Phosphorylation sites on calcium channel α1 and β subunits regulate ERK-dependent modulation of neuronal N-type calcium channels
Martin, S. and Butcher, A. and Berrow, N. and Richards, M.W. and Paddon, R. and Turner, D. and Doplhin, A. and Sihra, T. and Fitzgerald, E. (2006) Phosphorylation sites on calcium channel α1 and β subunits regulate ERK-dependent modulation of neuronal N-type calcium channels. Cell Calcium 39 (3), pp. 275-292. ISSN 0143-4160.
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 (Cav2.2) calcium channels and all four neuronal calcium channel β subunits (Cavβ), suggests that Cav2.2 and/or Cavβs may be ERK-phosphorylated. Here we report that GST-Cav2.2 I–II loop, and to a lesser extent Cavβ1b-His6, are substrates for ERK1/2 phosphorylation. Serine to alanine mutation of Ser-409 and/or Ser-447 on GST-Cav2.2 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 Cav2.2,β1b,α2δ1 versus mutant Cav2.2(S447A) or Cav2.2(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 Cavβ1b did not significantly reduce phosphorylation but did reduce UO126-induced current inhibition. Crucially, co-expression of Cav2.2(S447A) with Cavβ1b(S161,348A) had an additive effect, abolishing the action of UO126 on channel current, an effect not seen when Cavβ1b(S161,348A) was co-expressed with Cav2.2(S409A). Thus, Ser-447 on Cav2.2 and Ser-161 and Ser-348 of Cavβ1b 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 Cav2.2α1 and to a lesser extent possibly also Cavβ subunits.
|Keyword(s) / Subject(s):||Voltage-dependent calcium channel, Ras, Mitogen-activated protein kinase, small G protein|
|School:||Birkbeck Schools and Departments > School of Science > Biological Sciences|
|Date Deposited:||18 Aug 2011 10:30|
|Last Modified:||17 Apr 2013 12:21|
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