Eukaryotic-Prokaryotic Chimeras for Structure-Function Studies of the Intracellular Domain of Cys-Loop Receptors

Abstract

Receptors belonging to the Cys-loop gene superfamily of neurotransmittergated ion channels (e.g. acetylcholine, serotonin, GABA, and glycine receptors) mediate fast synaptic transmission. These receptors are targeted by a number of clinically used drugs (e.g. antiepileptics, antipsychotics, anesthetics). Cys-loop receptors contain three domains: extracellular (ECD), transmembrane (TMD), intracellular (ICD). The ECD and TMD have been studied in great detail and their 3-D structures have been determined. The recent identification of bacterial Cys-loop receptor homologues has propelled the structural knowledge to atomic resolution. However, all prokaryotic members lack the ICD. The ICD of metazoan Cys-loop receptors is the most diverse domain with respect to both length and amino-acid composition. The ICD therefore represents an attractive target for developing subtype-selective drugs with the promise of fewer side effects than drugs that target the highly-conserved ECD and TMD. We have engineered functional chimeras with ICDs from different eukaryotic receptors in the two-domain prokaryotic homologue GLIC (one chimera set published, Goyal, Salahudeen, Jansen, JBC 2011). Based on structure predictions others have hypothesized that the ICD is mainly unstructured, but contains an a-helical segment pre-TM4. We hypothesize based on computational results that cationic and anionic receptors have different ICD secondary structures, i.e., only cationic receptors contain an a-helical portion pre-TM4. For each eukaryotic ICD we generated 12 chimeras that differ by the linkers both N-and C-terminal to the inserted ICD. Interestingly, the number of functional chimeras in each set varies drastically between different ICDs. We are testing the functional consequences of inserting various ICDs using electrophysiological studies, and investigating the structure of the ICDs tethered to the TMD and at the lipid bilayer with spectroscopic and biochemical methods. 589-Pos Board B375Function of nAChRs Containing Alpha5 Subunits Neuronal nicotinic acetylcholine receptors (nAChRs) are abundantly expressed in the central nervous system. Neurons in several brain regions co-express alpha5, alpha4 and beta2 subunits. Changes in the receptor composition and stoichiometry of alpha4 and beta2 containing nAChRs are pertinent to disease states. Several genome-wide association and candidate gene studies have identified polymorphisms in the gene for the alpha5 subunit that are linked to an increased risk for nicotine addiction, alcohol addiction and lung cancer. However more information is required about the functional effects of incorporating a normal or mutated alpha5 subunit into the alpha4beta2 receptor pentamer. Here we examine differences in I-V relationships and dose-response relations between alpha4beta2 receptors and alpha4beta2 receptors including alpha5 subunits containing various mutations to the M2 domain expressed in Xenopus oocytes.