Alternative splicing controls G protein–dependent inhibition of N-type calcium channels in nociceptors

Abstract

Neurotransmitter release from mammalian sensory neurons is controlled by Ca(V)2.2 N-type calcium channels. N-type channels are a major target of neurotransmitters and drugs that inhibit calcium entry, transmitter release and nociception through their specific G protein-coupled receptors. G protein-coupled receptor inhibition of these channels is typically voltage-dependent and mediated by Gbetagamma, whereas N-type channels in sensory neurons are sensitive to a second G protein-coupled receptor pathway that inhibits the channel independent of voltage. Here we show that preferential inclusion in nociceptors of exon 37a in rat Cacna1b (encoding Ca(V)2.2) creates, de novo, a C-terminal module that mediates voltage-independent inhibition. This inhibitory pathway requires tyrosine kinase activation but not Gbetagamma. A tyrosine encoded within exon 37a constitutes a critical part of a molecular switch controlling N-type current density and G protein-mediated voltage-independent inhibition. Our data define the molecular origins of voltage-independent inhibition of N-type channels in the pain pathway.

GABAB receptors inhibit low-voltage activated and high-voltage activated Ca2+ channels in sensory neurons via distinct mechanisms

“…GABAB, similar to other Gi/o-coupled receptors, inhibit HVA currents using a 'fast' pathway that involves direct interaction of G protein with the channels [34,35,36,37,38] resulting in a conformational change of the channels complex that makes it harder to open by voltage [39,40]. There are also number of other pathways by which GPCR can inhibit VGCC, including phosphatidylinositol 4,5-bisphosphate (PIP2 depletion) [41,42], arachidonic acid [43] and Src kinase [15]. Inhibition of T-type channels by GABAB receptors in DRG neurons has been reported [44] but the mechanism of inhibition (and particularly if it is the same as for the inhibition of HVA currents) has not been investigated.…”

“…Therefore, we investigated effects of GABAB agonist baclofen on voltage-gated Ca 2+ currents in cultured small-diameter DRG neurons. DRG neurons express variety of VGCC subtypes, including high-voltage activated (HVA) N, P/Q and L-type channels [14,15,16,17,18] as well as low-voltage activated (LVA) T-type Ca 2+ channels [19,20,21].…”

G protein inhibition of CaV2 calcium channels

“…Voltage-independent inhibition is generally less well characterized and likely comprised of several distinct mechanisms including channel trafficking, phosphorylation and lipid signaling pathways (reviewed in ref. 34,35,40,[43][44][45][46][47][48]. The primary focus of this review is the direct, voltage-dependent inhibition of Ca V 2 channels.…”

“…107,108 Splice variants of the Ca V 2.2 C-terminus have also been demonstrated to introduce a novel regulatory site for tyrosine kinases that underlies one form of voltage-independent inhibition. 44 Studies investigating the role of the I-II linker in voltagedependent inhibition have at times come to somewhat differing conclusions about the relative importance of this region (reviewed in refs. 38 and 39).…”

Alternative Splicing Matters: N-Type Calcium Channels in Nociceptors

“…The inclusion of e37a creates a module that couples the N-type channel to a powerful form of G protein-dependent inhibition. 4 The inhibitory pathway that works through e37a is voltage-independent, requires G i/o and tyrosine kinase activation, and is used by mu opioid and GABA B receptors to downregulate N-type channel activity. Combined with our previous studies that show enrichment of e37a in nociceptors, 5 our data suggest a molecular basis for the high susceptibility of N-type currents in sensory neurons to voltage-independent inhibition following G protein activation.…”