Characterising the Ion Selectivity and Calcium Permeability of the Erwinia Ligand-Gated Ion Channel

Abstract

The Erwinia ligand-gated ion channel (ELIC) is a bacterial GABA-gated cation-selective channel that shares structural homology with vertebrate Cys-loop receptors[1]. Here we investigated the effects of Ca2+ on the functional properties of ELIC using wild-type and mutant receptors. Receptors were expressed in Xenopus oocytes and characterised using two electrode voltage-clamp electrophysiology. GABA concentration-response curves were recorded in ND96 buffer with 0, 1.8, 5 or 10 mM Ca2+. The data show that Ca2+ alters the rate of apparent desensitization and the GABA EC50. To probe ion selectivity the external NaCl concentration was altered and the effects on reversal potential measured by clamping cells between −60 and +100 mV in 10 mV steps. These data showed that ELIC is predominantly cation selective as previously reported[2]. The −1' (E229) residue has been identified as a critical region for controlling cation selectivity in Cys-loop receptors[3][4]. Consistent with this, an E229A mutation in ELIC decreased cation selectivity. When E229A was combined with a mutation at the N20' position (E229A/N250R) ELIC became predominantly anion selective, similar to experiments described for the 5-HT3 receptor[4]. This mutant eliminated the effects of Ca2+ on ELIC, demonstrating that Ca2+ effects are mediated either in the channel or in the intracellular domain.[1] Hilf & Dutzler., 2008, Nature, 452 (7185) 375-9.[2] Zimmermann et al., 2011 PLoS Biol, 9 (6).[3] Galzi et al., 1992 Nature, 359 (6395) 500-5.[4] Thompson & Lummis, 2003 Br J Pharmacol, 140 359-65.