Analysis of the potentiation mechanism of the α9α10 cholinergic nicotinic receptor by extracellular calcium.

SOFIA GALLINO; PATRICIO O. CRAIG; JUAN CARLOS BOFFI; PAOLA V. PLAZAS; A BELEN ELGOYHEN

Congreso; XXXVI Reunión Anual. de la. Sociedad Argentina de Investigación en Neurociencias; 2021

The α9α10 nicotinic cholinergic receptor (nAChR) is a pentameric cation-permeable ion channel that mediates the inhibitory synapse between efferent fibers and outer hair cells of the cochlea. Each nAChR subunit comprises a large extracellular amino-terminal domain, four transmembrane domains (TM1-TM4) and a long cytoplasmic loop between TM3 and TM4. 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 (Ca2+). While α9 nAChRs responses are blocked by Ca2+, ACh-evoked currents through α9α10 nAChRs are potentiated by Ca2+ in the micromolar range and blocked at millimolar concentrations. In order to identify the structural determinants responsible for Ca2+ potentiation, 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 Ca2+. Moreover, we identified 10 E45 and E175 as key residues involved in this potentiation. These studies are being complemented with molecular dynamics simulations of the interaction of Ca2+ with different nAChRs models to help in the structural interpretation of the results.