Large transient capacitive currents in wild-type lysosomal Cl-/H+ antiporter ClC-7 and residual transport activity in the proton glutamate mutant E312A.
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ABSTRACT:
SUBMITTER: Pusch M
PROVIDER: S-EPMC7681918 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A fundamental question concerning the ClC Cl-/H+ antiporters is the nature of their proton transport (PT) pathway. We addressed this issue by using a novel computational methodology capable of describing the explicit PT dynamics in the ClC-ec1 protein. The main result is that the Glu203 residue delivers a proton from the intracellular solution to the core of ClC-ec1 via a rotation of its side chain and subsequent acid dissociation. After reorientation of the Glu203 side chain, a transient water-mediated PT pathway between Glu203 and Glu148 is established that is able to receive and translocate the proton via Grotthuss shuttling after deprotonation of Glu203. A molecular-dynamics simulation of an explicit hydrated excess proton in this pathway suggests that a negatively charged Glu148 and the central Cl- ion act together to drive H+ to the extracellular side of the membrane. This finding is consistent with the experimental result that Cl- binding to the central site facilitates the proton movement. A calculation of the PT free-energy barrier for the ClC-ec1 E203V mutant also supports the proposal that a dissociable residue is required at this position for efficient delivery of H+ to the protein interior, in agreement with recent experimental results.
| S-EPMC2711357 | biostudies-literature
Project description:Using a reactive molecular dynamics simulation methodology, the free energy barrier for water-mediated proton transport between the two proton gating residues Glu(203) and Glu(148) in the ClC-ec1 antiporter, including the Grotthuss mechanism of proton hopping, was calculated. Three different chloride-binding states, with 1), both the central and internal Cl(-), 2), the central Cl(-) only, and 3), the internal Cl(-) only, were considered and the coupling to the H(+) transport studied. The results show that both the central and internal Cl(-) are essential for the proton transport from Glu(203) to Glu(148) to have a favorite free energy driving force. The rotation of the Glu(148) side chain was also found to be independent of the internal chloride binding state. These results emphasize the importance of the 2:1 stoichiometry of this well-studied Cl(-)/H(+) antiporter.
| S-EPMC3218320 | biostudies-literature
Project description:The ubiquitously expressed CLC chloride transporters are involved in a great variety of physiological functions. The CLC protein fold is shared by Cl- channels and 2Cl-:1H+ antiporters. The antiporters pump three charges per cycle across the membrane with two Cl ions moving in the opposite direction of one proton. Multiconformational continuum electrostatics was used to calculate the coupled thermodynamics of the protonation of the extracellular-facing gating Glu (Ex) and Cl- binding to the external (Sx) and central (Sc) sites in CLC-ec1, the Escherichia coli exchanger. Sx, Sc, and Ex are buried within the protein where the intersection of two helix N-termini creates a region with a strong, localized positive potential for anion binding. Our chemical potential titrations describe the thermodynamic linkage for binding the Cl- to each site and protons to Ex. We find that the 2Cl-:1H+ binding stoichiometry is a result of Cl- binding to Sx requiring H+ binding to Ex, whereas Cl- binding to Sc does not lead to proton uptake. When Sx binds a Cl-, the protonated Ex moves upward, out of the positive helix cage. The increasing Ex proton affinity on binding the first Cl- reduces the cost of binding the second Cl- at either Sx or Sc. Despite the repulsion among the anions, the lowest energy states have two anions bound in the helix cage. The state with no Cl- is not favored electrostatically, but relies on Ex blocking Sx and on the central residues Y445 and S107 blocking Sc.
| S-EPMC5658741 | biostudies-literature
Project description:X-ray crystal structures have been previously determined for three CLC-type transporter homologues, but the absolute unitary transport rate is known for only one of these. The Escherichia coli Cl(-)/H(+) antiporter (EC) moves ?2000 Cl(-) ions/s, an exceptionally high rate among membrane-transport proteins. It is not known whether such rapid turnover is characteristic of ClCs in general or if the E. coli homologue represents a functional outlier. Here, we characterize a CLC Cl(-)/H(+) antiporter from the cyanobacterium Synechocystis sp. PCC6803 (SY) and determine its crystal structure at 3.2 Å resolution. The structure of SY is nearly identical to that of EC, with all residues involved in Cl(-) binding and proton coupling structurally similar to their equivalents in EC. SY actively pumps protons into liposomes against a gradient and moves Cl(-) at ?20 s(-1), 1% of the EC rate. Electrostatic calculations, used to identify residues contributing to ion binding energetics in SY and EC, highlight two residues flanking the external binding site that are destabilizing for Cl(-) binding in SY and stabilizing in EC. Mutation of these two residues in SY to their counterparts in EC accelerates transport to ?150 s(-1), allowing measurement of Cl(-)/H(+) stoichiometry of 2/1. SY thus shares a similar structure and a common transport mechanism to EC, but it is by comparison slow, a result that refutes the idea that the transport mechanism of CLCs leads to intrinsically high rates.
| S-EPMC3164822 | biostudies-literature
Project description:Ion channels have historically been viewed as distinct from secondary active transporters. However, the recent discovery that the CLC 'chloride channel' family is made up of both channels and active transporters has led to the hypothesis that the ion-transport mechanisms of these two types of membrane proteins may be similar. Here we use single-channel analysis to demonstrate that ClC-0 channel gating (opening and closing) involves the transmembrane movement of protons. This result indicates that ClC-0 is a 'broken' Cl(-)/H(+) antiporter in which one of the conformational states has become leaky for chloride ions. This finding clarifies the evolutionary relationship between the channels and transporters and conveys that similar mechanisms and analogous protein movements are used by both.
| S-EPMC2559860 | biostudies-literature
Project description:Chloride-transporting membrane proteins of the CLC family appear in two distinct mechanistic flavors: H(+)-gated Cl(-) channels and Cl(-)/H(+) antiporters. Transmembrane H(+) movement is an essential feature of both types of CLC. X-ray crystal structures of CLC antiporters show the Cl(-) ion pathway through these proteins, but the H(+) pathway is known only inferentially by two conserved glutamate residues that act as way-stations for H(+) in its path through the protein. The extracellular-facing H(+) transfer glutamate becomes directly exposed to aqueous solution during the transport cycle, but the intracellular glutamate E203, Glu(in), is buried within the protein. Two regions, denoted "polar" and "interfacial," at the intracellular surface of the bacterial antiporter CLC-ec1 are examined here as possible pathways by which intracellular aqueous protons gain access to Glu(in). Mutations at multiple residues of the polar region have little effect on antiport rates. In contrast, mutation of E202, a conserved glutamate at the protein-water boundary of the interfacial region, leads to severe slowing of the Cl(-)/H(+) antiport rate. An X-ray crystal structure of E202Y, the most strongly inhibited of these substitutions, shows an aqueous portal leading to Glu(in) physically blocked by cross-subunit interactions; moreover, this mutation has only minimal effect on a monomeric CLC variant, which necessarily lacks such interactions. The several lines of experiments presented argue that E202 acts as a water-organizer that creates a proton conduit connecting intracellular solvent with Glu(in).
| S-EPMC3519907 | biostudies-literature
Project description:The acidic luminal pH of lysosomes, maintained within a narrow range, is essential for proper degrative function of the organelle and is generated by the action of a V-type H+ ATPase, but other pathways for ion movement are required to dissipate the voltage generated by this process. ClC-7, a Cl-/H+ antiporter responsible for lysosomal Cl- permeability, is a candidate to contribute to the acidification process as part of this 'counterion pathway' The signaling lipid PI(3,5)P2 modulates lysosomal dynamics, including by regulating lysosomal ion channels, raising the possibility that it could contribute to lysosomal pH regulation. Here, we demonstrate that depleting PI(3,5)P2 by inhibiting the kinase PIKfyve causes lysosomal hyperacidification, primarily via an effect on ClC-7. We further show that PI(3,5)P2 directly inhibits ClC-7 transport and that this inhibition is eliminated in a disease-causing gain-of-function ClC-7 mutation. Together, these observations suggest an intimate role for ClC-7 in lysosomal pH regulation.
| S-EPMC9242644 | biostudies-literature
Project description:Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl(-)/H(+) antiport current (I(ClC-3)) in HEK293 cells. The outward rectification and time dependence of I(ClC-3) closely resemble an endogenous HEK293 cell acid-activated Cl(-) current (ICl(acid)) seen at extracellular pH <or= 5.5. ICl(acid) was present in smooth muscle cells from wild-type but not ClC-3 null mice. We therefore sought to determine whether these currents were related. ICl(acid) was larger in cells expressing Ad-ClC-3. Protons shifted the reversal potential (E(rev)) of I(ClC-3) between pH 8.2 and 6.2, but not pH 6.2 and 5.2, suggesting that Cl(-) and H(+) transport become uncoupled at low pH. At pH 4.0 E(rev) was completely Cl(-) dependent (55.8 +/- 2.3 mV/decade). Several findings linked ClC-3 with native ICl(acid); 1) RNA interference directed at ClC-3 message reduced native ICl(acid); 2) removal of the extracellular "fast gate" (E224A) produced large currents that were pH-insensitive; and 3) wild-type I(ClC-3) and ICl(acid) were both inhibited by (2-sulfonatoethyl)methanethiosulfonate (MTSES; 10-500 microm)-induced alkanethiolation at exposed cysteine residues. However, a ClC-3 mutant lacking four extracellular cysteine residues (C103_P130del) was completely resistant to MTSES. C103_P130del currents were still acid-activated, but could be distinguished from wild-type I(ClC-3) and from native ICl(acid) by a much slower response to low pH. Thus, ClC-3 currents are activated by protons and ClC-3 protein may account for native ICl(acid). Low pH uncouples Cl(-)/H(+) transport so that at pH 4.0 ClC-3 behaves as an anion-selective channel. These findings have important implications for the biology of Cl(-)/H(+) antiporters and perhaps for pH regulation in highly acidic intracellular compartments.
| S-EPMC2807314 | biostudies-literature
Project description:Interactions between membrane protein interfaces in lipid bilayers play an important role in membrane protein folding but quantification of the strength of these interactions has been challenging. Studying dimerization of ClC-type transporters offers a new approach to the problem, as individual subunits adopt a stable and functionally verifiable fold that constrains the system to two states - monomer or dimer. Here, we use single-molecule photobleaching analysis to measure the probability of ClC-ec1 subunit capture into liposomes during extrusion of large, multilamellar membranes. The capture statistics describe a monomer to dimer transition that is dependent on the subunit/lipid mole fraction density and follows an equilibrium dimerization isotherm. This allows for the measurement of the free energy of ClC-ec1 dimerization in lipid bilayers, revealing that it is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes.
| S-EPMC5010387 | biostudies-literature
Project description:The CLC family of proteins are involved in a variety of physiological processes to control cellular chloride concentration. Two distinct classes of CLC proteins, Cl- channels and Cl-/H+ antiporters, have been functionally and structurally investigated over the last several decades. Previous studies have suggested that the conformational heterogeneity of the critical glutamate residue, Gluex, could explain the transport cycle of CLC-type Cl-/H+ antiporters. However, the presence of multiple conformations (Up, Middle, and Down) of the Gluex has been suggested from combined structural snapshots of 2 different CLC antiporters: CLC-ec1 from Escherichia coli and cmCLC from a thermophilic red alga, Cyanidioschyzon merolae Thus, we aimed to investigate further the heterogeneity of Gluex-conformations in CLC-ec1, the most deeply studied CLC antiporter, at both functional and structural levels. Here, we show that the crystal structures of the Gluex mutant E148D and wild-type CLC-ec1 with varying anion concentrations suggest a structural intermediate, the "Midlow" conformation. We also found that an extra anion can be located above the external Cl--binding site in the E148D mutant when the anion concentration is high. Moreover, we observed that a carboxylate in solution can occupy either the external or central Cl--binding site in the ungated E148A mutant using an anomalously detectable short carboxylic acid, bromoacetate. These results lend credibility to the idea that the Gluex can take at least 3 distinct conformational states during the transport cycle of a single CLC antiporter.
| S-EPMC6717299 | biostudies-literature