Phosphorylation sites on calcium channel α1 and β subunits regulate ERK-dependent modulation of neuronal N-type calcium channels

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.