Identification of calcium binding sites and structural determinants that regulate potentiation of α9α10 nicotinic cholinergic receptors.

SOFIA GALLINO; PAOLA V. PLAZAS; JUAN BOFFI; ANA BELÉN ELGOYHEN

Congreso; XXXIV Reunión Anual. de la. Sociedad Argentina de Investigación en Neurociencias; 2019

Nicotinic cholinergic receptors (nAChR) are pentameric cation-permeable ion channels activated by acetylcholine (ACh). Each nAChR subunit comprises a large extracellular amino-terminal domain, four transmembrane domains (TM1-TM4) and a long cytoplasmic loop between TM3 and TM4. The α9α10 nAChR mediates the inhibitory synapse between efferent fibers and outer hair cells of the cochlea. Expression of rat α9 and α10 nAChR subunits in Xenopus laevis oocytes yields functional α9 and α9α10 receptors, but not α10 homomeric nAChRs. One of the functional differences between α9 and α9α10 nAChRs is the modulation of their ACh-evoked responses by extracellular calcium (Ca+2). While α9 nAChRs responses are blocked by Ca2+, ACh-evoked currents through α9α10n AChRs are potentiated by Ca2+ in the micromolar range and blocked at millimolar concentrations. In order to identify the structural determinants responsible for these differences, we generated chimeric and mutant subunits, expressed them in Xenopus oocytes and performed electrophysiological recordings under two electrode voltage clamp. Our results suggest that the TM2-TM3 loop of the α10 subunit contains structural determinants responsible for the potentiation of the 910 nAChR by extracellular Ca2+. Moreover we identified 10 E45 and E175 as key residues of two potential Ca2+ binding sites involved in this potentiation.