Blocker protection in the pore of a voltage-gated K+ channel and its structural implications

The structure of the bacterial potassium channel KcsA has provided a framework for understanding the related voltage-gated potassium channels (Kv channels) that are used for signalling in neurons. Opening and closing of these Kv channels (gating) occurs at the intracellular entrance to the pore, and this is also the site at which many open channel blockers affect Kv channels. To learn more about the sites of blocker binding and about the structure of the open Kv channel, we investigated here the ability of blockers to protect against chemical modification of cysteines introduced at sites in transmembrane segment S6, which contributes to the intracellular entrance. Within the intracellular half of S6 we found an abrupt cessation of protection for both large and small blockers that is inconsistent with the narrow 'inner pore' seen in the KcsA structure. These and other results are most readily explained by supposing that the structure of Kv channels differs from that of the non-voltage-gated bacterial channel by the introduction of a sharp bend in the inner (S6) helices. This bend would occur at a Pro-X-Pro sequence that is highly conserved in Kv channels, near the site of activation gating.     

The twisted ion-permeation pathway of a resting voltage-sensing domain

Proteins containing voltage-sensing domains (VSDs) translate changes in membrane potential into changes in ion permeability or enzymatic activity. In channels, voltage change triggers a switch in conformation of the VSD, which drives gating in a separate pore domain, or, in channels lacking a pore domain, directly gates an ion pathway within the VSD. Neither mechanism is well understood. In the Shaker potassium channel, mutation of the first arginine residue of the S4 helix to a smaller uncharged residue makes the VSD permeable to ions ('omega current') in the resting conformation ('S4 down'). Here we perform a structure-guided perturbation analysis of the omega conductance to map its VSD permeation pathway. We find that there are four omega pores per channel, which is consistent with one conduction path per VSD. Permeating ions from the extracellular medium enter the VSD at its peripheral junction with the pore domain, and then plunge into the core of the VSD in a curved conduction pathway. Our results provide a model of the resting conformation of the VSD.     

A voltage-gated proton-selective channel lacking the pore domain

Voltage changes across the cell membrane control the gating of many cation-selective ion channels. Conserved from bacteria to humans, the voltage-gated-ligand superfamily of ion channels are encoded as polypeptide chains of six transmembrane-spanning segments (S1-S6). S1-S4 functions as a self-contained voltage-sensing domain (VSD), in essence a positively charged lever that moves in response to voltage changes. The VSD 'ligand' transmits force via a linker to the S5-S6 pore domain 'receptor', thereby opening or closing the channel. The ascidian VSD protein Ci-VSP gates a phosphatase activity rather than a channel pore, indicating that VSDs function independently of ion channels. Here we describe a mammalian VSD protein (H(V)1) that lacks a discernible pore domain but is sufficient for expression of a voltage-sensitive proton-selective ion channel activity. H(v)1 currents are activated at depolarizing voltages, sensitive to the transmembrane pH gradient, H+-selective, and Zn2+-sensitive. Mutagenesis of H(v)1 identified three arginine residues in S4 that regulate channel gating and two histidine residues that are required for extracellular inhibition of H(v)1 by Zn2+. H(v)1 is expressed in immune tissues and manifests the characteristic properties of native proton conductances (G(vH+)). In phagocytic leukocytes, G(vH+) are required to support the oxidative burst that underlies microbial killing by the innate immune system. The data presented here identify H(v)1 as a long-sought voltage-gated H+ channel and establish H(v)1 as the founding member of a family of mammalian VSD proteins.     

Voltage-sensing mechanism is conserved among ion channels gated by opposite voltages

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) ion channels are found in rhythmically firing cells in the brain and in the heart, where the cation current through HCN channels (called I(h) or I(f)) causes these cells to fire repeatedly. These channels are also found in non-pacing cells, where they control resting membrane properties, modulate synaptic transmission, mediate long-term potentiation, and limit extreme hyperpolarizations. HCN channels share sequence motifs with depolarization-activated potassium (Kv) channels, such as the fourth transmembrane segment S4. S4 is the main voltage sensor of Kv channels, in which transmembrane movement of S4 charges triggers the opening of the activation gate. Here, using cysteine accessibility methods, we investigate whether S4 moves in an HCN channel. We show that S4 movement is conserved between Kv and HCN channels, which indicates that S4 is also the voltage sensor in HCN channels. Our results suggest that a conserved voltage-sensing mechanism operates in the oppositely voltage-gated Kv and HCN channels, but that there are different coupling mechanisms between the voltage sensor and activation gate in the two different channels.     

Gating pore current in an inherited ion channelopathy

Ion channelopathies are inherited diseases in which alterations in control of ion conductance through the central pore of ion channels impair cell function, leading to periodic paralysis, cardiac arrhythmia, renal failure, epilepsy, migraine and ataxia. Here we show that, in contrast with this well-established paradigm, three mutations in gating-charge-carrying arginine residues in an S4 segment that cause hypokalaemic periodic paralysis induce a hyperpolarization-activated cationic leak through the voltage sensor of the skeletal muscle Na(V)1.4 channel. This 'gating pore current' is active at the resting membrane potential and closed by depolarizations that activate the voltage sensor. It has similar permeability to Na+, K+ and Cs+, but the organic monovalent cations tetraethylammonium and N-methyl-D-glucamine are much less permeant. The inorganic divalent cations Ba2+, Ca2+ and Zn2+ are not detectably permeant and block the gating pore at millimolar concentrations. Our results reveal gating pore current in naturally occurring disease mutations of an ion channel and show a clear correlation between mutations that cause gating pore current and hypokalaemic periodic paralysis. This gain-of-function gating pore current would contribute in an important way to the dominantly inherited membrane depolarization, action potential failure, flaccid paralysis and cytopathology that are characteristic of hypokalaemic periodic paralysis. A survey of other ion channelopathies reveals numerous examples of mutations that would be expected to cause gating pore current, raising the possibility of a broader impact of gating pore current in ion channelopathies.     

Removal of phospho-head groups of membrane lipids immobilizes voltage sensors of K+ channels

A fundamental question about the gating mechanism of voltage-activated K+ (Kv) channels is how five positively charged voltage-sensing residues in the fourth transmembrane segment are energetically stabilized, because they operate in a low-dielectric cell membrane. The simplest solution would be to pair them with negative charges. However, too few negatively charged channel residues are positioned for such a role. Recent studies suggest that some of the channel's positively charged residues are exposed to cell membrane phospholipids and interact with their head groups. A key question nevertheless remains: is the phospho-head of membrane lipids necessary for the proper function of the voltage sensor itself? Here we show that a given type of Kv channel may interact with several species of phospholipid and that enzymatic removal of their phospho-head creates an insuperable energy barrier for the positively charged voltage sensor to move through the initial gating step(s), thus immobilizing it, and also raises the energy barrier for the downstream step(s).     

Voltage-sensing residues in the S4 region of a mammalian K+ channel

The ability of ion-channel proteins to respond to a change of the transmembrane voltage is one of the basic mechanisms underlying electrical excitability of nerve and muscle membranes. The voltage sensor has been postulated to be the fourth putative transmembrane segment (S4) of voltage-activated Na+, Ca2+ and K+ channels. Mutations of positively charged residues within S4 alter gating of Na and Shaker-type K+ channels, but quantitative correlations between the charge or a residue in S4 and the gating valence of the channel have not yet been established. Here, with improved resolution of the voltage dependence of steady-state activation, we present estimates of the equivalent gating valence with sufficient precision to allow quantitative examination of the contribution of individual charged residues to the gating valence of a mammalian non-inactivating K+ channel. We conclude that at least part of the gating charge associated with channel activation is indeed contributed by charged residues within the S4 segment.     

Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment

Voltage-dependent K+ (Kv) channels repolarize the action potential in neurons and muscle. This type of channel is gated directly by membrane voltage through protein domains known as voltage sensors, which are molecular voltmeters that read the membrane voltage and regulate the pore. Here we describe the structure of a chimaeric voltage-dependent K+ channel, which we call the 'paddle-chimaera channel', in which the voltage-sensor paddle has been transferred from Kv2.1 to Kv1.2. Crystallized in complex with lipids, the complete structure at 2.4 ?ngstr?m resolution reveals the pore and voltage sensors embedded in a membrane-like arrangement of lipid molecules. The detailed structure, which can be compared directly to a large body of functional data, explains charge stabilization within the membrane and suggests a mechanism for voltage-sensor movements and pore gating.     

A proton pore in a potassium channel voltage sensor reveals a focused electric field

Voltage-dependent potassium channels are essential for the generation of nerve impulses. Voltage sensitivity is conferred by charged residues located mainly in the fourth transmembrane segment (S4) of each of the four identical subunits that make up the channel. These charged segments relocate when the potential difference across the membrane changes, controlling the ability of the pore to conduct ions. In the crystal structure of the Aeropyrum pernix potassium channel KvAP, the S4 and part of the third (S3B) transmembrane alpha-helices are connected by a hairpin turn in an arrangement termed the 'voltage-sensor paddle'. This structure was proposed to move through the lipid bilayer during channel activation, transporting positive charges across a large fraction of the membrane. Here we show that replacing the first S4 arginine by histidine in the Shaker potassium channel creates a proton pore when the cell is hyperpolarized. Formation of this pore does not support the paddle model, as protons would not have access to a lipid-buried histidine. We conclude that, at hyperpolarized potentials, water and protons from the internal and external solutions must be separated by a narrow barrier in the channel protein that focuses the electric field to a small voltage-sensitive region.     

Rotational movement during cyclic nucleotide-gated channel opening

Cyclic nucleotide-gated (CNG) channels are crucial components of visual, olfactory and gustatory signalling pathways. They open in response to direct binding of intracellular cyclic nucleotides and thus contribute to cellular control of both the membrane potential and intracellular Ca2+ levels. Cytosolic Ni2+ potentiates the rod channel (CNG1) response to cyclic nucleotides and inhibits the olfactory channel (CNG2) response. Modulation is due to coordination of Ni2+ by channel-specific histidines in the C-linker, between the S6 transmembrane segment and the cyclic nucleotide-binding domain. Here we report, using a histidine scan of the initial C-linker of the CNG1 channel, stripes of sites producing Ni2+ potentiation or Ni2+ inhibition, separated by 50 degrees on an alpha-helix. These results suggest a model for channel gating where rotation of the post-S6 region around the channel's central axis realigns the Ni2+-coordinating residues of multiple subunits. This rotation probably initiates movement of the S6 and pore opening.     

Gating charge displacement in voltage-gated ion channels involves limited transmembrane movement

Voltage-gated ion channels are responsible for generating electrical impulses in nerves and other excitable cells. The fourth transmembrane helix (S4) in voltage-gated channels is the primary voltage-sensing unit that mediates the response to a changing membrane electric field. The molecular mechanism of voltage sensing, particularly with respect to the magnitude of the transmembrane movement of S4, remains controversial. To determine the extent of this transmembrane movement, we use fluorescent resonance energy transfer between the S4 domain and a reference point in the lipid bilayer. The lipophilic ion dipicrylamine distributes on either side of the lipid bilayer depending on the membrane potential, and is used here as a resonance-energy-transfer acceptor from donor molecules attached to several positions in the Shaker K+ channel. A voltage-driven transmembrane movement of the donor should produce a transient fluorescence change because the acceptor also translocates as a function of voltage. In Shaker K+ channels no such transient fluorescence is observed, indicating that the S4 segment does not translocate across the lipid bilayer. Based on these observations, we propose a molecular model of voltage gating that can account for the observed 13e gating charge with limited transmembrane S4 movement.     

Mechanism of glutamate receptor desensitization

Ligand-gated ion channels transduce chemical signals into electrical impulses by opening a transmembrane pore in response to binding one or more neurotransmitter molecules. After activation, many ligand-gated ion channels enter a desensitized state in which the neurotransmitter remains bound but the ion channel is closed. Although receptor desensitization is crucial to the functioning of many ligand-gated ion channels in vivo, the molecular basis of this important process has until now defied analysis. Using the GluR2 AMPA-sensitive glutamate receptor, we show here that the ligand-binding cores form dimers and that stabilization of the intradimer interface by either mutations or allosteric modulators reduces desensitization. Perturbations that destabilize the interface enhance desensitization. Receptor activation involves conformational changes within each subunit that result in an increase in the separation of portions of the receptor that are linked to the ion channel. Our analysis defines the dimer interface in the resting and activated state, indicates how ligand binding is coupled to gating, and suggests modes of dimer dimer interaction in the assembled tetramer. Desensitization occurs through rearrangement of the dimer interface, which disengages the agonist-induced conformational change in the ligand-binding core from the ion channel gate.     

Crystal structure of a voltage-gated sodium channel in two potentially inactivated states

In excitable cells, voltage-gated sodium (Na(V)) channels activate to initiate action potentials and then undergo fast and slow inactivation processes that terminate their ionic conductance. Inactivation is a hallmark of Na(V) channel function and is critical for control of membrane excitability, but the structural basis for this process has remained elusive. Here we report crystallographic snapshots of the wild-type Na(V)Ab channel from Arcobacter butzleri captured in two potentially inactivated states at 3.2?? resolution. Compared to previous structures of Na(V)Ab channels with cysteine mutations in the pore-lining S6 helices (ref. 4), the S6 helices and the intracellular activation gate have undergone significant rearrangements: one pair of S6 helices has collapsed towards the central pore axis and the other S6 pair has moved outward to produce a striking dimer-of-dimers configuration. An increase in global structural asymmetry is observed throughout our wild-type Na(V)Ab models, reshaping the ion selectivity filter at the extracellular end of the pore, the central cavity and its residues that are analogous to the mammalian drug receptor site, and the lateral pore fenestrations. The voltage-sensing domains have also shifted around the perimeter of the pore module in wild-type Na(V)Ab, compared to the mutant channel, and local structural changes identify a conserved interaction network that connects distant molecular determinants involved in Na(V) channel gating and inactivation. These potential inactivated-state structures provide new insights into Na(V) channel gating and novel avenues to drug development and therapy for a range of debilitating Na(V) channelopathies.     

Small vertical movement of a K+ channel voltage sensor measured with luminescence energy transfer

Voltage-gated ion channels open and close in response to voltage changes across electrically excitable cell membranes. Voltage-gated potassium (Kv) channels are homotetramers with each subunit constructed from six transmembrane segments, S1-S6 (ref. 2). The voltage-sensing domain (segments S1-S4) contains charged arginine residues on S4 that move across the membrane electric field, modulating channel open probability. Understanding the physical movements of this voltage sensor is of fundamental importance and is the subject of controversy. Recently, the crystal structure of the KvAP channel motivated an unconventional 'paddle model' of S4 charge movement, indicating that the segments S3b and S4 might move as a unit through the lipid bilayer with a large (15-20-A) transmembrane displacement. Here we show that the voltage-sensor segments do not undergo significant transmembrane translation. We tested the movement of these segments in functional Shaker K+ channels by using luminescence resonance energy transfer to measure distances between the voltage sensors and a pore-bound scorpion toxin. Our results are consistent with a 2-A vertical displacement of S4, not the large excursion predicted by the paddle model. This small movement supports an alternative model in which the protein shapes the electric field profile, focusing it across a narrow region of S4 (ref. 6).     

The crystal structure of a voltage-gated sodium channel

Voltage-gated sodium (Na(V)) channels initiate electrical signalling in excitable cells and are the molecular targets for drugs and disease mutations, but the structural basis for their voltage-dependent activation, ion selectivity and drug block is unknown. Here we report the crystal structure of a voltage-gated Na(+) channel from Arcobacter butzleri (NavAb) captured in a closed-pore conformation with four activated voltage sensors at 2.7?? resolution. The arginine gating charges make multiple hydrophilic interactions within the voltage sensor, including unanticipated hydrogen bonds to the protein backbone. Comparisons to previous open-pore potassium channel structures indicate that the voltage-sensor domains and the S4-S5 linkers dilate the central pore by pivoting together around a hinge at the base of the pore module. The NavAb selectivity filter is short, ～4.6?? wide, and water filled, with four acidic side chains surrounding the narrowest part of the ion conduction pathway. This unique structure presents a high-field-strength anionic coordination site, which confers Na(+) selectivity through partial dehydration via direct interaction with glutamate side chains. Fenestrations in the sides of the pore module are unexpectedly penetrated by fatty acyl chains that extend into the central cavity, and these portals are large enough for the entry of small, hydrophobic pore-blocking drugs. This structure provides the template for understanding electrical signalling in excitable cells and the actions of drugs used for pain, epilepsy and cardiac arrhythmia at the atomic level.     

Molecular mechanism of ATP binding and ion channel activation in P2X receptors

P2X receptors are trimeric ATP-activated ion channels permeable to Na+, K+ and Ca2+. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body β-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.     

The principle of gating charge movement in a voltage-dependent K+ channel

The steep dependence of channel opening on membrane voltage allows voltage-dependent K+ channels to turn on almost like a switch. Opening is driven by the movement of gating charges that originate from arginine residues on helical S4 segments of the protein. Each S4 segment forms half of a 'voltage-sensor paddle' on the channel's outer perimeter. Here we show that the voltage-sensor paddles are positioned inside the membrane, near the intracellular surface, when the channel is closed, and that the paddles move a large distance across the membrane from inside to outside when the channel opens. KvAP channels were reconstituted into planar lipid membranes and studied using monoclonal Fab fragments, a voltage-sensor toxin, and avidin binding to tethered biotin. Our findings lead us to conclude that the voltage-sensor paddles operate somewhat like hydrophobic cations attached to levers, enabling the membrane electric field to open and close the pore.     

Open channel structure of MscL and the gating mechanism of mechanosensitive channels

Mechanosensitive channels act as membrane-embedded mechano-electrical switches, opening a large water-filled pore in response to lipid bilayer deformations. This process is critical to the response of living organisms to direct physical stimulation, such as in touch, hearing and osmoregulation. Here, we have determined the structural rearrangements that underlie these events in the large prokaryotic mechanosensitive channel (MscL) using electron paramagnetic resonance spectroscopy and site-directed spin labelling. MscL was trapped in both the open and in an intermediate closed state by modulating bilayer morphology. Transition to the intermediate state is characterized by small movements in the first transmembrane helix (TM1). Subsequent transitions to the open state are accompanied by massive rearrangements in both TM1 and TM2, as shown by large increases in probe dynamics, solvent accessibility and the elimination of all intersubunit spin-spin interactions. The open state is highly dynamic, supporting a water-filled pore of at least 25 A, lined mostly by TM1. These structures suggest a plausible molecular mechanism of gating in mechanosensitive channels.     

Molecular mechanism of cAMP modulation of HCN pacemaker channels.

Hyperpolarization-activated cation channels of the HCN gene family contribute to spontaneous rhythmic activity in both heart and brain. All four family members contain both a core transmembrane segment domain, homologous to the S1-S6 regions of voltage-gated K+ channels, and a carboxy-terminal 120 amino-acid cyclic nucleotide-binding domain (CNBD) motif. Homologous CNBDs are responsible for the direct activation of cyclic nucleotide-gated channels and for modulation of the HERG voltage-gated K+ channel--important for visual and olfactory signalling and for cardiac repolarization, respectively. The direct binding of cyclic AMP to the cytoplasmic site on HCN channels permits the channels to open more rapidly and completely after repolarization of the action potential, thereby accelerating rhythmogenesis. However, the mechanism by which cAMP binding modulates HCN channel gating and the basis for functional differences between HCN isoforms remain unknown. Here we demonstrate by constructing truncation mutants that the CNBD inhibits activation of the core transmembrane domain. cAMP binding relieves this inhibition. Differences in activation gating and extent of cAMP modulation between the HCN1 and HCN2 isoforms result largely from differences in the efficacy of CNBD inhibition.     

SOL-1 is a CUB-domain protein required for GLR-1 glutamate receptor function in C. elegans

Ionotropic glutamate receptors (iGluRs) mediate most excitatory synaptic signalling between neurons. Binding of the neurotransmitter glutamate causes a conformational change in these receptors that gates open a transmembrane pore through which ions can pass. The gating of iGluRs is crucially dependent on a conserved amino acid that was first identified in the 'lurcher' ataxic mouse. Through a screen for modifiers of iGluR function in a transgenic strain of Caenorhabditis elegans expressing a GLR-1 subunit containing the lurcher mutation, we identify suppressor of lurcher (sol-1). This gene encodes a transmembrane protein that is predicted to contain four extracellular beta-barrel-forming domains known as CUB domains. SOL-1 and GLR-1 are colocalized at the cell surface and can be co-immunoprecipitated. By recording from neurons expressing GLR-1, we show that SOL-1 is an accessory protein that is selectively required for glutamate-gated currents. We propose that SOL-1 participates in the gating of non-NMDA (N-methyl-D-aspartate) iGluRs, thereby providing a previously unknown mechanism of regulation for this important class of neurotransmitter receptor.