title: Phosphorylation sites on calcium channel alpha1 and beta subunits regulate ERK-dependent modulation of neuronal N-type calcium channels.
abstract: Voltage-dependent calcium channels (VDCCs) in sensory neurones are tonically up-regulated via Ras/extracellular signal regulated kinase (ERK) signalling. The presence of putative ERK consensus sites within the intracellular loop linking domains I and II of neuronal N-type (Ca(v)2.2) calcium channels and all four neuronal calcium channel beta subunits (Ca(v)beta), suggests that Ca(v)2.2 and/or Ca(v)betas may be ERK-phosphorylated. Here we report that GST-Ca(v)2.2 I-II loop, and to a lesser extent Ca(v)beta1b-His(6), are substrates for ERK1/2 phosphorylation. Serine to alanine mutation of Ser-409 and/or Ser-447 on GST-Ca(v)2.2 I-II loop significantly reduced phosphorylation. Loss of Ser-447 reduced phosphorylation to a greater extent than mutation of Ser-409. Patch-clamp recordings from wild-type Ca(v)2.2,beta1b,alpha2delta1 versus mutant Ca(v)2.2(S447A) or Ca(v)2.2(S409A) channels revealed that mutation of either site significantly reduced current inhibition by UO126, a MEK (ERK kinase)-specific inhibitor that down-regulates ERK activity. However, no additive effect was observed by mutating both residues together, suggesting some functional redundancy between these sites. Mutation of both Ser-161 and Ser-348 on Ca(v)beta1b did not significantly reduce phosphorylation but did reduce UO126-induced current inhibition. Crucially, co-expression of Ca(v)2.2(S447A) with Ca(v)beta1b(S161,348A) had an additive effect, abolishing the action of UO126 on channel current, an effect not seen when Ca(v)beta1b(S161,348A) was co-expressed with Ca(v)2.2(S409A). Thus, Ser-447 on Ca(v)2.2 and Ser-161 and Ser-348 of Ca(v)beta1b appear to be both necessary and sufficient for ERK-dependent modulation of these channels. Together, our data strongly suggest that modulation of neuronal N-type VDCCs by ERK involves phosphorylation of Ca(v)2.2alpha1 and to a lesser extent possibly also Ca(v)beta subunits.
authors.affiliation: Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK.
authors.forename: S W
authors.initials: SW
authors.lastname: Martin
authors.forename: A J
authors.initials: AJ
authors.lastname: Butcher
authors.forename: N S
authors.initials: NS
authors.lastname: Berrow
authors.forename: M W
authors.initials: MW
authors.lastname: Richards
authors.forename: R E
authors.initials: RE
authors.lastname: Paddon
authors.forename: D J
authors.initials: DJ
authors.lastname: Turner
authors.forename: A C
authors.initials: AC
authors.lastname: Dolphin
authors.forename: T S
authors.initials: TS
authors.lastname: Sihra
authors.forename: E M
authors.initials: EM
authors.lastname: Fitzgerald
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002070:Butadienes / Q000494:pharmacology; D019556:COS Cells; D020746:Calcium Channels, L-Type / Q000502:physiology; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000502:physiology*; D002522:Chlorocebus aethiops; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D017354:Point Mutation; D051381:Rats; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
chemical_list: D002070:Butadienes; C503279:Cacna1b protein, rat; D020746:Calcium Channels, L-Type; D020864:Calcium Channels, N-Type; C419385:L-type calcium channel alpha(1C); D009570:Nitriles; C113580:U 0126; D012694:Serine; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases