title: Ankyrin-B regulates Cav2.1 and Cav2.2 channel expression and targeting.
abstract: N-type and P/Q-type calcium channels are documented players in the regulation of synaptic function; however, the mechanisms underlying their expression and cellular targeting are poorly understood. Ankyrin polypeptides are essential for normal integral membrane protein expression in a number of cell types, including neurons, cardiomyocytes, epithelia, secretory cells, and erythrocytes. Ankyrin dysfunction has been linked to defects in integral protein expression, abnormal cellular function, and disease. Here, we demonstrate that ankyrin-B associates with Cav2.1 and Cav2.2 in cortex, cerebellum, and brain stem. Additionally, using in vitro and in vivo techniques, we demonstrate that ankyrin-B, via its membrane-binding domain, associates with a highly conserved motif in the DII/III loop domain of Cav2.1 and Cav2.2. Further, we demonstrate that this domain is necessary for proper targeting of Cav2.1 and Cav2.2 in a heterologous system. Finally, we demonstrate that mutation of a single conserved tyrosine residue in the ankyrin-binding motif of both Cav2.1 (Y797E) and Cav2.2 (Y788E) results in loss of association with ankyrin-B in vitro and in vivo. Collectively, our findings identify an interaction between ankyrin-B and both Cav2.1 and Cav2.2 at the amino acid level that is necessary for proper Cav2.1 and Cav2.2 targeting in vivo.
authors.affiliation: From the Dorothy M. Davis Heart and Lung Research Institute.
authors.forename: Crystal F
authors.initials: CF
authors.lastname: Kline
authors.forename: John
authors.initials: J
authors.lastname: Scott
authors.forename: Jerry
authors.initials: J
authors.lastname: Curran
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Hund
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Mohler
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000378:metabolism*; D001921:Brain; D020864:Calcium Channels, N-Type / Q000737:chemistry / Q000378:metabolism*; D017124:Conserved Sequence; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D011485:Protein Binding; D011689:Purkinje Cells / Q000378:metabolism; D051381:Rats; D014443:Tyrosine / Q000378:metabolism
chemical_list: D017487:Ankyrins; D020864:Calcium Channels, N-Type; D014443:Tyrosine; C498995:Ank2 protein, mouse; C094681:Ank3 protein, mouse; C500963:Cacna1b protein, mouse
keywords: Adaptor Proteins; Cytoskeleton; Ion Channels; Membrane Trafficking; Protein Sorting; Protein Targeting