Abstract

Elucidating the mechanisms that modulate calcium channels via opioid receptor activation is fundamental to our understanding of both pain perception and of how opioids modulate pain. Neuronal voltage-gated N-type (Ca_v2.2) calcium channels 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 co-expressed human μ-, δ- or κ-ORs with human Ca_v2.3 or Ca_v2.2 in human embryonic kidney (HEK293) 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 pre-pulses. In contrast, Ca_v2.3 modulation was voltage-independent and unaffected by depolarizing pre-pulses. However, Ca_v2.3 inhibition was sensitive to pertussis toxin and to intracellular application of GDP-β-S and GTP-γ-S. Co-expression 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.