Spatial Interaction of the L‐type Calcium Channel with α 5 β 1 Integrin

α 5 β 1 integrins play essential roles in modulating neuronal and vascular smooth muscle cell function by altering Ca 2+ entry through the L‐type calcium channel (Ca L ). We previously demonstrated that an increase in Ca 2+ current following α 5 β 1 integrin activation is mediated by PKA and Src phosphorylation of Ca L . Here, the spatial interaction of Ca L with α 5 β 1 integrin was examined using immunofluorescence (IF) confocal microscopy and co‐immunoprecipitation (IP). HEK293 cells expressing neuronal Ca L were plated on an extracellular matrix substrate prior to IF or IP. Co‐association of Ca L with β 1 integrin was only detectable by IP in cells plated on fibronectin (FN), not on poly‐l‐lysine. By IF, about 60% of wild type (WT) Ca L co‐localized with β 1 integrin on FN, using paxillin‐vinculin colocalization as a reference. Marginal decreases (~90% of WT) in the degree of Ca L colocalization with β 1 integrin were seen for Ca L constructs missing the proline‐rich domain (PRD) or the last 281 amino acid residues (Stop5). By IP, the degree of Ca L co‐association with β 1 integrin on FN was decreased to 59% and 79% of WT in the PRD and the Stop5 mutants, respectively. Significantly less co‐association (12% of WT) of Ca L with β 1 integrin was observed in a Ca L construct with altered phosphorylation sites for both PKA and Src. In patch clamp studies, α 5 β 1 integrin activation increased Ca L current by 107% in WT, 10% in the Stop5 mutant, and by 88% in the PRD mutant. Overall, our data suggest that the co‐association of Ca L with β 1 integrin depends on the activation of β 1 integrin by FN and subsequent phosphorylation of Ca L by PKA