Glic-Elic Chimeras have Unexpected Characteristics

Abstract

The prokaryotic ligand-gated ion channels from Gloeobacter violaceus (GLIC) and Erwinia chrysanthemi (ELIC) have provided useful information about the homologous Cys-loop family of neurotransmitter receptors. GLIC is of particular interest for the study of gating transitions, in that its structure has been elucidated in multiple conformational states under a number of conditions. However its mechanism of action is controversial - the protonation of His11’ in M2 has been shown to be essential for the activation of GLIC(1,2), yet a GLIC-glycine chimera retains proton sensitivity without the GLIC M2 domain(3). Here we create an ELIC-GLIC and a GLIC-ELIC chimera, express them in Xenopus oocytes, and test them for function using TEVC. The ELIC-GLIC chimera was not activated by 100mM GABA or pH 4. The GLIC-ELIC chimera was not activated by GABA, but was activated by protons with a pH50 of 6.6, similar to the GLIC-glycine chimera but not WT GLIC (pH50=5.5). We then tested the GLIC-ELIC chimera with crotonic acid and picrotoxin. Crotonic acid inhibits GLIC with an IC50 of 110μM; our data indicate it binds to the extracellular domain. Picrotoxin (IC50=2.6μM) blocks the GLIC pore(4); it likely cannot access the ELIC pore(5), but may bind to the extracellular domain (IC50=96μM;6). These compounds were less potent than expected in the chimera (IC50>300μM). Overall the data suggest that domain specific effects may not be accurately reproduced in complex chimeras with intercommunicating domains, such as an orthosteric binding site and a pore in ligand-gated ion channels.1. Rienzo et al. (2104) Chem.Biol. (in press); 2.Wang et al. (2012) J.Biol.Chem. 287: 6482; 3.Duret et al. (2011). PNAS 108: 12143; 4.Alqazzaz et al., (2011) Biophys.J. 101: 2912; 5.Ulens et al.(2014) Structure(in press). 6.Thompson et al. (2012) Neuropharm. 63:761.