved Mechanism for Gating in an Ionotropic Glutamate Receptor

Ionotropic glutamate receptor (iGluR) channels control synaptic activity. The crystallographic structure of GluA2, the prototypical iGluR, reveals a clamshell-like ligand-binding domain (LBD) that closes in the presence of glutamate to open a gate on the pore lining α-helix. How LBD closure leads to gate opening remains unclear. Here, we show that bending the pore helix at a highly conserved alanine residue (Ala-621) below the gate is responsible for channel opening. Substituting Ala-621 with the smaller more flexible glycine resulted in a basally active, nondesensitizing channel with ∼39-fold increase in glutamate potency without affecting surface expression or binding. On GluA2(A621G), the partial agonist kainate showed efficacy similar to a full agonist, and competitive antagonists CNQX and DNQX acted as a partial agonists. Met-629 in GluA2 sits above the gate and is critical in transmitting LBD closure to the gate. Substituting Met-629 with the flexible glycine resulted in reduced channel activity and glutamate potency. The pore regions in potassium channels are structurally similar to iGluRs. Whereas potassium channels typically use glycines as a hinge for gating, iGluRs use the less flexible alanine as a hinge at a similar position to maintain low basal activity allowing for ligand-mediated gating.     

State-dependent block of BK channels by synthesized shaker ball peptides

Crystal structures of potassium channels have strongly corroborated an earlier hypothetical picture based on functional studies, in which the channel gate was located on the cytoplasmic side of the pore. However, accessibility studies on several types of ligand-sensitive K(+) channels have suggested that their activation gates may be located near or within the selectivity filter instead. It remains to be determined to what extent the physical location of the gate is conserved across the large K(+) channel family. Direct evidence about the location of the gate in large conductance calcium-activated K(+) (BK) channels, which are gated by both voltage and ligand (calcium), has been scarce. Our earlier kinetic measurements of the block of BK channels by internal quaternary ammonium ions have raised the possibility that they may lack a cytoplasmic gate. We show in this study that a synthesized Shaker ball peptide (ShBP) homologue acts as a state-dependent blocker for BK channels when applied internally, suggesting a widening at the intracellular end of the channel pore upon gating. This is consistent with a gating-related conformational change at the cytoplasmic end of the pore-lining helices, as suggested by previous functional and structural studies on other K(+) channels. Furthermore, our results from two BK channel mutations demonstrate that similar types of interactions between ball peptides and channels are shared by BK and other K(+) channel types.     

Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels.

In cyclic nucleotide-gated channels (CNG), direct binding of cyclic nucleotides in the carboxy-terminal region is allosterically coupled to opening of the pore. A CNG1 channel pore was probed using site-directed cysteine substitution to elucidate conformational changes associated with channel opening. The effects of cysteine modification on permeation suggest a structural homology between CNG and KcsA pores. We found that intersubunit disulfide bonds form spontaneously between S399C residues in the helix bundle when channels are in the closed but not in the open state. While MTSET modification of pore-lining residues was state dependent, Ag(+) modification of V391C, in the inner vestibule, occurred at the same diffusion-limited rate in both open and closed states. Our results suggest that the helix bundle undergoes a conformational change associated with gating but is not the activation gate for CNG channels.     

Molecular compatibility of the channel gate and the N terminus of S5 segment for voltage-gated channel activity

Voltage-gated ion channels are modular proteins designed by the structural linkage of a voltage sensor and a pore domain. The functional coupling of these two protein modules is a subject of intense research. A major focus has been directed to decipher the role of the S4-S5 linker and the C-end of the inner pore helix in channel gating. However, the contribution of the cytosolic N terminus of S5 remains elusive. To address this issue, we used a chimeric subunit that linked the voltage sensor of the Shaker channel to the prokaryotic KcsA pore domain (denoted as Shaker-KcsA). This chimera preserved the Shaker sequences at both the N terminus of S5 and the C-end of S6. Chimeric Shaker-KcsA subunits did not form functional homomeric channels but were synthesized, folded, and trafficked to the cell surface, as evidenced by their co-assembly with Shaker wild type subunits. Sequential substitution of Shaker amino acids at the C-end of S6 and the N terminus of S5 by the corresponding KcsA created voltage-sensitive channels with voltage-dependent properties that asymptotically approached those of the wild type Shaker channel. Noteworthy, substitution of the region encompassing Phe(401)-Phe(404) at the N-end of Shaker S5 by KcsA residues resulted in a significant gain in voltage sensitivity of the chimeras. Furthermore, analysis of channel function at high [K(+)](o) revealed that the Phe(401)-Phe(404) region is an important molecular determinant for competent coupling of voltage sensing and pore opening. Taken together, these findings indicate that complete replacement of Shaker S5 and S6 by KcsA M1 and M2 is required for voltage-dependent gating of the prokaryotic channel. In addition, our results imply that the region encompassing Phe(401)-Phe(404) in Shaker is involved in protein-protein interactions with the voltage sensor, and signal to the Phe(401) in the S5 segment as a key molecular determinant to pair the voltage sensor and the pore domain.     

Massive endocytosis driven by lipidic forces originating in the outer plasmalemmal monolayer: a new approach to membrane recycling and lipid domains

The pore-lining amino acids of ion channel proteins reside on the interface between a polar (the pore) and a nonpolar environment (the rest of the protein). The structural dynamics of this region, which physically controls ionic flow, are essential components of channel gating. Using large-conductance, Ca(2+)-dependent K(+) (BK) channels, we devised a systematic charge-substitution method to probe conformational changes in the pore region during channel gating. We identified a deep-pore residue (314 in hSlo1) as a marker of structural dynamics. We manipulated the charge states of this residue by substituting amino acids with different valence and pKa, and by adjusting intracellular pH. We found that the charged states of the 314 residues stabilized an open state of the BK channel. With models based on known structures of related channels, we postulate a dynamic rearrangement of the deep-pore region during BK channel opening/closing, which involves a change of the degree of pore exposure for 314.     

KvAP-based model of the pore region of shaker potassium channel is consistent with cadmium- and ligand-binding experiments

Potassium channels play fundamental roles in excitable cells. X-ray structures of bacterial potassium channels show that the pore-lining inner helices obstruct the cytoplasmic entrance to the closed channel KcsA, but diverge in widely open channels MthK and KvAP, suggesting a gating-hinge role for a conserved Gly in the inner helix. A different location of the gating hinge and a narrower open pore were proposed for voltage-gated Shaker potassium channels that have the Pro-473-Val-Pro motif. Two major observations back the proposal: cadmium ions lock mutant Val-476-Cys in the open state by bridging Cys-476 and His-486 in adjacent helices, and cadmium blocks the locked-open double mutant Val-474-Cys/Val-476-Cys by binding to Cys-474 residues. Here we used molecular modeling to show that the open Shaker should be as wide as KvAP to accommodate an open-channel blocker, correolide. We further built KvAP-, MthK-, and KcsA-based models of the Shaker mutants and Monte-Carlo-minimized them with constraints Cys-476-Cd(2+)-His-486. The latter were consistent with the KvAP-based model, causing a small-bend N-terminal to the Pro-473-Val-Pro motif. The constraints significantly distorted the MthK-based structure, making it similar to KvAP. The KcsA structure resisted the constraints. Two Cd(2+) ions easily block the locked-open KvAP-based model at Cys-474 residues, whereas constraining a single cadmium ion to four Cys-474 caused large conformational changes and electrostatic imbalance. Although mutual disposition of the voltage-sensor and pore domains in the KvAP x-ray structure is currently disputed, our results suggest that the pore-region domain retains a nativelike conformation in the crystal.     

The pore helix is involved in stabilizing the open state of inwardly rectifying K+ channels

Ion channels can be gated by various extrinsic cues, such as voltage, pH, and second messengers. However, most ion channels display extrinsic cue-independent transitions as well. These events represent spontaneous conformational changes of the channel protein. The molecular basis for spontaneous gating and its relation to the mechanism by which channels undergo activation gating by extrinsic cue stimulation is not well understood. Here we show that the proximal pore helix of inwardly rectifying (Kir) channels is partially responsible for determining spontaneous gating characteristics, affecting the open state of the channel by stabilizing intraburst openings as well as the bursting state itself without affecting K(+) ion-channel interactions. The effect of the pore helix on the open state of the channel is qualitatively similar to that of two well-characterized mutations at the second transmembrane domain (TM2), which stabilize the channel in its activated state. However, the effects of the pore helix and the TM2 mutations on gating were additive and independent of each other. Moreover, in sharp contrast to the two TM2 mutations, the pore helix mutation did not affect the functionality of the agonist-responsive gate. Our results suggest that in Kir channels, the bottom of the pore helix and agonist-induced conformational transitions at the TM2 ultimately stabilize via different pathways the open conformation of the same gate.     

Open-state conformation of the KcsA K+ channel: Monte Carlo normal mode following simulations

Potassium channels fluctuate between closed and open states. The detailed mechanism of the conformational changes opening the intracellular pore in the K+ channel from Streptomyces lividans (KcsA) is unknown. Applying Monte Carlo normal mode following, we find that gating involves rotation and unwinding of the TM2 bundle, lateral movement of the TM2 helices away from the channel axis, and disappearance of the TM2 bundle. The open-state conformation of KcsA exhibits a very wide inner vestibule, with a radius approximately 5-7 A and inner helices bent at the A98-G99 hinge. Computed conformational changes demonstrate that spin labeling and X-ray experiments illuminate different stages in gating: transition begins with clockwise rotation of the TM2 helices ending at a final state with the TM2 bend hinged near residues A98-G99. The concordance between the computational and experimental results provides atomic-level insights into the structural rearrangements of the channel's inner pore.     

The (beta)gamma subunits of G proteins gate a K(+) channel by pivoted bending of a transmembrane segment

The molecular mechanism of ion channel gating remains unclear. Using approaches such as proline scanning mutagenesis and homology modeling, we localize the gate of the K(+) channels controlled by the (beta)gamma subunits of G proteins at the pore-lining bundle crossing of the second transmembrane (TM2) helices. We show that the flexibility afforded by a highly conserved glycine residue in the middle of TM2 is crucial for channel gating. In contrast, flexibility introduced immediately below the gate disrupts gating. We propose that the force produced by channel-G(beta)gamma interactions is transduced through the rigid region below the helix bundle crossing to bend TM2 at the glycine that serves as a hinge and open the gate.     

Molecular coupling in the human ether-a-go-go-related gene-1 (hERG1) K+ channel inactivation pathway

Emerging evidence suggests that K(+) channel inactivation involves coupling between residues in adjacent regions of the channel. Human ether-a-go-go-related gene-1 (hERG1) K(+) channels undergo a fast inactivation gating process that is crucial for maintaining electrical stability in the heart. The molecular mechanisms that drive inactivation in hERG1 channels are unknown. Using alanine scanning mutagenesis, we show that a pore helix residue (Thr-618) that points toward the S5 segment is critical for normal inactivation gating. Amino acid substitutions at position 618 modulate the free energy of inactivation gating, causing enhanced or reduced inactivation. Mutation of an S5 residue that is predicted to be adjacent to Thr-618 (W568L) abolishes inactivation and alters ion selectivity. The introduction of the Thr-618-equivalent residue in Kv1.5 enhances inactivation. Molecular dynamic simulations of the Kv1.2 tetramer reveal van der Waals coupling between hERG1 618- and 568-equivalent residues and a significant increase in interaction energies when threonine is introduced at the 618-equivalent position. We propose that coupling between the S5 segment and pore helix may participate in the inactivation process in hERG1 channels.     

The pore structure of the closed RyR1 channel

Using single particle electron cryomicroscopy, several helices in the membrane-spanning region of RyR1, including an inner transmembrane helix, a short pore helix, and a helix parallel to the membrane on the cytoplasmic side, have been clearly resolved. Our model places a highly conserved glycine (G4934) at the hinge position of the bent inner helix and two rings of negative charges at the luminal and cytoplasmic mouths of the pore. The kinked inner helix closely resembles the inner helix of the open MthK channel, suggesting that kinking alone does not open RyR1, as proposed for K+ channels.     

Binding of a gating modifier toxin induces intersubunit cooperativity early in the Shaker K channel's activation pathway

Potassium currents from voltage-gated Shaker K channels activate with a sigmoid rise. The degree of sigmoidicity in channel opening kinetics confirms that each subunit of the homotetrameric Shaker channel undergoes more than one conformational change before the channel opens. We have examined effects of two externally applied gating modifiers that reduce the sigmoidicity of channel opening. A toxin from gastropod mucus, 6-bromo-2-mercaptotryptamine (BrMT), and divalent zinc are both found to slow the same conformational changes early in Shaker's activation pathway. Sigmoidicity measurements suggest that zinc slows a conformational change independently in each channel subunit. Analysis of activation in BrMT reveals cooperativity among subunits during these same early steps. A lack of competition with either agitoxin or tetraethylammonium indicates that BrMT binds channel subunits outside of the external pore region in an allosterically cooperative fashion. Simulations including negatively cooperative BrMT binding account for its ability to induce gating cooperativity during activation. We conclude that cooperativity among K channel subunits can be greatly altered by experimental conditions.     

Identification and characterization of the slowly exchanging pH-dependent conformational rearrangement in KcsA

Gating of ion channels is strictly regulated by physiological conditions as well as intra/extracellular ligands. To understand the underlying structures mediating ion channel gating, we investigated the pH-dependent gating of the K(+) channel KcsA under near-physiological conditions, using solution-state NMR. In a series of (1)H(15)N-TROSY HSQC (transverse relaxation optimized spectroscopy-heteronuclear single quantum coherence) spectra measured at various pH values, significant chemical shift changes were detected between pH 3.9 and 5.2, reflecting a conformational rearrangement associated with the gating. The pH-dependent chemical shift changes were mainly observed for the resonances from the residues near the intracellular helix bundle, which has been considered to form the primary gate in the K(+) channel, as well as the intracellular extension of the inner helix. The substitution of His-25 by Ala abolished this pH-dependent conformational rearrangement, indicating that the residue serves as a "pH-sensor" for the channel. Although the electrophysiological open probability of KcsA is less than 10%, the conformations of the intracellular helix bundle between the acidic and neutral conditions seem to be remarkably different. This supports the recently proposed "dual gating" properties of the K(+) channel, in which the activation-coupled inactivation at the selectivity filter determines the channel open probability of the channel. Indeed, a pH-dependent chemical shift change was also observed for the signal from the Trp-67 indole, which is involved in a hydrogen bond network related to the activation-coupled inactivation. The slow kinetic parameter obtained for the intracellular bundle seems to fit better into the time scale for burst duration than very fast fluctuations within a burst period, indicating the existence of another gating element with faster kinetic properties.