Phosphorylation sites on calcium channel alpha 1 and beta 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 (Ca sub(v)2.2) calcium channels and all four neuronal calcium channel beta subunits (Ca sub(v) beta ), suggests that Ca sub(v)2.2 and/or Ca sub(v) beta s may be ERK-phosphorylated. Here we report that GST-Ca sub(v)2.2 I-II loop, and to a lesser extent Ca sub(v) beta 1b-His sub(6), are substrates for ERK1/2 phosphorylation. Serine to alanine mutation of Ser-409 and/or Ser-447 on GST-Ca sub(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 sub(v)2.2, beta 1b, alpha 2 delta 1 versus mutant Ca sub(v)2.2(S447A) or Ca sub(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 sub(v) beta 1b did not significantly reduce phosphorylation but did reduce UO126-induced current inhibition. Crucially, co-expression of Ca sub(v)2.2(S447A) with Ca sub(v) beta 1b(S161,348A) had an additive effect, abolishing the action of UO126 on channel current, an effect not seen when Ca sub(v) beta 1b(S161,348A) was co-expressed with Ca sub(v)2.2(S409A). Thus, Ser-447 on Ca sub(v)2.2 and Ser-161 and Ser-348 of Ca sub(v) beta 1b 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 sub(v)2.2 alpha 1 and to a lesser extent possibly also Ca sub(v) beta subunits.