FIGURE 6.

Ca_v2.1 and Ca_v2.2 DII/DIII motif associates with ankyrin-B. A and B, location of putative ABM in the DII/DIII cytoplasmic loop of Ca_v2.1 and Ca_v2.2 is shown. C and D, the putative ABM in Ca_v2.1 and Ca_v2.2 is conserved across species and harbors a central conserved tyrosine (bolded). E and F, three constructs were designed to evaluate the role of this amino acid sequence in ankyrin-B binding. These constructs include the entire DII/III loop, a portion of the loop that begins just prior to the putative ABM, and a portion of the loop that begins just after the putative ABM. G and H, in vitro binding assays demonstrate that, whereas the full-length DII/III loops of Ca_v2.1 and Ca_v2.2 and the smaller loop portion that contains the putative ABM are able to associate with GST-ankyrin-B MBD, the smaller loop portion that lacks this domain demonstrates no binding with GST-ankyrin-B MBD. I and J, Coomassie Blue staining demonstrating that equal loading of GST constructs was performed prior to imaging (denoted by asterisks).