Key role of the TM2-TM3 loop in calcium potentiation of the α9α10 nicotinic acetylcholine receptor

The α9α10 nicotinic cholinergic receptor (nAChR) is a ligand-gated pentameric cation-permeable ion channel that mediates synaptic transmission between descending efferent neurons and mechanosensory inner ear hair cells. When expressed in heterologous systems, α9 and α10 subunits can assemble into functional homomeric α9 and heteromeric α9α10 receptors. One of the differential properties between these 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. Using chimeric and mutant subunits, together with electrophysiological recordings under two-electrode voltage-clamp, we show that the TM2-TM3 loop of the rat α10 subunit contains key structural determinants responsible for the potentiation of the α9α10 nAChR by extracellular Ca²⁺. Moreover, molecular dynamics simulations reveal that the TM2-TM3 loop of α10 does not contribute to the Ca²⁺ potentiation phenotype through the formation of novel Ca²⁺ binding sites not present in the α9 receptor. These results suggest that the TM2-TM3 loop of α10 might act as a control element that facilitates the intramolecular rearrangements that follow ACh-evoked α9α10 nAChRs gating in response to local and transient changes of extracellular Ca²⁺ concentration. This finding might pave the way for the future rational design of drugs that target α9α10 nAChRs as otoprotectants.