Probing the trans-membrane domain of GLIC, a prokaryotic ligand-gated ion channel.

M AlQazzaz, SCRL Lummis. University of Cambridge, Cambridge, UK

Cys-loop receptors are responsible for the transmission of fast excitatory and inhibitory signals in the CNS and PNS. The absence of high-resolution structures for eukaryotic receptors hinders the rational design of more selective drug candidates. The Gloeobacter ligand-gated ion channel, GLIC, is a prokaryotic homologue of Cys-loop receptors whose structure was solved at high resolution using X-ray crystallography. GLIC has 28% overall structure identity with 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 is to investigate the GLIC M2 region, concentrating on those residues that are conserved across the Cys-loop receptor family, and thus providing information about the structural relevance of GLIC for eukaryotic receptors.

GLIC was functionally characterized using two-electrode voltage clamp (TEVC) as previously described (Alqazzaz et al., 2011). It shows half-maximal activation at pH 5.6±0.3, n=7, close to the pKa of histidine, where the role of His235 is essential in proton sensing linked to channel opening. The replacement of His235 with any other amino acid resulted in non-functional receptors. We further investigated the interaction between the M2 and M3 using the structures provided in the protein data bank of the presumable open conformation (3EAM) and the locally closed conformation (3TLT) as templates for our mutagenesis and functional studies. We identified the critical role of hydrogen bond between His235 and the backbone of the M3 domain, which provides a new mechanism of Cys-loop receptor channel opening/closing.

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, Thr226, Ser230, Leu232, Ile233, Ala234, Ile236, Ala237 and Phe238 using site-directed mutagenesis and tested their function TEVC. Our results show that Thr226, Ser230, Ile233 and His235 residues are all important for the production of a functional GLIC channel.

We conclude that His23 has a potential role in channel opening/closing, and the residues Thr226, Ser230, and Ile233 also play an important role in receptor function. Overall our data suggest the pore of GLIC is a suitable template to model Cys-loop receptor pores, but that GLICs mechanism of opening may differ from that of eukaryotic receptors.

References

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.