C-Terminal Modulation of Cav1.3 L-Type Calcium Channels Modifies their Gating Properties in Cochlear Inner Hair Cells

Abstract

The Ca2+ currents in inner hair cells (IHCs) are crucial for synaptic transmission and flow through voltage-gated calcium channels (VGCCs) formed by the α1 subunit Cav1.3. VGCCs exhibit a calmodulin (CaM) mediated calcium-dependent inactivation (CDI), via binding CAM to the channel's C-terminus. In IHCs, Cav1.3 exhibits unusually weak CDI, probably caused by calcium binding proteins (CaBP) competing with CaM. IHCs express long and short Cav1.3 splice variants either including (long variant, Cav1.3L) or excluding (short variants) a C-terminal modulatory domain. In expression systems - lacking CaBPs - the C-terminal modulatory mechanism (CTM) functions via intramolecular interaction of a proximal (PCRD) and a distal C-terminal regulatory domain (DCRD) by inhibiting CaM binding near the PCRD, thereby inhibiting CaM-mediated CDI (Bock et al., JBC 2011). Here, the role of the CTM for IHC VGCCs was investigated in Cav1.3L-DCRDHA/HA mice in which CTM was disrupted by partial replacement of the DCRD with an HA tag.Localization of HA-tagged Cav1.3 channels in IHCs was determined by immunohistochemistry. Channel properties were investigated by whole-cell patch-clamp recordings. Hearing was assessed using auditory brainstem responses (ABR) and distortion products of otoacoustic emissions (DPOAE).Anti-HA immunolabeling was present at all IHC ribbons. Patch-clamp recordings revealed significantly reduced CDI and increased amplitudes of Ca2+ and Ba2+ currents in Cav1.3L-DCRDHA/HA IHCs. Non-stationary fluctuation analysis showed unchanged numbers of Cav1.3 channels and single channel currents. Voltage dependence and activation kinetics of ICa and IBa, ABR thresholds and DPOAEs were unaffected.Our data demonstrate that the long Cav1.3 isoform is an intrinsic component of Cav1.3 clusters at all IHC ribbon synapses and that its DCRD is required for normal CDI and ICa amplitude.Funding:Austrian Science Fund (SFB-F4402), EC-project MRTN-CT-2006-35367 (“CavNet”), Innsbruck University, DFG (SFB-894)