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.

In dopaminergic (DA) substantia nigra (SN) neurons Cav2.3 R-type Ca2+-currents contribute to somatodendritic Ca2+-oscillations. This activity may contribute to the selective degeneration of these neurons in Parkinson's disease (PD) since Cav2.3-knockout is neuroprotective in a PD mouse model. Here we show that in tsA-201-cells the membrane-anchored β2-splice variants β2a and β2e are required to stabilize Cav2.3 gating properties allowing sustained Cav2.3 availability during simulated pacemaking and enhanced Ca2+-currents during bursts. We confirmed the expression of β2a- and β2e-subunit transcripts in the mouse SN and in identified SN DA neurons. Patch-clamp recordings of mouse DA midbrain neurons in culture and SN DA neurons in brain slices revealed SNX-482-sensitive R-type Ca2+-currents with voltage-dependent gating properties that suggest modulation by β2a- and/or β2e-subunits. Thus, β-subunit alternative splicing may prevent a fraction of Cav2.3 channels from inactivation in continuously active, highly vulnerable SN DA neurons, thereby also supporting Ca2+ signals contributing to the (patho)physiological role of Cav2.3 channels in PD.

and different types of learning and memory. Recently, large-scale genetic analysis revealed de-novo missense mutations in their pore-forming a 1 -subunit (CACNA1D gene) in 6 patients associated with a neurodevelopmental syndrome including varying degrees of sporadic autism spectrum disorder (ASD, G407R), intellectual disability (A749G), neurological manifestations (including seizures, V401L) and endocrine symptoms (G403D, I750M). A typical hallmark of these mutations are severe gating changes compatible with a gain-of-channel-function. Here we investigated if similar gating changes are observed in a de-novo CACNA1D mutation (IIS4-S5 linker, Ca v 1.3 a 1mut ) which could explain symptoms in a patient diagnosed with a severe developmental disorder of unknown cause. Methods: Mutant (Ca v 1.3 a 1mut ) and wild-type Ca v 1.3 a 1 were co-expressed together with b 3 and a 2 d-1 subunits in tsA-201 cells and calcium currents (15mM) were measured using the whole cell patch-clamp technique. Results: Very similar to the previously characterized mutation V401L (IS6), A749G and I750M (IIS6), Ca v 1.3 a 1mut dramatically shifted the voltagedependence of Ca v 1.3 steady-state activation and inactivation to more negative voltages ($20 mV) without slowing of inactivation. A complete biophysical analysis revealed that these changes are compatible with a mutational gainof-function phenotype. Conclusion: By demonstrating the typical gating changes previously shown by us for CACNA1D de-novo missense mutations we propose that Ca v 1.3 a 1mut also explains the symptoms in this patient with a severe developmental disorder. Patients carrying such mutations may benefit from treatment with already available L-type Ca 2þ -channel blockers, such as nimodipine. Such CACNA1D missense mutations are likely underreported in large-scale genetic analyses. Support: Austrian Science Fund (FWF F4402, W1101).

In dopaminergic (DA) substantia nigra (SN) neurons Cav2.3 R-type Ca2+-currents contribute to somatodendritic Ca2+-oscillations. These may contribute to the selective degeneration of these neurons in Parkinsons disease (PD) since Cav2.3-knockout is neuroprotective in a PD mouse model. However, the typical Cav2.3 gating would predict complete channel inactivation during SN DA neuronal firing. Here we show that in tsA-201-cells the membrane-anchored β2-splice variants β2a and β2e stabilize Cav2.3 gating properties allowing sustained Cav2.3 availability during simulated pacemaking and enhanced Ca2+-currents during bursts. We confirmed the expression of β2a and β2e-subunits in the SN and identified SN DA neurons. Patch-clamp recordings of SN DA neurons in mouse brain slices revealed R-type Ca2+-currents similar to β2a- or β2e-stabilized Cav2.3-currents and recordings in cultured murine DA neurons confirmed their activity during pacemaking. Taken together, our data support an important (patho)physiological role of β-subunit alternative splicing for Cav2.3 Ca2+-signaling in highly vulnerable SN DA neurons.

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