Modulation of Ca_v1.3 Ca²⁺ channel gating by Rab3 interacting molecule

Abstract

Neurotransmitter release and spontaneous action potentials during cochlear inner hair cell (IHC) development depend on the activity of Ca_v1.3 voltage-gated L-type Ca²⁺ channels. Their voltage- and Ca²⁺-dependent inactivation kinetics are slower than in other tissues but the underlying molecular mechanisms are not yet understood. We found that Rab3-interacting molecule-2α (RIM2α) mRNA is expressed in immature cochlear IHCs and the protein co-localizes with Ca_v1.3 in the same presynaptic compartment of IHCs. Expression of RIM proteins in tsA-201 cells revealed binding to the β-subunit of the channel complex and RIM-induced slowing of both Ca²⁺- and voltage-dependent inactivation of Ca_v1.3 channels. By inhibiting inactivation, RIM induced a non-inactivating current component typical for IHC Ca_v1.3 currents which should allow these channels to carry a substantial window current during prolonged depolarizations. These data suggest that RIM2 contributes to the stabilization of Ca_v1.3 gating kinetics in immature IHCs.

Introduction

Depolarization-induced Ca²⁺ entry through voltage-gated Ca²⁺ channels (VGCC) into electrically excitable cells is a key process regulating numerous physiological processes. Ten Ca²⁺ channel isoforms within three classes (Ca_v1–3) with different biophysical properties and subcellular localizations (Catterall et al., 2005) accomplish these diverse functions. Among isoforms gating is further fine-tuned by alternative splicing (Lipscombe and Raingo, 2007, Singh et al., 2008), accessory α2-δ and β-subunits (Davies et al., 2007, Dolphin, 2003) as well as by other channel associated proteins (Calin-Jageman and Lee, 2008, Dai et al., 2009). Among the high voltage activated Ca²⁺ channels Ca_v2 channels predominantly control presynaptic neurotransmitter release in neurons whereas postsynaptic Ca²⁺ influx through Ca_v1 (L-type) Ca²⁺ channels (LTCCs) modifies gene transcription and synaptic plasticity (Gomez-Ospina et al., 2006, Zhang et al., 2006). However, presynaptic neurotransmitter release at ribbon synapses from sensory cells of retinal photoreceptors and the cochlea is under the control of Ca_v1 rather than Ca_v2 channels. Tonic neurotransmitter release in response to light- or sound-evoked graded changes in membrane potential between −60 and −40 mV requires unusually slow inactivation of L-type Ca²⁺ currents in photoreceptors (Rabl and Thoreson, 2002) and cochlear inner hair cells (Grant and Fuchs, 2008, Johnson and Marcotti, 2008, Lee et al., 2007) and maintenance of window currents over prolonged time periods (McRory et al., 2004). Slow inactivation in IHCs is also a prerequisite to produce Ca²⁺ signals and spontaneous action potentials during IHC development (Marcotti et al., 2003).

In VGCCs inactivation during depolarizations is driven by the Ca²⁺ concentration sensed at the inner channel mouth (Ca²⁺-dependent inactivation, CDI) and by transmembrane voltage (voltage-dependent inactivation, VDI). To prevent efficient inactivation by these processes, LTCCs in photoreceptors developed special strategies. Photoreceptor L-type currents are largely carried by Ca_v1.4 LTCCs which auto-inhibit their own calmodulin (CaM)-dependent CDI by an intramolecular protein interaction within their C-terminus and their remaining VDI is intrinsically slow (Singh et al., 2006). Ca_v1.3 channels in the heart and brain display pronounced CDI and fast VDI (Koschak et al., 2001, Mangoni et al., 2003, Yang et al., 2006) but both processes are very slow in Ca_v1.3 channels of cochlear inner hair cells (Grant and Fuchs, 2008, Johnson and Marcotti, 2008, Marcotti et al., 2003). Strongly reduced CDI in IHCs can be explained by Ca²⁺ binding proteins, such as CaBP1 and CaBP4 (Cui et al., 2007, Lee et al., 2007, Striessnig, 2007, Yang et al., 2006), which compete with CaM binding and Ca²⁺ sensing to the Ca_v1.3 α1 subunit. Ca_vβ2 was recently shown to slightly affect CDI in Ca_vβ2 deficient IHCs, however VDI remained unaltered (Neef et al., 2009). Even if CDI is completely inhibited by these proteins, the remaining VDI of Ca_v1.3 currents in IHCs is still much slower than the VDI of Ca_v1.3 currents in heart, brain or heterologously expressed Ca_v1.3 channel complexes (Koschak et al., 2001, Mangoni et al., 2003, Platzer et al., 2000). So far the molecular basis for this physiologically relevant difference is unclear. The underlying mechanisms could be the expression of Ca_v1.3 α1 subunit splice variants with slow VDI in IHCs, stabilization of slow VDI by accessory subunits or by proteins associated with the presynaptic signaling complex at ribbon synapses. So far, no splice variants slowing the VDI have been reported in IHCs or in other adult tissues (Klugbauer et al., 2002) and none of the accessory subunits, not even β2a can explain this effect (Neef et al., 2009). Some modest reduction of VDI has been observed for CaBP1 (Cui et al., 2007) and syntaxin (Song et al., 2003) when co-expressed with the channel complex in HEK-293 cells but neither of them can account for the slow inactivation time constants observed in IHCs.

Recently the presynaptic Rab3 interacting molecule (RIM; Wang et al., 1997), a scaffold protein at the presynaptic active zone involved in Ca²⁺-induced neurotransmitter release in neurons (Sudhof, 2004, Schoch et al., 2006) has been identified as a Ca²⁺ channel modulator. It markedly suppressed the inactivation time course of presynaptic Ca_v2 channels and shifted the voltage-dependence of inactivation to more depolarized voltages (Kiyonaka et al., 2007). The modulatory effects of RIM are mediated via its tight binding to the β-subunit of the Ca²⁺ channel complexes. Due to its presynaptic location RIM effects were extensively studied on presynaptically localized Ca_v2 channels. Considering that Ca_v1.3 also represents a presynaptic VGCC in IHCs, RIM may be co-localized with LTCCs in the active zone of IHCs. In the present study we therefore investigated if RIM proteins are expressed in IHCs, if they are capable of modulating Ca_v1.3 function and to which extent they could contribute to the slow VDI and CDI of Ca_v1.3 currents in cochlear IHCs.