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The Gloeobacter ligand-gated ion channel, GLIC, has up to 28% sequence identity with eukaryotic Cys-loop receptors, and many key residues are conserved, especially in the 2nd trans-membrane pore lining region, M2. The M2 region is responsible for ion selectivity, ion flux, and binding a wide range of non-competitive inhibitors (Alqazzaz et al., 2011). The aim of this work was to investigate the GLIC M2 region, especially His235 (or 11'), which has been proposed as essential in proton sensing linked to channel opening (Wang et al., 2012), and residues that are conserved across the Cys-loop receptor family. To probe the roles of specific amino acids in GLIC M2 trans-membrane domain, we substituted M2 residues lining the ion pore and M2 residues facing M3 trans-membrane domains. We generated over 40 mutations at various positions including those at Glu 222(-2'), Thr 226 (2'), Ser 230 (6'), Leu 232 (8'), Ile 233 (9'), Ala 234 (10'), Ile 236 (12'), Ala 237 (13') and Phe 238 (14') using site-directed mutagenesis, and tested their function using two-electrode voltage clamp as previously described (Alqazzaz et al., 2011). Our results showed that Ser6', Ile9' and His11' residues are very sensitive to subsitution with all substituents resulting in non-functional receptors. We conclude that His 11' has a role in channel opening/closing, and the residues Thr226, Ser230, and Ile233 also play an important role in receptor function.Alqazzaz M, Thompson AJ, Price KL, Breitinger HG, Lummis SC (2011). Cys-loop receptor channel blockers also block GLIC. Biophysical journal 101(12): 2912-2918.Wang HL, Cheng X, Sine SM (2012). Intramembrane proton binding site linked to activation of bacterial pentameric ion channel. The Journal of biological chemistry 287(9): 6482-6489.

Several neuronal cell types co-express nicotinic acetylcholine receptors (nAChRs) and P2X receptors (P2XRs); and previous experiments from several laboratories show that these two ligand-gated channel subtypes interact with each other, both functionally and physically. We extended these studies to interactions between alpha6 subunit-containing (alpha6∗) nAChRs and P2X2 or P2X3 receptors (P2X2Rs, P2X3Rs). In Xenopus oocytes, alpha6 subunits mutated in the M2 region co-expressed with beta4 subunits displayed ACh-induced currents. P2X2 or P2X3 receptors expressed in the same oocytes displayed ATP-induced currents. When ACh and ATP were applied simultaneously, the total current was less than the sum of the individual currents_the conventional definition of cross-inhibition_for alpha6beta4-P2X2 oocytes. Several combinations of fluorescent protein (FP)-fused alpha6, beta4, P2X2, and P2X3 subunits were expressed in cultured mouse cortical cells and Neuro2a cells. In Forster resonance energy transfer (FRET) measurements using fluorescent lifetime imaging microscopy (FLIM) and normalized FRET (NFRET), we detected physical contact between alpha6beta2 nAChRs and P2X2Rs, between alpha6beta4 nAChRs and P2X2Rs, and between alpha6beta4 nAChRs and P2X3Rs. Thus, two lines of evidence show interactions between alpha6∗ nAChRs and P2XRs.Support: Beckman fellowship to CIR, NIH (NRSA to CIR, DA-19375, NS034407), Wellcome Trust, Yousef Jameel Award.