RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells

Abstract

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 CaMlike 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.

Excess extracellular K+ causes inner hair cell ribbon synapse degeneration

“…4 and 5 ). High-K + could depolarize IHCs to open Ca ++ channels 31 – 33 . Co ++ may block Ca ++ channels and consequently Ca ++ -dependent BK channels in IHCs.…”

“…In the IHC, Ca ++ can influx through Cav1.3 channels. High-K + depolarizes the IHC and can open Cav1.3 channels to influx Ca ++ 29 , 31 – 33 . Co ++ could block Ca ++ channels and eliminate such Ca ++ influx, therefore attenuating ribbon swelling (Figs.…”

Excess extracellular K+causes inner hair cell ribbon synapse degeneration

“…In this study, application of Co ++ attenuated high-K + induced ribbon degeneration (Figures 4 and 5). High-K + could depolarize IHCs to open Ca ++ channels (31)(32)(33). Co ++ may block Ca ++ channels and consequently Ca ++ -dependent BK channels in IHCs.…”

“…In the IHC, Ca ++ can influx through Cav1.3 channels. High-K + depolarizes the IHC and can open Cav1.3 channels to influx Ca ++ (29,(31)(32)(33). Co ++ could block Ca ++ channels and eliminate such Ca ++ influx, therefore attenuating ribbon swelling (Figures 4 and 5).…”

RIM2α/RBP2/β-subunit co-expression stabilizes slow Cav1.3 channel inactivation to improve auditory perception

“…In this issue, the Joerg Striessnig group (Ortner et al, 2020) shows with impressive details that co-expression of RBP2 together with RIM2α in heterologous tsA-201 cells dramatically slows the VDI of the Cav1.3L/β3 channel complex, mimicking the ultra-slow Ca 2+ current inactivation kinetics existing in IHCs [10]. Figure 1 (left) summarizes how VDI inactivation of Cav1.3L currents become progressively slower by co-expressing β3 or β2a with RIM2a and RBP2a to mimic closely the slow VDI in adult mouse IHC.…”