Probing Molecular Interactions in Erwinia Ligand-Gated Ion Channel (ELIC)

Abstract

The Erwinia ligand-gated ion channel (ELIC) is a bacterial pentameric ligand ion channel (pLGIC) originally identified in the enterobacterium Erwinia chrysanthemii. ELIC is closely related to a large class of neurotransmitter-gated ion channels (Cys-loop receptors) that underpin fast synaptic transmission in vertebrates and whose malfunction underlies a range of neurological disorders. As structures of Cys-loop receptors have lagged well behind those from prokaryotic pLGICs, ELIC, whose structure was solved in 2008, has proved to be an attractive model system. Here we explore molecular interactions in ELIC revealed by its structure. Of particular interest are intra-protein cation-π interactions, of which there are potentially six in each ELIC subunit: R48-W66; R99-F95; R105-Y38; R141-F142; R199-W160; and R301-W224. To probe these, the amino acids in each pair were swopped, using site directed mutagenesis, in addition to creating mutants with single amino acid changes to Ala and its pair. ELIC function was then probed following expression in HEKT cells using a membrane potential sensitive dye in a Flexstation. WT ELIC revealed GABA concentration dependent increases in fluorescence with a pEC50 of 2.97 + 0.01 (M, n > 6). The mutations had a range of effects from ablation of responses with all substituted amino acids (e.g. R105-Y38) to wild type responses with all substituted amino acids (e.g. R48-W66). The nature and importance of the interactions formed by these amino acids in ELIC, and whether the data can be extrapolated to better understand vertebrate Cys loop receptors, will be discussed.

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