Author Correction: Structure and electromechanical coupling of a voltage-gated Na+/H+ exchanger.
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ABSTRACT:
SUBMITTER: Yeo H
PROVIDER: S-EPMC10700128 | biostudies-literature | 2023 Dec
REPOSITORIES: biostudies-literature
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Author Correction: Structure and electromechanical coupling of a voltage-gated Na<sup>+</sup>/H<sup>+</sup> exchanger.
Yeo Hyunku H Mehta Ved V Gulati Ashutosh A Drew David D
Nature 20231201 7990
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Project description:Voltage-sensing domains control the activation of voltage-gated ion channels, with a few exceptions1. One such exception is the sperm-specific Na+/H+ exchanger SLC9C1, which is the only known transporter to be regulated by voltage-sensing domains2-5. After hyperpolarization of sperm flagella, SLC9C1 becomes active, causing pH alkalinization and CatSper Ca2+ channel activation, which drives chemotaxis2,6. SLC9C1 activation is further regulated by cAMP2,7, which is produced by soluble adenyl cyclase (sAC). SLC9C1 is therefore an essential component of the pH-sAC-cAMP signalling pathway in metazoa8,9, required for sperm motility and fertilization4. Despite its importance, the molecular basis of SLC9C1 voltage activation is unclear. Here we report cryo-electron microscopy (cryo-EM) structures of sea urchin SLC9C1 in detergent and nanodiscs. We show that the voltage-sensing domains are positioned in an unusual configuration, sandwiching each side of the SLC9C1 homodimer. The S4 segment is very long, 90 Å in length, and connects the voltage-sensing domains to the cytoplasmic cyclic-nucleotide-binding domains. The S4 segment is in the up configuration-the inactive state of SLC9C1. Consistently, although a negatively charged cavity is accessible for Na+ to bind to the ion-transporting domains of SLC9C1, an intracellular helix connected to S4 restricts their movement. On the basis of the differences in the cryo-EM structure of SLC9C1 in the presence of cAMP, we propose that, upon hyperpolarization, the S4 segment moves down, removing this constriction and enabling Na+/H+ exchange.
| S-EPMC10620092 | biostudies-literature
Project description:Voltage-gated ion channels play a central role in the generation of action potentials in the nervous system. They are selective for one type of ion - sodium, calcium, or potassium. Voltage-gated ion channels are composed of a central pore that allows ions to pass through the membrane and four peripheral voltage sensing domains that respond to changes in the membrane potential. Upon depolarization, voltage sensors in voltage-gated potassium channels (Kv) undergo conformational changes driven by positive charges in the S4 segment and aided by pairwise electrostatic interactions with the surrounding voltage sensor. Structure-function relations of Kv channels have been investigated in detail, and the resulting models on the movement of the voltage sensors now converge to a consensus; the S4 segment undergoes a combined movement of rotation, tilt, and vertical displacement in order to bring 3-4e(+) each through the electric field focused in this region. Nevertheless, the mechanism by which the voltage sensor movement leads to pore opening, the electromechanical coupling, is still not fully understood. Thus, recently, electromechanical coupling in different Kv channels has been investigated with a multitude of techniques including electrophysiology, 3D crystal structures, fluorescence spectroscopy, and molecular dynamics simulations. Evidently, the S4-S5 linker, the covalent link between the voltage sensor and pore, plays a crucial role. The linker transfers the energy from the voltage sensor movement to the pore domain via an interaction with the S6 C-termini, which are pulled open during gating. In addition, other contact regions have been proposed. This review aims to provide (i) an in-depth comparison of the molecular mechanisms of electromechanical coupling in different Kv channels; (ii) insight as to how the voltage sensor and pore domain influence one another; and (iii) theoretical predictions on the movement of the cytosolic face of the Kv channels during gating.
| S-EPMC3439648 | biostudies-literature
Project description:Inherited retinal degenerations (IRDs) are a group of untreatable and commonly blinding diseases characterized by progressive photoreceptor loss. IRD pathology has been linked to an excessive activation of cyclic nucleotide-gated channels (CNGC) leading to Na+- and Ca2+-influx, subsequent activation of voltage-gated Ca2+-channels (VGCC), and further Ca2+ influx. However, a connection between excessive Ca2+ influx and photoreceptor loss has yet to be proven.Here, we used whole-retina and single-cell RNA-sequencing to compare gene expression between the rd1 mouse model for IRD and wild-type (wt) mice. Differentially expressed genes indicated links to several Ca2+-signalling related pathways. To explore these, rd1 and wt organotypic retinal explant cultures were treated with the intracellular Ca2+-chelator BAPTA-AM or inhibitors of different Ca2+-permeable channels, including CNGC, L-type VGCC, T-type VGCC, Ca2+-release-activated channel (CRAC), and Na+/Ca2+ exchanger (NCX). Moreover, we employed the novel compound NA-184 to selectively inhibit the Ca2+-dependent protease calpain-2. Effects on the retinal activity of poly(ADP-ribose) polymerase (PARP), sirtuin-type histone-deacetylase, calpains, as well as on activation of calpain-1, and - 2 were monitored, cell death was assessed via the TUNEL assay.While rd1 photoreceptor cell death was reduced by BAPTA-AM, Ca2+-channel blockers had divergent effects: While inhibition of T-type VGCC and NCX promoted survival, blocking CNGCs and CRACs did not. The treatment-related activity patterns of calpains and PARPs corresponded to the extent of cell death. Remarkably, sirtuin activity and calpain-1 activation were linked to photoreceptor protection, while calpain-2 activity was related to degeneration. In support of this finding, the calpain-2 inhibitor NA-184 protected rd1 photoreceptors.These results suggest that Ca2+ overload in rd1 photoreceptors may be triggered by T-type VGCCs and NCX. High Ca2+-levels likely suppress protective activity of calpain-1 and promote retinal degeneration via activation of calpain-2. Overall, our study details the complexity of Ca2+-signalling in photoreceptors and emphasizes the importance of targeting degenerative processes specifically to achieve a therapeutic benefit for IRDs. Video Abstract.
| S-EPMC10836022 | biostudies-literature
Project description:Voltage-sensing (VSD) and cyclic nucleotide-binding domains (CNBD) gate ion channels for rapid electrical signaling. By contrast, solute carriers (SLCs) that passively redistribute substrates are gated by their substrates themselves. Here, we study the orphan sperm-specific solute carriers SLC9C1 that feature a unique tripartite structure: an exchanger domain, a VSD, and a CNBD. Voltage-clamp fluorimetry shows that SLC9C1 is a genuine Na+/H+ exchanger gated by voltage. The cellular messenger cAMP shifts the voltage range of activation. Mutations in the transport domain, the VSD, or the CNBD strongly affect Na+/H+ exchange, voltage gating, or cAMP sensitivity, respectively. Our results establish SLC9C1 as a phylogenetic chimaera that combines the ion-exchange mechanism of solute carriers with the gating mechanism of ion channels. Classic SLCs slowly readjust changes in the intra- and extracellular milieu, whereas voltage gating endows the Na+/H+ exchanger with the ability to produce a rapid pH response that enables downstream signaling events.
| S-EPMC6052114 | biostudies-literature
Project description:The hinged-lid model was long accepted as the canonical model for fast inactivation in Nav channels. It predicts that the hydrophobic IFM motif acts intracellularly as the gating particle that binds and occludes the pore during fast inactivation. However, the observation in recent high-resolution structures that the bound IFM motif is located far from the pore, contradicts this preconception. Here, we provide a mechanistic reinterpretation of fast inactivation based on structural analysis and ionic/gating current measurements. We demonstrate that in Nav1.4 the final inactivation gate is comprised of two hydrophobic rings at the bottom of S6 helices. These rings function in series and close downstream of IFM binding. Reducing the volume of the sidechain in both rings leads to a partially conductive, leaky inactivated state and decreases the selectivity for Na+ ion. Altogether, we present an alternative molecular framework to describe fast inactivation.
| S-EPMC10442390 | biostudies-literature
Project description:Fast Inactivation in voltage-gated Na + channels plays essential roles in numerous physiological functions. The canonical hinged-lid model has long predicted that a hydrophobic motif in the DIII-DIV linker (IFM) acts as the gating particle that occludes the permeation pathway during fast inactivation. However, the fact that the IFM motif is located far from the pore in recent high-resolution structures of Nav + channels contradicts this status quo model. The precise molecular determinants of fast inactivation gate once again, become an open question. Here, we provide a mechanistic reinterpretation of fast inactivation based on ionic and gating current data. In Nav1.4 the actual inactivation gate is comprised of two hydrophobic rings at the bottom of S6. These function in series and closing once the IFM motif binds. Reducing the volume of the sidechain in both rings led to a partially conductive inactivated state. Our experiments also point to a previously overlooked coupling pathway between the bottom of S6 and the selectivity filter.
| S-EPMC10168311 | biostudies-literature
Project description:Dent disease 1 (DD1) is caused by mutations in the CLCN5 gene encoding a voltage-gated electrogenic nCl-/H+ exchanger ClC-5. Using ion-selective microelectrodes and Xenopus oocytes, here we studied Cl-/H+ coupling properties of WT ClC-5 and four DD1-associated variants (S244L, R345W, Q629*, and T657S), along with trafficking and localization of ClC-5. WT ClC-5 had a 2Cl-/H+ exchange ratio at a Vh of +40 mV with a [Cl-]out of 104 mm, but the transport direction did not reverse with a [Cl-]out of 5 mm, indicating that ClC-5-mediated exchange of two Cl- out for one H+ in is not permissible. We hypothesized that ClC-5 and H+-ATPase are functionally coupled during H+-ATPase-mediated endosomal acidification, crucial for ClC-5 activation by depolarizing endosomes. ClC-5 transport that provides three net negative charges appeared self-inhibitory because of ClC-5's voltage-gated properties, but shunt conductance facilitated further H+-ATPase-mediated endosomal acidification. Thus, an on-and-off "burst" of ClC-5 activity was crucial for preventing Cl- exit from endosomes. The subcellular distribution of the ClC-5:S244L variant was comparable with that of WT ClC-5, but the variant had a much slower Cl- and H+ transport and displayed an altered stoichiometry of 1.6:1. The ClC-5:R345W variant exhibited slightly higher Cl-/H+ transport than ClC-5:S244L, but co-localized with early endosomes, suggesting decreased ClC-5:R345W membrane trafficking is perhaps in a fully functional form. The truncated ClC-5:Q629* variant displayed the lowest Cl-/H+ exchange and was retained in the endoplasmic reticulum and cis-Golgi, but not in early endosomes, suggesting the nonsense mutation affects ClC-5 maturation and trafficking.
| S-EPMC7008381 | biostudies-literature
Project description:Propofol is widely used in the clinic for the induction and maintenance of general anesthesia. As with most general anesthetics, however, our understanding of its mechanism of action remains incomplete. Local and general anesthetics largely inhibit voltage-gated Na+ channels (Navs) by inducing an apparent stabilization of the inactivated state, associated in some instances with pore block. To determine the biophysical and molecular basis of propofol action in Navs, we investigated NaChBac and NavMs, two prokaryotic Navs with distinct voltage dependencies and gating kinetics, by whole-cell patch clamp electrophysiology in the absence and presence of propofol at clinically relevant concentrations (2-10 µM). In both Navs, propofol induced a hyperpolarizing shift of the pre-pulse inactivation curve without any significant effects on recovery from inactivation at strongly hyperpolarized voltages, demonstrating that propofol does not stabilize the inactivated state. Moreover, there was no evidence of fast or slow pore block by propofol in a non-inactivating NaChBac mutant (T220A). Propofol also induced hyperpolarizing shifts of the conductance-voltage relationships with negligible effects on the time constants of deactivation at hyperpolarized voltages, indicating that propofol does not stabilize the open state. Instead, propofol decreases the time constants of macroscopic activation and inactivation. Adopting a kinetic scheme of Nav gating that assumes preferential closed-state recovery from inactivation, a 1.7-fold acceleration of the rate constant of activation and a 1.4-fold acceleration of the rate constant of inactivation were sufficient to reproduce experimental observations with computer simulations. In addition, molecular dynamics simulations and molecular docking suggest that propofol binding involves interactions with gating machinery in the S4-S5 linker and external pore regions. Our findings show that propofol is primarily a positive gating modulator of prokaryotic Navs, which ultimately inhibits the channels by promoting activation-coupled inactivation.
| S-EPMC6122921 | biostudies-literature
Project description:Ca2+ homeostasis is essential for cell function and survival. As such, the cytosolic Ca2+ concentration is tightly controlled by a wide number of specialized Ca2+ handling proteins. One among them is the Na+ -Ca2+ exchanger (NCX), a ubiquitous plasma membrane transporter that exploits the electrochemical gradient of Na+ to drive Ca2+ out of the cell, against its concentration gradient. In this critical role, this secondary transporter guides vital physiological processes such as Ca2+ homeostasis, muscle contraction, bone formation, and memory to name a few. Herein, we review the progress made in recent years about the structure of the mammalian NCX and how it relates to function. Particular emphasis will be given to the mammalian cardiac isoform, NCX1.1, due to the extensive studies conducted on this protein. Given the degree of conservation among the eukaryotic exchangers, the information highlighted herein will provide a foundation for our understanding of this transporter family. We will discuss gene structure, alternative splicing, topology, regulatory mechanisms, and NCX's functional role on cardiac physiology. Throughout this article, we will attempt to highlight important milestones in the field and controversial topics where future studies are required. © 2021 American Physiological Society. Compr Physiol 12:1-37, 2021.
| S-EPMC8773166 | biostudies-literature
Project description:Human cortical pyramidal neurons are large, have extensive dendritic trees, and yet have unexpectedly fast input-output properties: Rapid subthreshold synaptic membrane potential changes are reliably encoded in timing of action potentials (APs). Here, we tested whether biophysical properties of voltage-gated sodium (Na+) and potassium (K+) currents in human pyramidal neurons can explain their fast input-output properties. Human Na+ and K+ currents exhibited more depolarized voltage dependence, slower inactivation, and faster recovery from inactivation compared with their mouse counterparts. Computational modeling showed that despite lower Na+ channel densities in human neurons, the biophysical properties of Na+ channels resulted in higher channel availability and contributed to fast AP kinetics stability. Last, human Na+ channel properties also resulted in a larger dynamic range for encoding of subthreshold membrane potential changes. Thus, biophysical adaptations of voltage-gated Na+ and K+ channels enable fast input-output properties of large human pyramidal neurons.
| S-EPMC10569700 | biostudies-literature