title: Differential Cav2.1 and Cav2.3 channel inhibition by baclofen and α-conotoxin Vc1.1 via GABAB receptor activation.
abstract: 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 Ba(2+) 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.
authors.affiliation: Health Innovations Research Institute, RMIT University, Melbourne, Victoria 3083, Australia.
authors.forename: Géza
authors.initials: G
authors.lastname: Berecki
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: McArthur
authors.forename: Hartmut
authors.initials: H
authors.lastname: Cuny
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Clark
authors.forename: David J
authors.initials: DJ
authors.lastname: Adams
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001418:Baclofen / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000096:biosynthesis* / Q000235:genetics; D020908:Calcium Channels, R-Type / Q000096:biosynthesis* / Q000235:genetics; D027682:Cation Transport Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000235:genetics; D020916:Conotoxins / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D058786:GABA-B Receptor Agonists / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D051381:Rats; D018080:Receptors, GABA-B / Q000378:metabolism
chemical_list: D001418:Baclofen; D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D020908:Calcium Channels, R-Type; D027682:Cation Transport Proteins; D020916:Conotoxins; D058786:GABA-B Receptor Agonists; D018080:Receptors, GABA-B; C423009:CACNA1E protein, human; C507383:alpha-conotoxin Vc1.1