Differential CaV2.1 and CaV2.3 Channel Inhibition by Baclofen and α-Conotoxin Vc1.1 via GABAB Receptor Activation

Abstract

In the nervous system, Cav2.1 (P/Q-type), Cav2.2 (N-type) and Cav2.3 (R-type) calcium channels contribute to synaptic transmission and are modulated via G protein-coupled receptor pathways. The analgesic α-conotoxin Vc1.1 has been identified as a selective inhibitor of Cav2.2 channels acting via GABAB receptors. We investigated baclofen and Vc1.1 modulation of human Cav2.1 or Cav2.3 channels via human GABAB receptor activation in HEK cells. Baclofen (50 µM) inhibited Cav2.1 and Cav2.3 channel Ba2+ currents by ∼ 40%, whereas Vc1.1 did not affect Cav2.1, but potently inhibited Cav2.3, with a half-maximal inhibitory concentration of ∼ 300 pM. Depolarizing paired-pulses revealed that ∼ 75% of the baclofen inhibition of Cav2.1 was voltage-dependent, and could be relieved by strong depolarization. In contrast, baclofen or Vc1.1 inhibition of Cav2.3 channels was solely mediated via voltage-independent pathways that could be disrupted by pertussis toxin, GDP-β-S or the GABAB receptor antagonist, CGP55845. Over-expression of c-Src kinase significantly increased inhibition of Cav2.3 by Vc1.1. Conversely, co-expression of a double mutant c-Src or pre-treatment with a phosphorylated pp60c-Src peptide abolished the effect of Vc1.1. Site-directed mutational analyses of Cav2.3 demonstrated that tyrosines 1761 and 1765 within exon 37 are critical for mediating the inhibition by Vc1.1 and are involved in baclofen inhibition of these channels. Remarkably, point mutations introducing specific c-Src phosphorylation sites into human Cav2.1 channels conveyed Vc1.1 sensitivity. Our findings demonstrate that Vc1.1 inhibition of Cav2.3 is due to specific c-Src phosphorylation sites in the C-teminus, which defines Cav2.3 channels as potential targets for analgesic α-conotoxins.