ROLE OF TM3-TM3 LOOP IN THE POTENTIATION MECHANISM OF THE Α9Α10 CHOLINERGIC NICOTINIC RECEPTOR BY EXTRACELLULAR CALCIUM

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

Congreso; Society for Neuroscience 52nd Annual Meeting; 2022

The α9α10 nicotinic acetylcholine receptor (nAChR) is expressed in cochlear hair cells. This nAChR mediates the inhibitory synapse between efferent fibers and outer hair cells. The inhibition results from calcium entry through the nAChR, in the presence of acetylcholine (ACh), followed by the activation of a Ca2+ dependent potassium current. The α9α10 nAChR plays a key role in auditory neural circuits at the post-hearing onset. Hence, a deep understanding of the modulatory effects of this cholinergic receptor in auditory circuits would be extremely useful for the development strategies towards hearing loss.The α9α10 nAChR is a pentameric cation-permeable ion channel that is composed of α9 and α10 subunits. Each nAChR subunit comprises a large extracellular amino-terminal domain, four transmembrane domains (TM1-TM4), a long cytoplasmic loop between TM3 and TM4 and a C-terminal domain. 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 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 several chimeric and mutant α10 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 E71 and E 202 as possible key residues of two potential Ca2+ binding sites involved in this potentiation.Moreover, to elucidate the mechanism of this potentiation by extracellular Ca2+we performed molecular dynamics simulations of the interaction of Ca2+ with different nAChRs models (9 and 10 homomeric receptors, and α9α10 heteromeric receptor in both grouped and alternated configurations). The result of this study shows that both heteromeric 9α10 and homomeric 9 nAChRs exhibit similar calcium binding in the environment of their TM2-TM3 loops. Therefore, our hypothesis is that the TM2-TM3 loop of the 10 subunit contributes with structural determinants that are key for the gating of the a9a10 nAChR, once ACh binding has occurred in the presence of extracellular Ca2+.