A prokaryotic proton-gated ion channel from the nicotinic acetylcholine receptor family

Abstract

Ligand-gated ion channels (LGICs) mediate excitatory and inhibitory transmission in the nervous system. Among them, the pentameric or 'Cys-loop' receptors (pLGICs) compose a family that until recently was found in only eukaryotes. Yet a recent genome search identified putative homologues of these proteins in several bacterial species. Here we report the cloning, expression and functional identification of one of these putative homologues from the cyanobacterium Gloeobacter violaceus. It was expressed as a homo-oligomer in HEK 293 cells and Xenopus oocytes, generating a transmembrane cationic channel that is opened by extracellular protons and shows slow kinetics of activation, no desensitization and a single channel conductance of 8 pS. Electron microscopy and cross-linking experiments of the protein fused to the maltose-binding protein and expressed in Escherichia coli are consistent with a homo-pentameric organization. Sequence comparison shows that it possesses a compact structure, with the absence of the amino-terminal helix, the canonical disulphide bridge and the large cytoplasmic domain found in eukaryotic pLGICs. Therefore it embodies a minimal structure required for signal transduction. These data establish the prokaryotic origin of the family. Because Gloeobacter violaceus carries out photosynthesis and proton transport at the cytoplasmic membrane, this new proton-gated ion channel might contribute to adaptation to pH change.

The M4 Transmembrane α-Helix Contributes Differently to Both the Maturation and Function of Two Prokaryotic Pentameric Ligand-gated Ion Channels

“…The injection of wild type GLIC cRNA into Xenopus oocytes led to the expression of proton-activated channels on the oocyte surface that responded to protons in a dose-dependent manner (Fig. 1), with a pH 50 value of 5.03 Ϯ 0.08 (n ϭ 38), consistent with published data (46). Twenty-five of the 26 M4 Ala mutants gave robust proton-activated currents, with the mutations displaying either no (⌬pH 50 Յ 0.09 for 8/26 mutants) or statistically significant changes in pH 50 (0.09 Ͻ ⌬pH 50 Յ 0.6 for 17/26 mutants, p Ͻ 0.05) relative to wild type.…”

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

“…of the LGIC structures is still debatable, a fundamental difference in the ECD-TMD interface in ELIC, GLIC, and GluCL in comparison to nAChR is the relative positioning of the interfacial loops and the extent of interaction between them (1,3,5,7,54,60). Several factors may contribute to the observed structural differences between these channels (17,61).…”

Allosteric and hyperekplexic mutant phenotypes investigated on an α 1 glycine receptor transmembrane structure

“…At pH 5, application of the channel blocker picrotoxinin (100 μM) yielded a 91 ± 7% inhibition of the Lily-His currents showing the specificity of the response. For the mutational studies shown below, we recorded Lily and GLIC in BHK cells with a C-terminal HA tag (16) that does not alter the electrophysiological response (17), yielding EC 50 s of 3.3 ± 0.1 × 10 −7 M (pH 6.5) and 4.8 ± 0.6 × 10 −6 M (pH 5.3), respectively.…”