RBP2 stabilizes slow Cav1.3 Ca(2+) channel inactivation properties of cochlear inner hair cells

Cav1.3 L-type Ca(2+) channels (LTCCs) in cochlear inner hair cells (IHCs) are essential for hearing as they convert sound-induced graded receptor potentials into tonic postsynaptic glutamate release. To enable fast and indefatigable presynaptic Ca(2+) signaling, IHC Cav1.3 channels exhibit a negative activation voltage range and uniquely slow inactivation kinetics. Interaction with CaM-like Ca(2+)-binding proteins inhibits Ca(2+)-dependent inactivation, while the mechanisms underlying slow voltage-dependent inactivation (VDI) are not completely understood. Here we studied if the complex formation of Cav1.3 LTCCs with the presynaptic active zone proteins RIM2α and RIM-binding protein 2 (RBP2) can stabilize slow VDI. We detected both RIM2α and RBP isoforms in adult mouse IHCs, where they co-localized with Cav1.3 and synaptic ribbons. Using whole-cell patch-clamp recordings (tsA-201 cells), we assessed their effect on the VDI of the C-terminal full-length Cav1.3 (Cav1.3(L)) and a short splice variant (Cav1.3(42A)) that lacks the C-terminal RBP2 interaction site. When co-expressed with the auxiliary β3 subunit, RIM2α alone (Cav1.3(42A)) or RIM2α/RBP2 (Cav1.3(L)) reduced Cav1.3 VDI to a similar extent as observed in IHCs. Membrane-anchored β2 variants (β2a, β2e) that inhibit inactivation on their own allowed no further modulation of inactivation kinetics by RIM2α/RBP2. Moreover, association with RIM2α and/or RBP2 consolidated the negative Cav1.3 voltage operating range by shifting the channel’s activation threshold toward more hyperpolarized potentials. Taken together, the association with “slow” β subunits (β2a, β2e) or presynaptic scaffolding proteins such as RIM2α and RBP2 stabilizes physiological gating properties of IHC Cav1.3 LTCCs in a splice variant-dependent manner ensuring proper IHC function. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00424-019-02338-4) contains supplementary material, which is available to authorized users.