Phosphorylation of the CaV3.1 channel in vitro at Serine 2234.

A) Amino acid sequence alignment of a small region within the CaV3.1 channel C-terminus showing a consensus Cdk5 phosphorylation site conserved among three species. The box marks the sequence that matches the Cdk5 consensus site with the phosphorylatable serine at position 2234. Direct DNA sequencing of PCR products confirmed that the construct generated by site-directed mutagenesis of the Cdk5 consensus phosphorylation site contained the mutant sequence. B) For in vitro Cdk5-mediated phosphorylation assays GST-CaV3.1 fusion wild-type (WT) and S2234A mutant proteins were purified on glutathione agarose beads, fractionated by SDS-PAGE and stained with Coomassie blue. C) Purified recombinant CaV3.1 wild-type and S2234A constructs (amino acid residues 2154 to 2254) were subjected to phosphorylation by purified Cdk5/p25 complex in vitro. D) Comparison of the relative in vitro phosphorylation levels GST-CaV3.1 fusion WT and S2234A mutant proteins (n = 3).