Role of the KcsA channel cytoplasmic domain in pH-dependent gating

The KcsA channel is a representative potassium channel that is activated by changes in pH. Previous studies suggested that the region that senses pH is entirely within its transmembrane segments. However, we recently revealed that the cytoplasmic domain also has an important role, because its conformation was observed to change dramatically in response to pH changes. Here, to investigate the effects of the cytoplasmic domain on pH-dependent gating, we made a chimera mutant channel consisting of the cytoplasmic domain of the KcsA channel and the transmembrane region of the MthK channel. The chimera showed a pH dependency similar to that of KcsA, indicating that the cytoplasmic domain can act as a pH sensor. To identify how this region detects pH, we substituted certain cytoplasmic domain amino acids that are normally negatively charged at pH 7 for neutral ones in the KcsA channels. These mutants opened independently of pH, suggesting that electrostatic charges have a major role in the cytoplasmic domain's ability to sense and respond to pH.     

Surface structure and its dynamic rearrangements of the KcsA potassium channel upon gating and tetrabutylammonium blocking

KcsA is the first potassium channel for which the molecular structure was revealed. However, the high resolution structural information is limited to the transmembrane domain, and the dynamic picture of the full KcsA channel remains unsolved. We have developed a new approach to investigate the surface structure of proteins, and we applied this method to investigate the full length of the KcsA channel. Single-cysteine substitution was introduced into 25 sites, and specific reaction of these mutated channels to a bare surface of a flat gold plate was evaluated by surface plasmon resonance measurements. The surface plasmon resonance signals revealed the highest exposure for the mutant of the C-terminal end. When the gate of the KcsA channel is kept closed at pH 7.5, the extent of exposure showed periodic patterns for the consecutive sites located in the cytoplasmic (CP) and N-terminal domain. This suggests that these stretches take the alpha-helical structure. When the channel was actively gated at pH 4.0, many sites in the CP domain became exposed. Compared with the rigid structure in pH 7.5, these results indicate that the CP domain became loosely packed upon active gating. The C-terminal end of the M2 helix is a moving part of the gate, and it is exposed to the outer surface slightly at pH 4.0. By adding a channel blocker, tetrabutylammonium, the gate is further exposed. This suggests that in the active gating tetrabutylammonium keeps the gate open rather than being trapped in the central cavity.     

A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel

Voltage-gated K+ channels contain a central pore domain and four surrounding voltage-sensing domains. How and where changes in the structure of the voltage-sensing domains couple to the pore domain so as to gate ion conduction is not understood. The crystal structure of KcsA, a bacterial K+ channel homologous to the pore domain of voltage-gated K+ channels, provides a starting point for addressing this question. Guided by this structure, we used tryptophan-scanning mutagenesis on the transmembrane shell of the pore domain in the Shaker voltage-gated K+ channel to localize potential protein-protein and protein-lipid interfaces. Some mutants cause only minor changes in gating and when mapped onto the KcsA structure cluster away from the interface between pore domain subunits. In contrast, mutants producing large changes in gating tend to cluster near this interface. These results imply that voltage-sensing domains interact with localized regions near the interface between adjacent pore domain subunits.     

A KcsA/MloK1 Chimeric Ion Channel Has Lipid-dependent Ligand-binding Energetics

Cyclic nucleotide-modulated ion channels play crucial roles in signal transduction in eukaryotes. The molecular mechanism by which ligand binding leads to channel opening remains poorly understood, due in part to the lack of a robust method for preparing sufficient amounts of purified, stable protein required for structural and biochemical characterization. To overcome this limitation, we designed a stable, highly expressed chimeric ion channel consisting of the transmembrane domains of the well characterized potassium channel KcsA and the cyclic nucleotide-binding domains of the prokaryotic cyclic nucleotide-modulated channel MloK1. This chimera demonstrates KcsA-like pH-sensitive activity which is modulated by cAMP, reminiscent of the dual modulation in hyperpolarization-activated and cyclic nucleotide-gated channels that display voltage-dependent activity that is also modulated by cAMP. Using this chimeric construct, we were able to measure for the first time the binding thermodynamics of cAMP to an intact cyclic nucleotide-modulated ion channel using isothermal titration calorimetry. The energetics of ligand binding to channels reconstituted in lipid bilayers are substantially different from those observed in detergent micelles, suggesting that the conformation of the chimera''s transmembrane domain is sensitive to its (lipid or lipid-mimetic) environment and that ligand binding induces conformational changes in the transmembrane domain. Nevertheless, because cAMP on its own does not activate these chimeric channels, cAMP binding likely has a smaller energetic contribution to gating than proton binding suggesting that there is only a small difference in cAMP binding energy between the open and closed states of the channel.     

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.     

Conformational dynamics of M2 helices in KirBac channels: helix flexibility in relation to gating via molecular dynamics simulations

KirBac1.1 and 3.1 are bacterial homologues of mammalian inward rectifier K channels. We have performed extended molecular dynamics simulations (five simulations, each of >20 ns duration) of the transmembrane domain of KirBac in two membrane environments, a palmitoyl oleoyl phosphatidylcholine bilayer and an octane slab. Analysis of these simulations has focused on the conformational dynamics of the pore-lining M2 helices, which form the cytoplasmic hydrophobic gate of the channel. Principal components analysis reveals bending of M2, with a molecular hinge at the conserved glycine (Gly134 in KirBac1.1, Gly120 in KirBac3.1). More detailed analysis reveals a dimer-of-dimers type motion. The first two eigenvectors describing the motions of M2 correspond to helix kink and swivel motions. The conformational flexibility of M2 seen in these simulations correlates with differences in M2 conformation between that seen in the X-ray structures of closed channels (KcsA and KirBac) in which the helix is undistorted, and in open channels (e.g. MthK) in which the M2 helix is kinked. Thus, the simulations, albeit on a time scale substantially shorter than that required for channel gating, suggest a gating model in which the intrinsic flexibility of M2 about a molecular hinge is coupled to conformational transitions of an intracellular 'gatekeeper' domain, the latter changing conformation in response to ligand binding.     

Relationship between pore occupancy and gating in BK potassium channels

Permeant ions can have significant effects on ion channel conformational changes. To further understand the relationship between ion occupancy and gating conformational changes, we have studied macroscopic and single-channel gating of BK potassium channels with different permeant monovalent cations. While the slopes of the conductance-voltage curve were reduced with respect to potassium for all permeant ions, BK channels required stronger depolarization to open only when thallium was the permeant ion. Thallium also slowed the activation and deactivation kinetics. Both the change in kinetics and the shift in the GV curve were dependent on the thallium passing through the permeation pathway, as well as on the concentration of thallium. There was a decrease in the mean open time and an increase in the number of short flicker closing events with thallium as the permeating ion. Mean closed durations were unaffected. Application of previously established allosteric gating models indicated that thallium specifically alters the opening and closing transition of the channel and does not alter the calcium activation or voltage activation pathways. Addition of a closed flicker state into the allosteric model can account for the effect of thallium on gating. Consideration of the thallium concentration dependence of the gating effects suggests that the flicker state may correspond to the collapsed selectivity filter seen in crystal structures of the KcsA potassium channel under the condition of low permeant ion concentration.     

Calculation of rigid-body conformational changes using restraint-driven Cartesian transformations

We present an approach for calculating conformational changes in membrane proteins using limited distance information. The method, named restraint-driven Cartesian transformations, involves 1) the use of relative distance changes; 2) the systematic sampling of rigid body movements in Cartesian space; 3) a penalty evaluation; and 4) model refinement using energy minimization. As a test case, we have analyzed the structural basis of activation gating in the Streptomyces lividans potassium channel (KcsA). A total of 10 pairs of distance restraints derived from site-directed spin labeling and electron paramagnetic resonance (SDSL-EPR) spectra were used to calculate the open conformation of the second transmembrane domains of KcsA (TM2). The SDSL-EPR based structure reveals a gating mechanism consistent with a scissoring-type motion of the TM2 segments that includes a pivot point near middle of the helix. The present approach considerably reduces the amount of time and effort required to establish the overall nature of conformational changes in membrane proteins. It is expected that this approach can be implemented into restrained molecular dynamics protocol to calculate the structure and conformational changes in a variety of membrane protein systems.     

Yeast screen for constitutively active mutant G protein-activated potassium channels

GIRK2 is a major contributor to G protein-activated inward rectifier potassium channels in the mammalian brain. How GIRK channels open upon contact with Gbetagamma remains unknown. Using a yeast genetic screen to select constitutively active mutants from a randomly mutagenized GIRK2 library, we identified five gating mutations at four residues in the transmembrane domain. Further mutagenesis indicates that GIRK channel opening involves a rotation of the transmembrane segments, bringing one of these residues (V188) to a pore-lining position in the open conformation. Combined with double-mutant studies, these findings suggest that GIRK channels gate by moving from the open conformation inferred from our yeast study of Kir2.1 to a closed conformation perhaps resembling the known KcsA structure.     

Structure of the KcsA channel intracellular gate in the open state

Ion channels catalyze the selective transfer of ions across the membrane in response to a variety of stimuli. These channels gate by controlling the access of ions to a centrally located water-filled pore. The crystal structure of the Streptomyces lividans potassium channel (KcsA) has allowed a molecular exploration of this mechanism. Electron paramagnetic resonance (EPR) studies have uncovered significant conformational changes at the intracellular end of the second transmembrane helix (TM2) upon gating. We have used site-directed spin labeling (SDSL) and EPR spectroscopy in an attempt to quantify the structural rearrangements of the KcsA TM2 bundle underlying the transition from the closed to the open state. Under conditions favoring the closed and open conformations, 10 intersubunit distances were obtained across TM2 segments from tandem dimer constructs. Analysis of these data points to a mechanism in which each TM2 helix tilts away from the permeation pathway, towards the membrane plane, and rotates about its helical axis, supporting a scissoring-type motion with a pivot point near residues 107-108. These movements are accompanied by a large increase in the diameter of the vestibule below the central water-filled cavity.     

Mixed modes in opening of KcsA potassium channel from a targeted molecular dynamics simulation

Potassium channels conduct K⁺ flow selectively across the membrane through a central pore. During a process called gating, the potassium channels undergo a conformational change that opens or closes the ion-conducting pore. The potassium channel KcsA has been structurally determined in its closed state. However, the dynamic mechanism of the gating transition of the KcsA channel is still being investigated. Here, a targeted molecular dynamics simulation up to 150 ns is performed to investigate the detailed opening process of the KcsA channel with an open Kv1.2 structure serving as the target. The channel arrived at a self-determined quasi-stable state within 60 ns. The rigid-body and hinge-bending modes are observed mixed together in the remaining 90 ns long quasi-stable state. The mixed-mode movement seems come from the competition between the helix rigidity and the biased-applied gating force.     

Hydropathic analysis and comparison of KcsA and Shaker potassium channels

The similarity in structure of potassium (K(+)) channels from different families has been revealed by only recently available crystallographic 3D structural data. The hydropathic analysis presented in this work illuminates whether homologous residues perform the same functions in channels that use different gating mechanisms. We calculated and compared the hydropathic profiles of two K(+) channels, KcsA and Kv1.2 (the latter a member of the Shaker family), at their pore-forming domain. Quantitative information describing important interactions stabilizing the protein beyond obvious secondary-structure elements was extracted from the analysis and applied as a template for subsequent molecular-dynamics (MD) analyses. For example, two key groups of interactions, defining the turns that connect the transmembrane helices and responsible for the orientation of the pore helix, were identified. Our results also indicate that Asp(80) and Asp(379) play a similar role in stabilizing the P-loop of KcsA and Kv1.2, respectively, but to significantly different extents.     

Clustering and coupled gating modulate the activity in KcsA, a potassium channel model

Different patterns of channel activity have been detected by patch clamping excised membrane patches from reconstituted giant liposomes containing purified KcsA, a potassium channel from prokaryotes. The more frequent pattern has a characteristic low channel opening probability and exhibits many other features reported for KcsA reconstituted into planar lipid bilayers, including a moderate voltage dependence, blockade by Na(+), and a strict dependence on acidic pH for channel opening. The predominant gating event in this low channel opening probability pattern corresponds to the positive coupling of two KcsA channels. However, other activity patterns have been detected as well, which are characterized by a high channel opening probability (HOP patterns), positive coupling of mostly five concerted channels, and profound changes in other KcsA features, including a different voltage dependence, channel opening at neutral pH, and lack of Na(+) blockade. The above functional diversity occurs correlatively to the heterogeneous supramolecular assembly of KcsA into clusters. Clustering of KcsA depends on protein concentration and occurs both in detergent solution and more markedly in reconstituted membranes, including giant liposomes, where some of the clusters are large enough (up to micrometer size) to be observed by confocal microscopy. As in the allosteric conformational spread responses observed in receptor clustering (Bray, D. and Duke, T. (2004) Annu. Rev. Biophys. Biomol. Struct. 33, 53-73) our tenet is that physical clustering of KcsA channels is behind the observed multiple coupled gating and diverse functional responses.     

Three-dimensional structure of a voltage-gated potassium channel at 2.5 nm resolution

BACKGROUND: The voltage-gated potassium channel Shaker from Drosophila consists of a tetramer of identical subunits, each containing six transmembrane segments. The atomic structure of a bacterial homolog, the potassium channel KcsA, is much smaller than Shaker. It does not have a voltage sensor and other important domains like the N-terminal tetramerization (T1) domain. The structure of these additional elements has to be studied in the more complex voltage-gated channels. RESULTS: We determined the three-dimensional structure of the entire Shaker channel at 2.5 nm resolution using electron microscopy. The four-fold symmetric structure shows a large and a small domain linked by thin 2 nm long connectors. To interpret the structure, we used the crystal structures of the isolated T1 domain and the KcsA channel. A unique density assignment was made based on the symmetry and dimensions of the crystal structures and domains, identifying the smaller domain as the cytoplasmic mass of Shaker containing T1 and the larger domain as embedded in the membrane. CONCLUSIONS: The two-domain architecture of the Shaker channel is consistent with the recently proposed "hanging gondola" model for the T1 domain, putting the T1 domain at a distance from the membrane domain but attached to it by thin connectors. The space between the two domains is sufficient to permit cytoplasmic access of ions and the N-terminal inactivation domain to the pore region. A hanging gondola architecture has also been observed in the nicotinic acetylcholine receptor and the KcsA structure, suggesting that it is a common element of ion channels.     

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.     

Forced gating motions by a substituted titratable side chain at the bundle crossing of a potassium channel

Numerous inwardly rectifying potassium (Kir) channels possess an aromatic residue in the helix bundle crossing region, forming the narrowest pore constriction in crystal structures. However, the role of the Kir channel bundle crossing as a functional gate remains uncertain. We report a unique phenotype of Kir6.2 channels mutated to encode glutamate at this position (F168E). Despite a prediction of four glutamates in close proximity, Kir6.2(F168E) channels are predominantly closed at physiological pH, whereas alkalization causes rapid and reversible channel activation. These findings suggest that F168E glutamates are uncharged at physiological pH but become deprotonated at alkaline pH, forcing channel opening due to mutual repulsion of nearby negatively charged side chains. The potassium channel pore scaffold likely brings these glutamates close together, causing a significant pK(a) shift relative to the free side chain (as seen in the KcsA selectivity filter). Alkalization also shifts the apparent ATP sensitivity of the channel, indicating that forced motion of the bundle crossing is coupled to the ATP-binding site and may resemble conformational changes involved in wild-type Kir6.2 gating. The study demonstrates a novel mechanism for engineering extrinsic control of channel gating by pH and shows that conformational changes in the bundle crossing region are involved in ligand-dependent gating of Kir channels.     

Interaction between the cytoplasmic and transmembrane domains of the mechanosensitive channel MscS

The bacterial mechanosensitive channel MscS protects the bacteria from rupture on hypoosmotic shock. MscS is composed of a transmembrane domain with an ion permeation pore and a large cytoplasmic vestibule that undergoes significant conformational changes on gating. In this study, we investigated whether specific residues in the transmembrane and cytoplasmic domains of MscS influence each other during gating. When Asp-62, a negatively charged residue located in the loop that connects the first and second transmembrane helices, was replaced with either a neutral (Cys or Asn) or basic (Arg) amino acid, increases in both the gating threshold and inactivation rate were observed. Similar effects were observed after neutralization or reversal of the charge of either Arg-128 or Arg-131, which are both located near Asp-62 on the upper surface of the cytoplasmic domain. Interestingly, the effects of replacing Asp-62 with arginine were complemented by reversing the charge of Arg-131. Complementation was not observed after simultaneous neutralization of the charge of these residues. These findings suggest that the cytoplasmic domain of MscS affects both the mechanosensitive gating and the channel inactivation rate through the electrostatic interaction between Asp-62 and Arg-131.     

A prokaryotic potassium ion channel with two predicted transmembrane segments from Streptomyces lividans.

We report the identification, functional expression, purification, reconstitution and electrophysiological characterization of an up to now unique prokaryotic potassium ion channel (KcsA). It has a rectifying current-voltage relationship and displays subconductance states, the largest of which amounts to A approximately equal to 90 pS. The channel is blocked by Cs- ions and gating requires the presence of Mg2+ ions. The kcsA gene has been identified in the gram-positive soil bacterium Streptomyces lividans. It encodes a predicted 17.6 kDa protein with two potential membrane-spanning helices linked by a central domain which shares a high degree of similarity with the H5 segment conserved among eukaryotic ion channels. Multiple alignments of deduced amino acids suggest that the novel channel has the closest kinship to the S5, H5 and S6 regions of voltage-gated K+ channel families, mainly to the subfamily represented by the Shaker protein from Drosophila melanogaster. Moreover, KcsA is most distantly related to eukaryotic inwardly rectifying channels with two putative predicted transmembrane segments.     

A chimeric prokaryotic pentameric ligand–gated channel reveals distinct pathways of activation

Recent high resolution structures of several pentameric ligand–gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron–electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand–gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand–gated ion channel, which is activated by protons. We found that the chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators.