Non-conductive Conformation Research Articles

Physical mechanism for gating and mechanosensitivity of the human TRAAK K+ channel.

SummaryActivation of mechanosensitive ion channels by physical force underlies many physiological processes including the sensation of touch, hearing and pain1–5. TRAAK ion channels are neuronally expressed members of the two-pore domain K+ (K2P) channel family and are mechanosensitive6. They are involved in controlling mechanical and temperature nociception in mice7. Mechanosensitivity of TRAAK is mediated directly through the lipid bilayer: it is a membrane tension gated channel8. However, the molecular mechanism of TRAAK channel gating and mechanosensitivity is unknown. Here we present crystal structures of TRAAK in conductive and nonconductive conformations defined by the presence of permeant ions along the conduction pathway. In the nonconductive state, a lipid acyl chain accesses the channel cavity through a 5 Å-wide lateral opening in the membrane inner leaflet and physically blocks ion passage. In the conductive state, rotation of a transmembrane helix (TM4) about a central hinge seals the intramembrane opening, preventing lipid block of the cavity and permitting ion entry. Additional rotation of a membrane interacting TM2-TM3 segment, unique to mechanosensitive K2Ps, against TM4 may further stabilize the conductive conformation. Comparison of the structures reveals a biophysical explanation for TRAAK mechanosensitivity: an expansion in cross sectional area up to 2.7 nm2 in the conductive state is expected to create a membrane tension-dependent energy difference between conformations that promotes force activation. Our results show how tension of the lipid bilayer can be harnessed to control gating and mechanosensitivity of a eukaryotic ion channel.

The Cardiac Ryanodine Receptor (RyR2): Investigating Mechanisms of Gating at the Selectivity Filter

Ryanodine receptors (RyR) provide the pathway for release of intracellular calcium ions (Ca2+) that initiate muscle contraction. Mutations in cardiac RyRs, (RyR2) underlie arrhythmia and sudden cardiac death. RyRs are therefore an attractive therapeutic target, however more information regarding structure and function is required. The enormous size of RyRs precludes detailed structural analysis. Models, based on potassium channel templates, exist for the RyR pore-forming region (PFR) providing an invaluable framework in which predictions of specific processes of ion translocation and gating mechanisms can be tested.Potassium channels have a conserved gating mechanism involving a distinct hydrogen-bonding network of residues at the selectivity filter that is responsible for holding the filter in an inactive, non-conducting conformation. This study examines interactions of equivalent residues in RyR2 to ascertain whether a similar gating mechanism exists.Three alanine (D4829A, Y4813A, Y4839A) and one conserved tryptophan (Y4839W) RyR2 mutations were constructed to assess a proposed hydrogen-bonding network. Mutated channels formed functional homotetramers in vivo whereby they released Ca2+ upon caffeine addition. Differences in [3H]-ryanodine binding on isolated WT and mutant mix membrane populations revealed 1) altered ryanodine binding site and/or 2) altered calcium sensitivity for mutant channels. Preliminary single-channel experiments assessing ion handling and gating properties under steady state conditions suggest that the selectivity filer has a role in channel gating. Conductance and open probability (Po) for Y4839A was reduced by 15 % and 76 % respectively compared to WT RyR2. No single-channel experiments were performed for Y4813A due to inherent protein instability when purified. Four unique gating modes including subconductance states for D4829A were observed. Further experiments are required to assess the role of the selectivity filter in gating.Study supported by the British Heart Foundation.

Pore Helices Play a Dynamic Role as Integrators of Domain Motion during Kv11.1 Channel Inactivation Gating

Proteins that form ion-selective pores in the membrane of cells are integral to many rapid signaling processes, including regulating the rhythm of the heartbeat. In potassium channels, the selectivity filter is critical for both endowing an exquisite selectivity for potassium ions, as well as for controlling the flow of ions through the pore. Subtle rearrangements in the complex hydrogen-bond network that link the selectivity filter to the surrounding pore helices differentiate conducting (open) from nonconducting (inactivated) conformations of the channel. Recent studies suggest that beyond the selectivity filter, inactivation involves widespread rearrangements of the channel protein. Here, we use rate equilibrium free energy relationship analysis to probe the structural changes that occur during selectivity filter gating in Kv11.1 channels, at near atomic resolution. We show that the pore helix plays a crucial dynamic role as a bidirectional interface during selectivity filter gating. We also define the molecular bases of the energetic coupling between the pore helix and outer helix of the pore domain that occurs early in the transition from open to inactivated states, as well as the coupling between the pore helix and inner helix late in the transition. Our data demonstrate that the pore helices are more than just static structural elements supporting the integrity of the selectivity filter; instead they play a crucial dynamic role during selectivity filter gating.

Pore Determinants of KCNQ3 K+ Current Expression

KCNQ3 homomeric channels yield very small macroscopic currents compared with other KCNQ channels or KCNQ2/3 heteromers. Two disparate regions of the channels—the C-terminus and the pore region—have been implicated in governing KCNQ current amplitudes. We previously showed that the C-terminus plays a secondary role compared with the pore region. Here, we confirm the critical role of the pore region in determining KCNQ3 currents. We find that mutations at the 312 position in the pore helix of KCNQ3 (I312E, I312K, and I312R) dramatically decreased KCNQ3 homomeric currents as well as heteromeric KCNQ2/3 currents. Evidence that these mutants were expressed in the heteromers includes shifted TEA sensitivity compared with KCNQ2 homomers. To test for differential membrane protein expression, we performed total internal reflection fluorescence imaging, which revealed only small differences that do not underlie the differences in macroscopic currents. To determine whether this mechanism generalizes to other KCNQ channels, we tested the effects of analogous mutations at the conserved I273 position in KCNQ2, with similar results. Finally, we performed homology modeling of the pore region of wild-type and mutant KCNQ3 channels to investigate the putative structural mechanism mediating these results. The modeling suggests that the lack of current in I312E, I312K, and I312R KCNQ3 channels is due to pore helix-selectivity filter interactions that lock the selectivity filter in a nonconductive conformation.

Contribution of Ion Binding Affinity to Ion Selectivity and Permeation in KcsA, a Model Potassium Channel

Ion permeation and selectivity, key features in ion channel function, are believed to arise from a complex ensemble of energetic and kinetic variables. Here we evaluate the contribution of pore cation binding to ion permeation and selectivity features of KcsA, a model potassium channel. For this, we used E71A and M96V KcsA mutants in which the equilibrium between conductive and nonconductive conformations of the channel is differently shifted. E71A KcsA is a noninactivating channel mutant. Binding of K(+) to this mutant reveals a single set of low-affinity K(+) binding sites, similar to that seen in the binding of K(+) to wild-type KcsA that produces a conductive, low-affinity complex. This seems consistent with the observed K(+) permeation in E71A. Nonetheless, the E71A mutant retains K(+) selectivity, which cannot be explained on the basis of just its low affinity for this ion. At variance, M96V KcsA is a rapidly inactivating mutant that has lost selectivity for K(+) and also conducts Na(+). Here, low-affinity binding and high-affinity binding of both cations are detected, seemingly in agreement with both being permeating species in this mutant channel. In conclusion, binding of the ion to the channel protein seemingly explains certain gating, ion selectivity, and permeation properties. Ion binding stabilizes greatly the channel and, depending upon ion type and concentration, leads to different conformations and ion binding affinities. High-affinity states guarantee binding of specific ions and mediate ion selectivity but are nonconductive. Conversely, low-affinity states would not discriminate well among different ions but allow permeation to occur.

A Prokaryotic Perspective on Pentameric Ligand-Gated Ion Channel Structure and Function

The X-ray structures of two prokaryotic pentameric ligand-gated ion channels (pLGICs) have provided first structural insight into the family at high resolution. The structure of GLIC, a proton-activated channel from the cyanobacterium Gloebacter violaceous shows an open conformation of the pore. The channel conducts cations with similar properties as the nicotinic acetylcholine receptor and is inhibited by the same set of open channel blockers. The transmembrane pore is funnel-shaped with a wide hydrophobic entrance at the extracellular side that narrows to a hydrophilic intracellular opening. In this region conserved residues coordinate ions which have lost a large part of their hydration shell.The structure of ELIC, a pLGIC from the plant pathogen Erwinia chrysanthemi shows a non-conducting conformation of the channel that was obtained in the absence of ligands. In its structure the extracellular half of the pore is occluded by bulky hydrophobic residues that likely prevent ion conduction. ELIC is activated by a set of primary amines that include the neurotransmitter GABA. The protein forms cation selective channels with large single channel conductance that slowly desensitize in the presence of ligands.The strong structural similarity to their eukaryotic counterparts combined with their comparably simpler functional behavior make ELIC and GLIC important model systems for the pLGIC family that will ultimately allow a detailed comprehension of mechanistic properties that are still only poorly understood.

External Ba2+ Block of the Two-pore Domain Potassium Channel TREK-1 Defines Conformational Transition in Its Selectivity Filter

TREK-1 is a member of the two-pore domain potassium channel family that is known as a leak channel and plays a key role in many physiological and pathological processes. The conformational transition of the selectivity filter is considered as an effective strategy for potassium channels to control the course of potassium efflux. It is well known that TREK-1 is regulated by a large volume of extracellular and intracellular signals. However, until now, little was known about the selectivity filter gating mechanism of the channel. In this research, it was found that Ba(2+) blocked the TREK-1 channel in a concentration- and time-dependent manner. A mutagenesis analysis showed that overlapped binding of Ba(2+) at the assumed K(+) binding site 4 (S4) within the selectivity filter was responsible for the inhibitory effects on TREK-1. Then, Ba(2+) was used as a probe to explore the conformational transition in the selectivity filter of the channel. It was confirmed that collapsed conformations were induced by extracellular K(+)-free and acidification at the selectivity filters, leading to nonconductive to permeable ions. Further detailed characterization demonstrated that the two conformations presented different properties. Additionally, the N-terminal truncated isoform (ΔN41), a product derived from alternative translation initiation, was identified as a constitutively nonconductive variant. Together, these results illustrate the important role of selectivity filter gating in the regulation of TREK-1 by the extracellular K(+) and proton.

Assigning Functional States to Structural Models of Nicotinic-Receptor Type ion Channels

Aromatic-aromatic interactions are a prominent feature of the crystal structure of ELIC a bacterial member of the nicotinic-receptor superfamily of ion channels where five pore-facing phenylalanines come together to form a structure akin to a narrow iris that occludes the transmembrane pore. To identify the functional state of the channel that this structure represents, we engineered phenylalanines at various pore-facing positions of the muscle acetylcholine receptor, including the position that aligns with the native phenylalanine 246 of ELIC, and assessed the consequences of such mutations using electrophysiological and toxin-binding assays. From our experiments, we conclude that the interaction among the side chains of pore-facing phenylalanines leads to the formation of a non-conductive conformation that is unresponsive to the application of acetylcholine and is highly stable even in the absence of ligand. Moreover, electrophysiological recordings from a GLIC channel (another bacterial member of the superfamily) engineered to have a ring of phenylalanines at the corresponding pore-facing position suggest that this novel refractory state is distinct from the well-known desensitized state. It seems reasonable to propose, then, that it is in this peculiar non-conductive conformation that the ELIC channel was crystallized. It seems also reasonable to propose that, in the absence of rings of pore-facing aromatic side chains, such stable conformation may never be attained by the acetylcholine receptor. Incidentally, we also noticed that the response of the proton-gated, wild-type GLIC channel to a fast change in pH from 7.4 to 4.5 (on the extracellular side) is only transient, with the evoked current fading completely in a matter of seconds. This raises the possibility that the crystal structures of GLIC obtained at pH 4.0 and pH 4.6 correspond to the (well-known) desensitized state.

Mapping the sequence of conformational changes underlying selectivity filter gating in the Kv11.1 potassium channel

The potassium channel selectivity filter both discriminates between K(+) and sodium ions and contributes to gating of ion flow. Static structures of conducting (open) and nonconducting (inactivated) conformations of this filter are known; however, the sequence of protein rearrangements that connect these two states is not. We show that closure of the selectivity filter gate in the human K(v)11.1 K(+) channel (also known as hERG, for ether-a-go-go-related gene), a key regulator of the rhythm of the heartbeat, is initiated by K(+) exit, followed in sequence by conformational rearrangements of the pore domain outer helix, extracellular turret region, voltage sensor domain, intracellular domains and pore domain inner helix. In contrast to the simple wave-like sequence of events proposed for opening of ligand-gated ion channels, a complex spatial and temporal sequence of widespread domain motions connect the open and inactivated states of the K(v)11.1 K(+) channel.

A Molecular Switch between the Outer and the Inner Vestibules of the Voltage-gated Na+ Channel

Voltage-gated ion channels are transmembrane proteins that undergo complex conformational changes during their gating transitions. Both functional and structural data from K+ channels suggest that extracellular and intracellular parts of the pore communicate with each other via a trajectory of interacting amino acids. No crystal structures are available for voltage-gated Na+ channels, but functional data suggest a similar intramolecular communication involving the inner and outer vestibules. However, the mechanism of such communication is unknown. Here, we report that amino acid Ile-1575 in the middle of transmembrane segment 6 of domain IV (DIV-S6) in the adult rat skeletal muscle isoform of the voltage-gated sodium channel (rNaV1.4) may act as molecular switch allowing for interaction between outer and inner vestibules. Cysteine scanning mutagenesis of the internal part of DIV-S6 revealed that only mutations at site 1575 rescued the channel from a unique kinetic state (“ultra-slow inactivation,” IUS) produced by the mutation K1237E in the selectivity filter. A similar effect was seen with I1575A. Previously, we reported that conformational changes of both the internal and the external vestibule are involved in the generation of IUS. The fact that mutations at site 1575 modulate IUS produced by K1237E strongly suggests an interaction between these sites. Our data confirm a previously published molecular model in which Ile-1575 of DIV-S6 is in close proximity to Lys-1237 of the selectivity filter. Furthermore, these functional data define the position of the selectivity filter relative to the adjacent DIV-S6 segment within the ionic permeation pathway.

Bridging the Gap between Structural Models of Nicotinic Receptor Superfamily Ion Channels and Their Corresponding Functional States

Aromatic–aromatic interactions are a prominent feature of the crystal structure of ELIC [Protein Data Bank (PDB) code 2VL0], a bacterial member of the nicotinic receptor superfamily of ion channels where five pore-facing phenylalanines come together to form a structure akin to a narrow iris that occludes the transmembrane pore. To identify the functional state of the channel that this structure represents, we engineered phenylalanines at various pore-facing positions of the muscle acetylcholine (ACh) receptor (one position at a time), including the position that aligns with the native phenylalanine 246 of ELIC, and assessed the consequences of such mutations using electrophysiological and toxin-binding assays. From our experiments, we conclude that the interaction among the side chains of pore-facing phenylalanines, rather than the accumulation of their independent effects, leads to the formation of a nonconductive conformation that is unresponsive to the application of ACh and is highly stable even in the absence of ligand. Moreover, electrophysiological recordings from a GLIC channel (another bacterial member of the superfamily) engineered to have a ring of phenylalanines at the corresponding pore-facing position suggest that this novel refractory state is distinct from the well-known desensitized state. It seems reasonable to propose then that it is in this peculiar nonconductive conformation that the ELIC channel was crystallized. It seems also reasonable to propose that, in the absence of rings of pore-facing aromatic side chains, such stable conformation may never be attained by the ACh receptor. Incidentally, we also noticed that the response of the proton-gated wild-type GLIC channel to a fast change in pH from pH 7.4 to pH 4.5 (on the extracellular side) is only transient, with the evoked current fading completely in a matter of seconds. This raises the possibility that the crystal structures of GLIC obtained at pH 4.0 (PDB code 3EHZ) and pH 4.6 (PDB code 3EAM) correspond to the to the (well-known) desensitized state.

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 Twist on Potassium Channel Gating

The selectivity filter of potassium channels, the narrowest segment of the pore, permits only potassium ions to diffuse through the pore. Clarke et al. (2010) now present 11 structures of an inwardly rectifying potassium channel, providing evidence that the selectivity filter functions in channel gating and conformational changes in the cytoplasmic domains correlate with pore opening.

Principles of conduction and hydrophobic gating in K + channels

We present the first atomic-resolution observations of permeation and gating in a K(+) channel, based on molecular dynamics simulations of the Kv1.2 pore domain. Analysis of hundreds of simulated permeation events revealed a detailed conduction mechanism, resembling the Hodgkin-Keynes "knock-on" model, in which translocation of two selectivity filter-bound ions is driven by a third ion; formation of this knock-on intermediate is rate determining. In addition, at reverse or zero voltages, we observed pore closure by a novel "hydrophobic gating" mechanism: A dewetting transition of the hydrophobic pore cavity-fastest when K(+) was not bound in selectivity filter sites nearest the cavity-caused the open, conducting pore to collapse into a closed, nonconducting conformation. Such pore closure corroborates the idea that voltage sensors can act to prevent pore collapse into the intrinsically more stable, closed conformation, and it further suggests that molecular-scale dewetting facilitates a specific biological function: K(+) channel gating. Existing experimental data support our hypothesis that hydrophobic gating may be a fundamental principle underlying the gating of voltage-sensitive K(+) channels. We suggest that hydrophobic gating explains, in part, why diverse ion channels conserve hydrophobic pore cavities, and we speculate that modulation of cavity hydration could enable structural determination of both open and closed channels.

Gαi and Gβγ Jointly Regulate the Conformations of a Gβγ Effector, the Neuronal G Protein-activated K+ Channel (GIRK)

Stable complexes among G proteins and effectors are an emerging concept in cell signaling. The prototypical G betagamma effector G protein-activated K(+) channel (GIRK; Kir3) physically interacts with G betagamma but also with G alpha(i/o). Whether and how G alpha(i/o) subunits regulate GIRK in vivo is unclear. We studied triple interactions among GIRK subunits 1 and 2, G alpha(i3) and G betagamma. We used in vitro protein interaction assays and in vivo intramolecular Förster resonance energy transfer (i-FRET) between fluorophores attached to N and C termini of either GIRK1 or GIRK2 subunit. We demonstrate, for the first time, that G betagamma and G alpha(i3) distinctly and interdependently alter the conformational states of the heterotetrameric GIRK1/2 channel. Biochemical experiments show that G betagamma greatly enhances the binding of GIRK1 subunit to G alpha(i3)(GDP) and, unexpectedly, to G alpha(i3)(GTP). i-FRET showed that both G alpha(i3) and G betagamma induced distinct conformational changes in GIRK1 and GIRK2. Moreover, GIRK1 and GIRK2 subunits assumed unique, distinct conformations when coexpressed with a "constitutively active" G alpha(i3) mutant and G betagamma together. These conformations differ from those assumed by GIRK1 or GIRK2 after separate coexpression of either G alpha(i3) or G betagamma. Both biochemical and i-FRET data suggest that GIRK acts as the nucleator of the GIRK-G alpha-G betagamma signaling complex and mediates allosteric interactions between G alpha(i)(GTP) and G betagamma. Our findings imply that G alpha(i/o) and the G alpha(i) betagamma heterotrimer can regulate a G betagamma effector both before and after activation by neurotransmitters.

High-Resolution Crystallographic Analysis of the Kcsa Gating Cycle from Cysteine-Trapped Open Channels

The K+ channel pore domain contains all the elements necessary to catalyze the selective permeation of K+ ions, in addition to regulate events underlying activation and inactivation gating. In KcsA, an inactivation process related to C-type inactivation in eukaryotic channels has been attributed to putative conformational changes at the selectivity filter (SF)[1]. Previously, we have provided crystallographic evidence for the conformational changes associated to C-type inactivation, albeit at relatively low-resolution [2]. Here, we have taken advantage of a cysteine-bridged locked open KcsA-mutant to study the structural changes at the selectivity filter when the activation gate (AG) is open and the filter transitions between its conductive and non-conductive conformations. We report the structures of KcsA for the non-inactivating mutantE71A at 2.1 A; the fully inactivated mutant Y82A at 2.32 A; and the non-inactivating mutant F103A at 2.64 A, where the allosteric coupling between the two gates (AG and SF) has been impaired. This set of high-resolution structures for different KcsA kinetic states represent a sharp improvement over the resolution of non-cysteine trapped mutants and will be interpreted in relation to their complementary functional characterization.1. Cordero-Morales, J.F., et al., Molecular determinants of gating at the potassium-channel selectivity filter. Nat Struct Mol Biol, 2006. 13(4): p. 311-8.2. Cuello, L.G., et al. (2008). Structural basis of K+ channel C-type inactivation: Crystal structure of KcsA in the Open/C-type inactivated conformation. 52nd Annual meeting of the Biophysical Society. Mini-symposium

Rearrangements at the Heterodimer Interface are Integral to NMDA Receptor Activation

Upon binding the neurotransmitter glutamate and the obligatory co-agonist glycine, NMDA receptors activate by opening a membrane permeable pore or desensitize by switching into a high-affinity non-conducting conformation. Both activation and desensitization require that the agonist-binding clamshell within each subunit closes to engulf the agonists. It has been hypothesized that this movement strains the contacts between agonist-binding domains of GluN1 and GluN2 and rupture of this interface causes receptor desensitization by disengaging agonist-binding from pore-opening. To investigate the role of inter-subunit contacts in NMDA receptor gating, we cross-linked the dimer interface by introducing cysteine residues at positions predicted to interact across subunits: N521 and L777 of GluN1 and E516 and L780 of GluN2A, respectively. Steady-state single-channel recordings indicated that cross-linked receptors had drastically reduced open probabilities (∼200-fold, Po = 0.0032) due to ∼5-fold shorter openings and ∼100-fold longer closures (means, SEM): MOT = 1.8 ± 0.2 ms, MCT = 792 ± 213 ms (n = 6; 80,028 events). However, the mean duration of closed intervals associated with desensitization remained unaltered (wt, tauD=2700 ms; mut, tauD=3150). Reduction of the disulfide bonds (10 mM DTT) significantly potentiated single channel currents (means, SEM: Po = 0.14 ± 0.02) by restoring the mean duration of openings (11.7 ± 1.6 ms) and significantly shortening mean closed durations (90 ± 19 ms), but had no discernible effects on microscopic desensitization (n = 7; 262,396 events). Based on these data, we propose that flexibility in the heterodimer interface at the level of agonist-binding domains represents an integral part of NMDA receptor activation.

Structure of a tetrameric MscL in an expanded intermediate state

The ability of cells to sense and respond to mechanical force underlies diverse processes such as touch and hearing in animals, gravitropism in plants, and bacterial osmoregulation1, 2. In bacteria, mechanosensation is mediated by the mechanosensitive channels of large (MscL), small (MscS), potassium-dependent (MscK), and mini (MscM) conductances. These channels act as “emergency relief valves” protecting bacteria from lysis upon acute osmotic downshock3. Among them, MscL has been intensively studied since the original identification and characterization 15 years ago by Kung and co-workers4. MscL is reversibly and directly gated by changes in membrane tension. In the open state, MscL forms a nonselective 3 nS-conductance channel which gates at tensions close to the lytic limit of the bacterial membrane. An earlier crystal structure at 3.5 Å resolution of a pentameric MscL from Mycobacterium tuberculosis (TbMscL) represents a closed-state or nonconducting conformation5, 6. MscL has a complex gating behaviour; it exhibits several intermediates between the closed and open states, including one putative nonconductive expanded state and at least three sub-conducting states7. Although our understanding of the closed5, 6 and open8-10 states of MscL has been increasing, little is known about the structures of the intermediate states despite their importance in elucidating the complete gating process of MscL. Here we present the crystal structure of a truncation mutant (Δ95-120) of MscL from Staphylococcus aureus (SaMscL-CΔ26) at 3.8 Å resolution. Strikingly, SaMscL-CΔ26 forms a tetrameric channel with both transmembrane (TM) helices tilted away from the membrane normal at angles close to that inferred for the open state9, likely corresponding to a nonconductive but partially expanded intermediate state.

Structural Basis For The Coupling Between Activation And Inactivation Gating In Potassium Channels

We have known the structure for the closed-state of a potassium channel pore domain (PD) for more than a decade. However, major progress in understanding the molecular basis for activation and inactivation gating in K-channels had to wait until high-resolution structural information of the channel in the open state became available. Recently, we solved the structure KcsA in it fully open conformation, as well four others partial openings, which richly illustrated the channel activation-inactivation pathway. Analysis of these open structures suggested that residue F103 in TM2 interacts with the c-terminal end of the pore helix, compressing the pitch of its first helical turn. As a consequence, the distance between E71-D80 side chains is shortened, strengthening the carboxyl-carboxylate interaction that leads to a non-conductive conformation of the selectivity filter. Perturbation mutagenesis at position 103, affected gating kinetics as predicted from our structural analysis: small side chain substitutions F103A and F103C severely impaired inactivation kinetics, suggesting an allosteric coupling between the inner helical bundle and the selectivity filter. Free energy calculations show strong open state interaction-energies between F103 and surrounding residues. Similar interactions were probed in the Shaker K-channel by mutating highly conserved I470, equivalent to F103, to a smaller side chain. In the mutant I470A, inactivation was abrogated, suggesting that a similar mechanism underlies inactivation coupling in eukaryotic potassium channels. A crystallography study of these mutants in the open KcsA will be reported.

Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel

The X-ray structure of a pentameric ligand-gated ion channel from Erwinia chrysanthemi (ELIC) has recently provided structural insight into this family of ion channels at high resolution. The structure shows a homo-pentameric protein with a barrel-stave architecture that defines an ion-conduction pore located on the fivefold axis of symmetry. In this structure, the wide aqueous vestibule that is encircled by the extracellular ligand-binding domains of the five subunits narrows to a discontinuous pore that spans the lipid bilayer. The pore is constricted by bulky hydrophobic residues towards the extracellular side, which probably serve as barriers that prevent the diffusion of ions. This interrupted pore architecture in ELIC thus depicts a non-conducting conformation of a pentameric ligand-gated ion channel, the thermodynamically stable state in the absence of bound ligand. As ligand binding promotes pore opening in these ion channels and the specific ligand for ELIC has not yet been identified, we have turned our attention towards a homologous protein from the cyanobacterium Gloebacter violaceus (GLIC). GLIC was shown to form proton-gated channels that are activated by a pH decrease on the extracellular side and that do not desensitize after activation. Both prokaryotic proteins, ELIC and GLIC form ion channels that are selective for cations over anions with poor discrimination among monovalent cations, characteristics that resemble the conduction properties of the cation-selective branch of the family that includes acetylcholine and serotonin receptors. Here we present the X-ray structure of GLIC at 3.1 A resolution. The structure reveals a conformation of the channel that is distinct from ELIC and that probably resembles the open state. In combination, both structures suggest a novel gating mechanism for pentameric ligand-gated ion channels where channel opening proceeds by a change in the tilt of the pore-forming helices.