Structure of a prokaryotic sodium channel pore reveals essential gating elements and an outer ion binding site common to eukaryotic channels.
ABSTRACT: Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail "neck", are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the "outer ion" site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies, shows that this site forms a previously unknown determinant of CaV high-affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily.
Project description:Cyclic-nucleotide-gated channels are essential for vision and olfaction. They belong to the voltage-gated ion channel superfamily but their activities are controlled by intracellular cyclic nucleotides instead of transmembrane voltage. Here we report a 3.5-Å-resolution single-particle electron cryo-microscopy structure of a cyclic-nucleotide-gated channel from Caenorhabditis elegans in the cyclic guanosine monophosphate (cGMP)-bound open state. The channel has an unusual voltage-sensor-like domain, accounting for its deficient voltage dependence. A carboxy-terminal linker connecting S6 and the cyclic-nucleotide-binding domain interacts directly with both the voltage-sensor-like domain and the pore domain, forming a gating ring that couples conformational changes triggered by cyclic nucleotide binding to the gate. The selectivity filter is lined by the carboxylate side chains of a functionally important glutamate and three rings of backbone carbonyls. This structure provides a new framework for understanding mechanisms of ion permeation, gating and channelopathy of cyclic-nucleotide-gated channels and cyclic nucleotide modulation of related channels.
Project description:Understanding the gating mechanism of ion channel proteins is key to understanding the regulation of cell signaling through these channels. Channel opening and closing are regulated by diverse environmental factors that include temperature, electrical voltage across the channel, and proton concentration. Low permeability in voltage-gated potassium ion channels (Kv) is intimately correlated with the prolonged action potential duration observed in many acidosis diseases. The Kv channels consist of voltage-sensing domains (S1-S4 helices) and central pore domains (S5-S6 helices) that include a selectivity filter and water-filled cavity. The voltage-sensing domain is responsible for the voltage-gating of Kv channels. While the low permeability of Kv channels to potassium ion is highly correlated with the cellular proton concentration, it is unclear how an intracellular acidic condition drives their closure, which may indicate an additional pH-dependent gating mechanism of the Kv family. Here, we show that two residues E327 and H418 in the proximity of the water cavity of Kv1.2 play crucial roles as a pH switch. In addition, we present a structural and molecular concept of the pH-dependent gating of Kv1.2 in atomic detail, showing that the protonation of E327 and H418 disrupts the electrostatic balance around the S6 helices, which leads to a straightening transition in the shape of their axes and causes dewetting of the water-filled cavity and closure of the channel. Our work offers a conceptual advancement to the regulation of the pH-dependent gating of various voltage-gated ion channels and their related biological functions.
Project description:Four-domain voltage-gated Ca(2+) and Na(+) channels (CaV, NaV) underpin nervous system function and likely emerged upon intragenic duplication of a primordial two-domain precursor. To investigate if two-pore channels (TPCs) may represent an intermediate in this evolutionary transition, we performed molecular docking simulations with a homology model of TPC1, which suggested that the pore region could bind antagonists of CaV or NaV. CaV or NaV antagonists blocked NAADP (nicotinic acid adenine dinucleotide phosphate)-evoked Ca(2+) signals in sea urchin egg preparations and in intact cells that overexpressed TPC1. By sequence analysis and inspection of the model, we predicted a noncanonical selectivity filter in animal TPCs in which the carbonyl groups of conserved asparagine residues are positioned to coordinate cations. In contrast, a distinct clade of TPCs [TPCR (for TPC-related)] in several unicellular species had ion selectivity filters with acidic residues more akin to CaV. TPCRs were predicted to interact strongly with CaV antagonists. Our data suggest that acquisition of a "blueprint" pharmacological profile and changes in ion selectivity within four-domain voltage-gated ion channels may have predated intragenic duplication of an ancient two-domain ancestor.
Project description:Homotetrameric bacterial voltage-gated sodium channels share major biophysical features with their more complex eukaryotic counterparts, including a slow-inactivation mechanism that reduces ion-conductance activity during prolonged depolarization through conformational changes in the pore. The bacterial sodium channel NaVAb activates at very negative membrane potentials and inactivates through a multiphase slow-inactivation mechanism. Early voltage-dependent inactivation during one depolarization is followed by late use-dependent inactivation during repetitive depolarization. Mutations that change the molecular volume of Thr206 in the pore-lining S6 segment can enhance or strongly block early voltage-dependent inactivation, suggesting that this residue serves as a molecular hub controlling the coupling of activation to inactivation. In contrast, truncation of the C-terminal tail enhances the early phase of inactivation yet completely blocks late use-dependent inactivation. Determination of the structure of a C-terminal tail truncation mutant and molecular modeling of conformational changes at Thr206 and the S6 activation gate led to a two-step model of these gating processes. First, bending of the S6 segment, local protein interactions dependent on the size of Thr206, and exchange of hydrogen-bonding partners at the level of Thr206 trigger pore opening followed by the early phase of voltage-dependent inactivation. Thereafter, conformational changes in the C-terminal tail lead to late use-dependent inactivation. These results have important implications for the sequence of conformational changes that lead to multiphase inactivation of NaVAb and other sodium channels.
Project description:The activation gate of ion channels controls the transmembrane flux of permeant ions. In voltage-gated K(+) channels, the aperture formed by the S6 bundle crossing can widen to open or narrow to close the ion permeation pathway, whereas the selectivity filter gates ion flux in cyclic-nucleotide gated (CNG) and Slo1 channels. Here we explore the structural basis of the activation gate for Slo2.1, a weakly voltage-dependent K(+) channel that is activated by intracellular Na(+) and Cl(-). Slo2.1 channels were heterologously expressed in Xenopus laevis oocytes and activated by elevated [NaCl]i or extracellular application of niflumic acid. In contrast to other voltage-gated channels, Slo2.1 was blocked by verapamil in an activation-independent manner, implying that the S6 bundle crossing does not gate the access of verapamil to its central cavity binding site. The structural basis of Slo2.1 activation was probed by Ala scanning mutagenesis of the S6 segment and by mutation of selected residues in the pore helix and S5 segment. Mutation to Ala of three S6 residues caused reduced trafficking of channels to the cell surface and partial (K256A, I263A, Q273A) or complete loss (E275A) of channel function. P271A Slo2.1 channels trafficked normally, but were nonfunctional. Further mutagenesis and intragenic rescue by second site mutations suggest that Pro271 and Glu275 maintain the inner pore in an open configuration by preventing formation of a tight S6 bundle crossing. Mutation of several residues in S6 and S5 predicted by homology modeling to contact residues in the pore helix induced a gain of channel function. Substitution of the pore helix residue Phe240 with polar residues induced constitutive channel activation. Together these findings suggest that (1) the selectivity filter and not the bundle crossing gates ion permeation and (2) dynamic coupling between the pore helix and the S5 and S6 segments mediates Slo2.1 channel activation.
Project description:Many voltage-gated ion channel (VGIC) superfamily members contain six-transmembrane segments in which the first four form a voltage-sensing domain (VSD) and the last two form the pore domain (PD). Studies of potassium channels from the VGIC superfamily together with identification of voltage-sensor only proteins have suggested that the VSD and the PD can fold independently. Whether such transmembrane modularity is common to other VGIC superfamily members has remained untested. Here we show, using protein dissection, that the Silicibacter pomeroyi voltage-gated sodium channel (Na(V)Sp1) PD forms a stand-alone, ion selective pore (Na(V)Sp1p) that is tetrameric, ?-helical, and that forms functional, sodium-selective channels when reconstituted into lipid bilayers. Mutation of the Na(V)Sp1p selectivity filter from LESWSM to LDDWSD, a change similar to that previously shown to alter ion selectivity of the bacterial sodium channel Na(V)Bh1 (NaChBac), creates a calcium-selective pore-only channel, Ca(V)Sp1p. We further show that production of PDs can be generalized by making pore-only proteins from two other extremophile Na(V)s: one from the hydrocarbon degrader Alcanivorax borkumensis (Na(V)Ab1p), and one from the arsenite oxidizer Alkalilimnicola ehrlichei (Na(V)Ae1p). Together, our data establish a family of active pore-only ion channels that should be excellent model systems for study of the factors that govern both sodium and calcium selectivity and permeability. Further, our findings suggest that similar dissection approaches may be applicable to a wide range of VGICs and, thus, serve as a means to simplify and accelerate biophysical, structural, and drug development efforts.
Project description: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 (rNa(V)1.4) 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," I(US)) 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 I(US). The fact that mutations at site 1575 modulate I(US) 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.
Project description:Activity of cyclic nucleotide-gated (CNG) cation channels underlies signal transduction in vertebrate visual receptors. These highly specialized receptor channels open when they bind cyclic GMP (cGMP). Here, we find that certain mutations restricted to the region around the ion selectivity filter render the channels essentially fully voltage gated, in such a manner that the channels remain mostly closed at physiological voltages, even in the presence of saturating concentrations of cGMP. This voltage-dependent gating resembles the selectivity filter-based mechanism seen in KcsA K(+) channels, not the S4-based mechanism of voltage-gated K(+) channels. Mutations that render CNG channels gated by voltage loosen the attachment of the selectivity filter to its surrounding structure, thereby shifting the channel's gating equilibrium toward closed conformations. Significant pore opening in mutant channels occurs only when positive voltages drive the pore from a low-probability open conformation toward a second open conformation to increase the channels' open probability. Thus, the structure surrounding the selectivity filter has evolved to (nearly completely) suppress the expression of inherent voltage-dependent gating of CNGA1, ensuring that the binding of cGMP by itself suffices to open the channels at physiological voltages.
Project description:Cyclic nucleotide-gated channels mediate transduction of light into electric signals in vertebrate photoreceptors. These channels are primarily controlled by the binding of intracellular cyclic GMP (cGMP). Glutamate residue 363 near the extracellular end of the ion selectivity filter interacts with the pore helix and helps anchor the filter to the helix. Disruption of this interaction by mutations renders the channels essentially fully voltage gated in the presence of saturating concentrations of cGMP. Here, we find that lowering extracellular pH makes the channels conduct in an extremely outwardly rectifying manner, as does a neutral glutamine substitution at E363. A pair of cysteine mutations, E363C and L356C (the latter located midway the pore helix), largely eliminates current rectification at low pH. Therefore, this low pH-induced rectification primarily reflects voltage-dependent gating involving the ion selectivity filter rather than altered electrostatics around the external opening of the ion pore and thus ion conduction. It then follows that protonation of E363, like the E363Q mutation, disrupts the attachment of the selectivity filter to the pore helix. Loosening the selectivity filter from its surrounding structure shifts the gating equilibrium toward closed states. At low extracellular pH, significant channel opening occurs only when positive voltages drive the pore from a low probability open conformation to a second open conformation. Consequently, at low extracellular pH the channels become practically fully voltage gated, even in the presence of a saturating concentration of cGMP.
Project description: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.