Neuronal Cav2.1 (P/Q-type), Cav2.2 (N-type), and Cav2.3 (R-type) calcium channels contribute to synaptic transmission and are modulated through G protein–coupled receptor pathways. The analgesic α-conotoxin Vc1.1 acts through γ-aminobutyric acid type B (GABAB) receptors (GABABRs) to inhibit Cav2.2 channels. We investigated GABABR-mediated modulation by Vc1.1, a cyclized form of Vc1.1 (c-Vc1.1), and the GABABR agonist baclofen of human Cav2.1 or Cav2.3 channels heterologously expressed in human embryonic kidney cells. 50 µM baclofen inhibited Cav2.1 and Cav2.3 channel Ba2+ currents by ∼40%, whereas c-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 through voltage-independent pathways that could be disrupted by pertussis toxin, guanosine 5′-[β-thio]diphosphate trilithium salt, or the GABABR antagonist CGP55845. Overexpression of the kinase c-Src significantly increased inhibition of Cav2.3 by c-Vc1.1. Conversely, coexpression of a catalytically inactive double mutant form of c-Src or pretreatment with a phosphorylated pp60c-Src peptide abolished the effect of c-Vc1.1. Site-directed mutational analyses of Cav2.3 demonstrated that tyrosines 1761 and 1765 within exon 37 are critical for inhibition of Cav2.3 by c-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 conferred c-Vc1.1 sensitivity. Our findings show that Vc1.1 inhibition of Cav2.3, which defines Cav2.3 channels as potential targets for analgesic α-conotoxins, is caused by specific c-Src phosphorylation sites in the C terminus.

Elucidating the mechanisms that modulate calcium channels via opioid receptor activation is fundamental to our understanding of both pain perception and how opioids modulate pain. Neuronal voltage-gated N-type calcium channels (Ca_v2.2) are inhibited by activation of G protein–coupled opioid receptors (ORs). However, inhibition of R-type (Ca_v2.3) channels by μ- or κ-ORs is poorly defined and has not been reported for δ-ORs. To investigate such interactions, we coexpressed human μ-, δ-, or κ-ORs with human Ca_v2.3 or Ca_v2.2 in human embryonic kidney 293 cells and measured depolarization-activated Ba²⁺ currents (I_Ba). Selective agonists of μ-, δ-, and κ-ORs inhibited I_Ba through Ca_v2.3 channels by 35%. Ca_v2.2 channels were inhibited to a similar extent by κ-ORs, but more potently (60%) via μ- and δ-ORs. Antagonists of δ- and κ-ORs potentiated I_Ba amplitude mediated by Ca_v2.3 and Ca_v2.2 channels. Consistent with G protein βγ (Gβγ) interaction, modulation of Ca_v2.2 was primarily voltage-dependent and transiently relieved by depolarizing prepulses. In contrast, Ca_v2.3 modulation was voltage-independent and unaffected by depolarizing prepulses. However, Ca_v2.3 inhibition was sensitive to pertussis toxin and to intracellular application of guanosine 5′-[β-thio]diphosphate trilithium salt and guanosine 5′-[γ-thio]triphosphate tetralithium salt. Coexpression of Gβγ-specific scavengers—namely, the carboxyl terminus of the G protein–coupled receptor kinase 2 or membrane-targeted myristoylated-phosducin—attenuated or abolished Ca_v2.3 modulation. Our study reveals the diversity of OR-mediated signaling at Ca_v2 channels and identifies neuronal Ca_v2.3 channels as potential targets for opioid analgesics. Their novel modulation is dependent on pre-existing OR activity and mediated by membrane-delimited Gβγ subunits in a voltage-independent manner.