The nonconducting W434F mutant adopts upon membrane depolarization an inactivated-like state that differs from wild-type Shaker-IR potassium channels.

Voltage-gated K+ (Kv) channels mediate the flow of K+ across the cell membrane by regulating the conductive state of their activation gate (AG). Several Kv channels display slow C-type inactivation, a process whereby their selectivity filter (SF) becomes less or nonconductive. It has been proposed that, in the fast inactivation-removed Shaker-IR channel, the W434F mutation epitomizes the C-type inactivated state because it functionally accelerates this process. By introducing another pore mutation that prevents AG closure, P475D, we found a way to record ionic currents of the Shaker-IR-W434F-P475D mutant at hyperpolarized membrane potentials as the W434F-mutant SF recovers from its inactivated state. This W434F conductive state lost its high K+ over Na+ selectivity, and even NMDG+ can permeate, features not observed in a wild-type SF. This indicates that, at least during recovery from inactivation, the W434F-mutant SF transitions to a widened and noncationic specific conformation.

Structure, function, and ion-binding properties of a K+ channel stabilized in the 2,4-ion-bound configuration.

Here, we present the atomic resolution crystallographic structure, the function, and the ion-binding properties of the KcsA mutants, G77A and G77C, that stabilize the 2,4-ion-bound configuration (i.e., water, K+, water, K+-ion-bound configuration) of the K+ channel's selectivity filter. A full functional and thermodynamic characterization of the G77A mutant revealed wild-type-like ion selectivity and apparent K+-binding affinity, in addition to showing a lack of C-type inactivation gating and a marked reduction in its single-channel conductance. These structures validate, from a structural point of view, the notion that 2 isoenergetic ion-bound configurations coexist within a K+ channel's selectivity filter, which fully agrees with the water-K+-ion-coupled transport detected by streaming potential measurements.

Inverted allosteric coupling between activation and inactivation gates in K+ channels.

The selectivity filter and the activation gate in potassium channels are functionally and structurally coupled. An allosteric coupling underlies C-type inactivation coupled to activation gating in this ion-channel family (i.e., opening of the activation gate triggers the collapse of the channel's selectivity filter). We have identified the second Threonine residue within the TTVGYGD signature sequence of K+ channels as a crucial residue for this allosteric communication. A Threonine to Alanine substitution at this position was studied in three representative members of the K+-channel family. Interestingly, all of the mutant channels exhibited lack of C-type inactivation gating and an inversion of their allosteric coupling (i.e., closing of the activation gate collapses the channel's selectivity filter). A state-dependent crystallographic study of KcsA-T75A proves that, on activation, the selectivity filter transitions from a nonconductive and deep C-type inactivated conformation to a conductive one. Finally, we provide a crystallographic demonstration that closed-state inactivation can be achieved by the structural collapse of the channel's selectivity filter.

Structural Dynamics of the MscL C-terminal Domain.

The large conductance mechanosensitive channel (MscL), acts as an osmoprotective emergency valve in bacteria by opening a large, water-filled pore in response to changes in membrane tension. In its closed configuration, the last 36 residues at the C-terminus form a bundle of five α-helices co-linear with the five-fold axis of symmetry. Here, we examined the structural dynamics of the C-terminus of EcMscL using site-directed spin labelling electron paramagnetic resonance (SDSL EPR) spectroscopy. These experiments were complemented with computational modelling including molecular dynamics (MD) simulations and finite element (FE) modelling. Our results show that under physiological conditions, the C-terminus is indeed an α-helical bundle, located near the five-fold symmetry axis of the molecule. Both experiments and computational modelling demonstrate that only the top part of the C-terminal domain (from the residue A110 to E118) dissociates during the channel gating, while the rest of the C-terminus stays assembled. This result is consistent with the view that the C-terminus functions as a molecular sieve and stabilizer of the oligomeric MscL structure as previously suggested.

The gating cycle of a K+ channel at atomic resolution.

C-type inactivation in potassium channels helps fine-tune long-term channel activity through conformational changes at the selectivity filter. Here, through the use of cross-linked constitutively open constructs, we determined the structures of KcsA's mutants that stabilize the selectivity filter in its conductive (E71A, at 2.25 Å) and deep C-type inactivated (Y82A at 2.4 Å) conformations. These structural snapshots represent KcsA's transient open-conductive (O/O) and the stable open deep C-type inactivated states (O/I), respectively. The present structures provide an unprecedented view of the selectivity filter backbone in its collapsed deep C-type inactivated conformation, highlighting the close interactions with structural waters and the local allosteric interactions that couple activation and inactivation gating. Together with the structures associated with the closed-inactivated state (C/I) and in the well-known closed conductive state (C/O), this work recapitulates, at atomic resolution, the key conformational changes of a potassium channel pore domain as it progresses along its gating cycle.

Hysteresis of KcsA potassium channel's activation- deactivation gating is caused by structural changes at the channel's selectivity filter.

Mode-shift or hysteresis has been reported in ion channels. Voltage-shift for gating currents is well documented for voltage-gated cation channels (VGCC), and it is considered a voltage-sensing domain's (VSD) intrinsic property. However, uncoupling the Shaker K+ channel's pore domain (PD) from the VSD prevented the mode-shift of the gating currents. Consequently, it was proposed that an open-state stabilization of the PD imposes a mechanical load on the VSD, which causes its mode-shift. Furthermore, the mode-shift displayed by hyperpolarization-gated cation channels is likely caused by structural changes at the channel's PD similar to those underlying C-type inactivation. To demonstrate that the PD of VGCC undergoes hysteresis, it is imperative to study its gating process in the absence of the VSD. A back-door strategy is to use KcsA (a K+ channel from the bacteria Streptomyces lividans) as a surrogate because it lacks a VSD and exhibits an activation coupled to C-type inactivation. By directly measuring KcsA's activation gate opening and closing in conditions that promote or halt C-type inactivation, we have found (i) that KcsA undergoes mode-shift of gating when having K+ as the permeant ion; (ii) that Cs+ or Rb+, known to halt C-inactivation, prevented mode-shift of gating; and (iii) that, in the total absence of C-type inactivation, KcsA's mode-shift was prevented. Finally, our results demonstrate that an allosteric communication causes KcsA's activation gate to "remember" the conformation of the selectivity filter, and hence KcsA requires a different amount of energy for opening than for closing.

A cost-effective protocol for the over-expression and purification of fully-functional and more stable Erwinia chrysanthemi ligand-gated ion channel.

The Erwinia chrysanthemi ligand-gated ion channel, ELIC, is considered an excellent structural and functional surrogate for the whole pentameric ligand-gated ion channel family. Despite its simplicity, ELIC is structurally capable of undergoing ligand-dependent activation and a concomitant desensitization process. To determine at the molecular level the structural changes underlying ELIC's function, it is desirable to produce large quantities of protein. This protein should be properly folded, fully-functional and amenable to structural determinations. In the current paper, we report a completely new protocol for the expression and purification of milligram quantities of fully-functional, more stable and crystallizable ELIC. The use of an autoinduction media and inexpensive detergents during ELIC extraction, in addition to the high-quality and large quantity of the purified channel, are the highlights of this improved biochemical protocol.

An Escherichia coli-Based Assay to Assess the Function of Recombinant Human Hemichannels.

Connexins form the gap junctional channels that mediate cell-to-cell communication, and also form hemichannels present at the plasma membrane. Hemichannels are permeable to small hydrophilic compounds, including molecules involved in autocrine and paracrine signaling. An abnormal hemichannel opening causes or contributes to cell damage in common human disorders (e.g., cardiac infarct, cerebrovascular accidents, deafness, skin diseases, and cataracts) and is therefore a potential pharmacological target. The discovery of useful hemichannels inhibitors has been hampered in part by the lack of suitable high-throughput functional assays. Here, we developed and characterized an assay useful to assess the function of hemichannels formed by human connexins expressed in a genetically modified Escherichia coli strain. The LB2003 cells, devoid of three key K+ uptake transport mechanisms, cannot grow in low-[K+] medium, but expression of Cx26, Cx43, or Cx46 rescues their growth defect (growth complementation). We developed a protocol for a simple, inexpensive, easily scalable, reproducible, and sensitive assay that should be useful for the discovery of new and better hemichannel inhibitors based on the analysis of small-compound libraries.

The role of MscL amphipathic N terminus indicates a blueprint for bilayer-mediated gating of mechanosensitive channels.

The bacterial mechanosensitive channel MscL gates in response to membrane tension as a result of mechanical force transmitted directly to the channel from the lipid bilayer. MscL represents an excellent model system to study the basic biophysical principles of mechanosensory transduction. However, understanding of the essential structural components that transduce bilayer tension into channel gating remains incomplete. Here using multiple experimental and computational approaches, we demonstrate that the amphipathic N-terminal helix of MscL acts as a crucial structural element during tension-induced gating, both stabilizing the closed state and coupling the channel to the membrane. We propose that this may also represent a common principle in the gating cycle of unrelated mechanosensitive ion channels, allowing the coupling of channel conformation to membrane dynamics.

Functional hemichannels formed by human connexin 26 expressed in bacteria.

Gap-junction channels (GJCs) communicate the cytoplasm of adjacent cells and are formed by head-to-head association of two hemichannels (HCs), one from each of the neighbouring cells. GJCs mediate electrical and chemical communication between cells, whereas undocked HCs participate in paracrine signalling because of their permeability to molecules such as ATP. Sustained opening of HCs under pathological conditions results in water and solute fluxes that cannot be compensated by membrane transport and therefore lead to cell damage. Mutations of Cx26 (connexin 26) are the most frequent cause of genetic deafness and it is therefore important to understand the structure-function relationship of wild-type and deafness-associated mutants. Currently available connexin HC expression systems severely limit the pace of structural studies and there is no simple high-throughput HC functional assay. The Escherichia coli-based expression system presented in the present study yields milligram amounts of purified Cx26 HCs suitable for functional and structural studies. We also show evidence of functional activity of recombinant Cx26 HCs in intact bacteria using a new growth complementation assay. The E. coli-based expression system has high potential for structural studies and high-throughput functional screening of HCs.

Expression, purification, and reconstitution of the voltage-sensing domain from Ci-VSP.

The voltage-sensing domain (VSD) is the common scaffold responsible for the functional behavior of voltage-gated ion channels, voltage sensitive enzymes, and proton channels. Because of the position of the voltage dependence of the available VSD structures, at present, they all represent the activated state of the sensor. Yet in the absence of a consensus resting state structure, the mechanistic details of voltage sensing remain controversial. The voltage dependence of the VSD from Ci-VSP (Ci-VSD) is dramatically right shifted, so that at 0 mV it presumably populates the putative resting state. Appropriate biochemical methods are an essential prerequisite for generating sufficient amounts of Ci-VSD protein for high-resolution structural studies. Here, we present a simple and robust protocol for the expression of eukaryotic Ci-VSD in Escherichia coli at milligram levels. The protein is pure, homogeneous, monodisperse, and well-folded after solubilization in Anzergent 3-14 at the analyzed concentration (~0.3 mg/mL). Ci-VSD can be reconstituted into liposomes of various compositions, and initial site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopic measurements indicate its first transmembrane segment folds into an α-helix, in agreement with the homologous region of other VSDs. On the basis of our results and enhanced relaxation EPR spectroscopy measurement, Ci-VSD reconstitutes essentially randomly in proteoliposomes, precluding straightforward application of transmembrane voltages in combination with spectroscopic methods. Nevertheless, these results represent an initial step that makes the resting state of a VSD accessible to a variety of biophysical and structural approaches, including X-ray crystallography, spectroscopic methods, and electrophysiology in lipid bilayers.

Mechanism of activation gating in the full-length KcsA K+ channel.

Using a constitutively active channel mutant, we solved the structure of full-length KcsA in the open conformation at 3.9 Å. The structure reveals that the activation gate expands about 20 Å, exerting a strain on the bulge helices in the C-terminal domain and generating side windows large enough to accommodate hydrated K(+) ions. Functional and spectroscopic analysis of the gating transition provides direct insight into the allosteric coupling between the activation gate and the selectivity filter. We show that the movement of the inner gate helix is transmitted to the C-terminus as a straightforward expansion, leading to an upward movement and the insertion of the top third of the bulge helix into the membrane. We suggest that by limiting the extent to which the inner gate can open, the cytoplasmic domain also modulates the level of inactivation occurring at the selectivity filter.

On the structural basis of modal gating behavior in K(+) channels.

Modal-gating shifts represent an effective regulatory mechanism by which ion channels control the extent and time course of ionic fluxes. Under steady-state conditions, the K(+) channel KcsA shows three distinct gating modes, high-P(o), low-P(o) and a high-frequency flicker mode, each with about an order of magnitude difference in their mean open times. Here we show that in the absence of C-type inactivation, mutations at the pore-helix position Glu71 unmask a series of kinetically distinct modes of gating in a side chain-specific way. These gating modes mirror those seen in wild-type channels and suggest that specific interactions in the side chain network surrounding the selectivity filter, in concert with ion occupancy, alter the relative stability of pre-existing conformational states of the pore. The present results highlight the key role of the selectivity filter in regulating modal gating behavior in K(+) channels.

Structural dynamics of the magnesium-bound conformation of CorA in a lipid bilayer.

The transmembrane conformation of Thermotoga maritima CorA, a magnesium transport system, has been studied in its Mg(2+)-bound form by site-directed spin labeling and electron paramagnetic resonance spectroscopy. Probe mobility together with accessibility data were used to evaluate the overall dynamics and relative arrangement of individual transmembrane segments TM1 and TM2. TM1 extends toward the cytoplasmic side creating a water-filled cavity, while TM2 is located in the periphery of the oligomer, contacting the lipid bilayer. A structural model for the conserved extracellular loop was generated based on EPR data and MD simulations, in which residue E316 is located toward the five-fold symmetry axis in position to electrostatically influence divalent ion translocation. Electrostatic analysis of our model suggest that, in agreement with the crystal structure, Mg(2+) -bound CorA is in a closed conformation. The present results suggest that long-range structural rearrangements are necessary to allow Mg(2+) translocation.

Structural mechanism of C-type inactivation in K(+) channels.

Interconversion between conductive and non-conductive forms of the K(+) channel selectivity filter underlies a variety of gating events, from flicker transitions (at the microsecond timescale) to C-type inactivation (millisecond to second timescale). Here we report the crystal structure of the Streptomyces lividans K(+) channel KcsA in its open-inactivated conformation and investigate the mechanism of C-type inactivation gating at the selectivity filter from channels 'trapped' in a series of partially open conformations. Five conformer classes were identified with openings ranging from 12 A in closed KcsA (Calpha-Calpha distances at Thr 112) to 32 A when fully open. They revealed a remarkable correlation between the degree of gate opening and the conformation and ion occupancy of the selectivity filter. We show that a gradual filter backbone reorientation leads first to a loss of the S2 ion binding site and a subsequent loss of the S3 binding site, presumably abrogating ion conduction. These structures indicate a molecular basis for C-type inactivation in K(+) channels.

Structural basis for the coupling between activation and inactivation gates in K(+) channels.

The coupled interplay between activation and inactivation gating is a functional hallmark of K(+) channels. This coupling has been experimentally demonstrated through ion interaction effects and cysteine accessibility, and is associated with a well defined boundary of energetically coupled residues. The structure of the K(+) channel KcsA in its fully open conformation, in addition to four other partial channel openings, richly illustrates the structural basis of activation-inactivation gating. Here, we identify the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that, as a consequence of the hinge-bending and rotation of the TM2 helix, the aromatic ring of Phe 103 tilts towards residues Thr 74 and Thr 75 in the pore-helix and towards Ile 100 in the neighbouring subunit. This allows the network of hydrogen bonds among residues Trp 67, Glu 71 and Asp 80 to destabilize the selectivity filter, allowing entry to its non-conductive conformation. Mutations at position 103 have a size-dependent effect on gating kinetics: small side-chain substitutions F103A and F103C severely impair inactivation kinetics, whereas larger side chains such as F103W have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open-state interaction-energies between Phe 103 and the surrounding residues. We probed similar interactions in the Shaker K(+) channel where inactivation was impaired in the mutant I470A. We propose that side-chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K(+) channels.

A molecular mechanism for proton-dependent gating in KcsA.

Activation gating in KcsA is elicited by changes in intracellular proton concentration. Thompson et al. identified a charge cluster around the inner gate that plays a key role in defining proton activation in KcsA. Here, through functional and spectroscopic approaches, we confirmed the role of this charge cluster and now provide a mechanism of pH-dependent gating. Channel opening is driven by a set of electrostatic interactions that include R117, E120 and E118 at the bottom of TM2 and H25 at the end of TM1. We propose that electrostatic compensation in this charge cluster stabilizes the closed conformation at neutral pH and that its disruption at low pH facilitates the transition to the open conformation by means of helix-helix repulsion.

Design and characterization of a constitutively open KcsA.

The molecular nature of the structure responsible for proton sensitivity in KcsA has been identified as a charge cluster that surrounds the inner helical bundle gate. Here, we show that this proton sensor can be modified to engineer a constitutively open form of KcsA, amenable to functional, spectroscopic and structural analyses. By combining charge neutralizations for all acidic and basic residues in the cluster at positions 25, 117-122 and 124 (but not E118), a mutant KcsA is generated that displays constitutively open channel activity up to pH 9. The structure of this mutant revealed that full opening appears to be inhibited by lattice forces since the activation gate seems to be only on the early stages of opening.

Three-dimensional architecture of membrane-embedded MscS in the closed conformation.

The mechanosensitive channel of small conductance (MscS) is part of a coordinated response to osmotic challenges in Escherichia coli. MscS opens as a result of membrane tension changes, thereby releasing small solutes and effectively acting as an osmotic safety valve. Both the functional state depicted by its crystal structure and its gating mechanism remain unclear. Here, we combine site-directed spin labeling, electron paramagnetic resonance spectroscopy, and molecular dynamics simulations with novel energy restraints based on experimental electron paramagnetic resonance data to investigate the native transmembrane (TM) and periplasmic molecular architecture of closed MscS in a lipid bilayer. In the closed conformation, MscS shows a more compact TM domain than in the crystal structure, characterized by a realignment of the TM segments towards the normal of the membrane. The previously unresolved NH(2)-terminus forms a short helical hairpin capping the extracellular ends of TM1 and TM2 and is in close interaction with the bilayer interface. The present three-dimensional model of membrane-embedded MscS in the closed state represents a key step in determining the molecular mechanism of MscS gating.

Structural dynamics of an isolated voltage-sensor domain in a lipid bilayer.

A strong interplay between the voltage-sensor domain (VSD) and the pore domain (PD) underlies voltage-gated channel functions. In a few voltage-sensitive proteins, the VSD has been shown to function without a canonical PD, although its structure and oligomeric state remain unknown. Here, using EPR spectroscopy, we show that the isolated VSD of KvAP can remain monomeric in a reconstituted bilayer and retain a transmembrane conformation. We find that water-filled crevices extending deep into the membrane around S3, a scaffold conducive to transport of protons/cations, are intrinsic to the VSD. Differences in solvent accessibility in comparison to the full-length KvAP allowed us to define an interacting footprint of the PD on the VSD. This interaction is centered around S1 and S2 and suggests a rotation of 70 degrees -100 degrees relative to Kv1.2-Kv2.1 chimera. Sequence-conservation patterns in Kv channels, Hv channels, and voltage-sensitive phosphatases reveal several near-universal features suggesting a common molecular architecture for all VSDs.