Study of the structural determinants involved in the potentiation of the α9α10 cholinergic nicotinic receptor by extracellular calcium

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

Congreso; Neuroscience 2021; 2021

Society for Neuroscience

The α9α10 nicotinic cholinergic receptor (nAChR) mediates the inhibitory synapse between medial olivocochlear efferent neurons and outer hair cells of the cochlea. The inhibition results from calcium (Ca2+) entry through the α9α10 nAChR, and the subsequent activation of a small-conductance SK2 Ca2+-dependent potassium channel. 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 of new translational strategies towards hearing loss and hyperacusis.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 E 45 and E 175 as possible key residues of two potential Ca2+ binding sites involved in this potentiation.These studies are being complemented with molecular dynamics simulations of the interaction of Ca2+ with different nAChRs models (9 and 10 homomeric receptors, and α9α10 heteromeric receptor in grouped or alternated subunit spatial arrangements) to help in the structural interpretation of the experimental results obtained.