Voltage-dependent K+ channels are members of the family of voltage-dependent cation (K+, Na+ and Ca2+) channels that open and allow ion conduction in response to changes in cell membrane voltage. This form of gating underlies the generation of nerve and muscle action potentials, among other processes. Here we present the structure of KvAP, a voltage-dependent K+ channel from Aeropyrum pernix. We have determined a crystal structure of the full-length channel at a resolution of 3.2 A, and of the isolated voltage-sensor domain at 1.9 A, both in complex with monoclonal Fab fragments. The channel contains a central ion-conduction pore surrounded by voltage sensors, which form what we call 'voltage-sensor paddles'-hydrophobic, cationic, helix-turn-helix structures on the channel's outer perimeter. Flexible hinges suggest that the voltage-sensor paddles move in response to membrane voltage changes, carrying their positive charge across the membrane.

X-ray structure of a voltage-dependent K+ channel

The ClC chloride channels catalyse the selective flow of Cl- ions across cell membranes, thereby regulating electrical excitation in skeletal muscle and the flow of salt and water across epithelial barriers. Genetic defects in ClC Cl- channels underlie several familial muscle and kidney diseases. Here we present the X-ray structures of two prokaryotic ClC Cl- channels from Salmonella enterica serovar typhimurium and Escherichia coli at 3.0 and 3.5 A, respectively. Both structures reveal two identical pores, each pore being formed by a separate subunit contained within a homodimeric membrane protein. Individual subunits are composed of two roughly repeated halves that span the membrane with opposite orientations. This antiparallel architecture defines a selectivity filter in which a Cl- ion is stabilized by electrostatic interactions with alpha-helix dipoles and by chemical coordination with nitrogen atoms and hydroxyl groups. These findings provide a structural basis for further understanding the function of ClC Cl- channels, and establish the physical and chemical basis of their anion selectivity.

/pdf/x-ray-structure-of-a-clc-chloride-channel-at-3-0-a-reveals-1wk74mj1p6.pdf

X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity.

Cl− channels reside both in the plasma membrane and in intracellular organelles Their functions range from ion homeostasis to cell volume regulation, transepithelial transport, and regulation of e

https://www.leibniz-fmp.de/fileadmin/user_upload/Physiology_and_Pathology_of_Ion_Transport/documents/PhysRev2002.pdf

Molecular Structure and Physiological Function of Chloride Channels

There are two classes of glucose transporters involved in glucose homeostasis in the body, the facilitated transporters or uniporters (GLUTs) and the active transporters or symporters (SGLTs). The ...

Biology of Human Sodium Glucose Transporters

Lysosomes, the terminal organelles on the endocytic pathway, digest macromolecules and make their components available to the cell as nutrients. Hydrolytic enzymes specific to a wide range of targets reside within the lysosome; these enzymes are activated by the highly acidic pH (between 4.5 and 5.0) in the organelles' interior. Lysosomes generate and maintain their pH gradients by using the activity of a proton-pumping V-type ATPase, which uses metabolic energy in the form of ATP to pump protons into the lysosome lumen. Because this activity separates electric charge and generates a transmembrane voltage, another ion must move to dissipate this voltage for net pumping to occur. This so-called counterion may be either a cation (moving out of the lysosome) or an anion (moving into the lysosome). Recent data support the involvement of ClC-7, a Cl(-)/H(+) antiporter, in this process, although many open questions remain as to this transporter's involvement. Although functional results also point to a cation transporter, its molecular identity remains uncertain. Both the V-ATPase and the counterion transporter are likely to be important players in the mechanisms determining the steady-state pH of the lysosome interior. Exciting new results suggest that lysosomal pH may be dynamically regulated in some cell types.

/pdf/lysosomal-acidification-mechanisms-53xx1ofvg0.pdf

Lysosomal Acidification Mechanisms

To reach the mammalian gut, enteric bacteria must pass through the stomach. Many such organisms survive exposure to the harsh gastric environment (pH 1.5-4) by mounting extreme acid-resistance responses, one of which, the arginine-dependent system of Escherichia coli, has been studied at levels of cellular physiology, molecular genetics and protein biochemistry. This multiprotein system keeps the cytoplasm above pH 5 during acid challenge by continually pumping protons out of the cell using the free energy of arginine decarboxylation. At the heart of the process is a 'virtual proton pump' in the inner membrane, called AdiC, that imports L-arginine from the gastric juice and exports its decarboxylation product agmatine. AdiC belongs to the APC superfamily of membrane proteins, which transports amino acids, polyamines and organic cations in a multitude of biological roles, including delivery of arginine for nitric oxide synthesis, facilitation of insulin release from pancreatic beta-cells, and, when inappropriately overexpressed, provisioning of certain fast-growing neoplastic cells with amino acids. High-resolution structures and detailed transport mechanisms of APC transporters are currently unknown. Here we describe a crystal structure of AdiC at 3.2 A resolution. The protein is captured in an outward-open, substrate-free conformation with transmembrane architecture remarkably similar to that seen in four other families of apparently unrelated transport proteins.

/pdf/structure-of-a-prokaryotic-virtual-proton-pump-at-3-2-a-4mwichzkkm.pdf

Structure of a prokaryotic virtual proton pump at 3.2 A resolution.

CLC-ec1 is a prokaryotic CLC-type Cl−/H+ exchange transporter Little is known about the mechanism of H+ coupling to Cl− A critical glutamate residue, E148, was previously shown to be required for Cl−/H+ exchange by mediating proton transfer between the protein and the extracellular solution To test whether an analogous H+ acceptor exists near the intracellular side of the protein, we performed a mutagenesis scan of inward-facing carboxyl-bearing residues and identified E203 as the unique residue whose neutralization abolishes H+ coupling to Cl− transport Glutamate at this position is strictly conserved in all known CLCs of the transporter subclass, while valine is always found here in CLC channels The x-ray crystal structure of the E203Q mutant is similar to that of the wild-type protein Cl− transport rate in E203Q is inhibited at neutral pH, and the double mutant, E148A/E203Q, shows maximal Cl− transport, independent of pH, as does the single mutant E148A The results argue that substrate exchange by CLC-ec1 involves two separate but partially overlapping permeation pathways, one for Cl− and one for H+ These pathways are congruent from the protein's extracellular surface to E148, and they diverge beyond this point toward the intracellular side This picture demands a transport mechanism fundamentally different from familiar alternating-access schemes

https://rupress.org/jgp/article-pdf/126/6/563/1221523/jgp1266563.pdf

Separate ion pathways in a Cl-/H+ exchanger

The process of arginine-dependent extreme acid resistance (XAR) is one of several decarboxylase-antiporter systems that protects Escherichia coli and possibly other enteric bacteria from exposure to the strong acid environment of the stomach. Arginine-dependent acid resistance depends on an intracellular proton-utilizing arginine α-decarboxylase and a membrane transport protein necessary for delivering arginine to and removing agmatine, its decarboxylation product, from the cytoplasm. The arginine system afforded significant protection to wild-type E. coli cells in our acid shock experiments. The gene coding for the transport protein is identified here as a putative membrane protein of unknown function, YjdE, which we now name adiC. Strains from which this gene is deleted fail to mount arginine-dependent XAR, and they cannot perform coupled transport of arginine and agmatine. Homologues of this gene are found in other bacteria in close proximity to homologues of the arginine decarboxylase in a gene arrangement pattern similar to that in E coli. Evidence for a lysine-dependent XAR system in E. coli is also presented. The protection by lysine, however, is milder than that by arginine.

Arginine-Agmatine Antiporter in Extreme Acid Resistance in Escherichia coli

CLC-ec1 is an E. coli homologue of the CLC family of Cl− channels, which are widespread throughout eukaryotic organisms. The structure of this membrane protein is known, and its physiological role has been described, but our knowledge of its functional characteristics is severely limited by the absence of electrophysiological recordings. High-density reconstitution and incorporation of crystallization-quality CLC-ec1 in planar lipid bilayers failed to yield measurable CLC-ec1 currents due to porin contamination. A procedure developed to prepare the protein at a very high level of purity allowed us to measure macroscopic CLC-ec1 currents in lipid bilayers. The current is Cl− selective, and its pH dependence mimics that observed with a 36Cl− flux assay in reconstituted liposomes. The unitary conductance is estimated to be <0.2 pS. Surprisingly, the currents have a subnernstian reversal potential in a KCl gradient, indicating imperfect selectivity for anions over cations. Mutation of a conserved glutamate residue found in the selectivity filter eliminates the pH-dependence of both currents and 36Cl− flux and appears to trap CLC-ec1 in a constitutively active state. These effects correlate well with known characteristics of eukaryotic CLC channels. The E148A mutant displays nearly ideal Cl− selectivity.

https://rupress.org/jgp/article-pdf/123/2/109/1218996/jgp1232109.pdf

Ionic currents mediated by a prokaryotic homologue of CLC Cl- channels.

The CLC-family protein CLC-ec1, a bacterial homologue of known structure, stoichiometrically exchanges two Cl− for one H+ via an unknown membrane transport mechanism. This study examines mutations at a conserved tyrosine residue, Y445, that directly coordinates a Cl− ion located near the center of the membrane. Mutations at this position lead to “uncoupling,” such that the H+/Cl− transport ratio decreases roughly with the volume of the substituted side chain. The uncoupled proteins are still able to pump protons uphill when driven by a Cl− gradient, but the extent and rate of this H+ pumping is weaker in the more uncoupled variants. Uncoupling is accompanied by conductive Cl− transport that is not linked to counter-movement of H+, i.e., a “leak.” The unitary Cl− transport rate, measured in reconstituted liposomes by both a conventional initial-velocity method and a novel Poisson dilution approach, is ∼4,000 s−1 for wild-type protein, and the uncoupled mutants transport Cl− at similar rates.

/pdf/uncoupling-and-turnover-in-a-cl-h-exchange-transporter-5d5nowxoxc.pdf

Carole Williams

Papers

Structure of a prokaryotic virtual proton pump at 3.2 A resolution.

Separate ion pathways in a Cl-/H+ exchanger

Arginine-Agmatine Antiporter in Extreme Acid Resistance in Escherichia coli

Ionic currents mediated by a prokaryotic homologue of CLC Cl- channels.

Uncoupling and turnover in a Cl-/H+ exchange transporter