Protein-protein interaction between cPLA2 and splice variants of -subunit of BK channels

Li J, Al-Khalili O, Ramosevac S, Eaton DC, Denson DD. Proteinprotein interaction between cPLA2 and splice variants of -subunit of BK channels. Am J Physiol Cell Physiol 298: C251–C262, 2010. First published November 25, 2009; doi:10.1152/ajpcell.00221.2009.—Altering the splice variant composition of large-conductance Ca -activated potassium (BK) channels can alter their activity and apparent sensitivity to Ca and other regulators of activity. We hypothesized that differences in the responsiveness to arachidonic acid of GH3 and GH4 cells was due to a difference in two splice variants, one present in GH3 cells and the other in GH4 cells. The sequences of the two splice variants differ from one another in several ways, but the largest difference is the presence or absence of 27 amino acids in the COOH terminus of the BK -subunit. Open probability of the variant containing the 27 amino acids is significantly increased by arachidonic acid, while the variant lacking the 27 amino acids is insensitive to arachidonic acid. In addition, sensitivity of BK channels to arachidonic acid depends on cytosolic phospholipase A2 (cPLA2). Here we used the Mammalian Matchmaker two-hybrid assay and two BK -subunit constructs with [rSlo(27)] and without [rSlo(0)] the 27amino acid motif to determine whether cPLA2 associates with one construct [rSlo(27)] and not the other. We hypothesized that differential association of cPLA2 might explain the differing responsiveness of the two constructs and GH3 and GH4 cells to arachidonic acid. We found that cPLA2 is strongly associated with the COOH terminus of rSlo(27) and only very weakly associated with rSlo(0). We also found that arachidonic acid has a lower affinity for rSlo(0) than for rSlo(27). We conclude that the lack of response of BK channels in GH4 cells to arachidonic acid can be explained, in part, by the poor binding of cPLA2 to the COOH terminus of the rSlo(0) -subunit, which is very similar to the splice variant found in the arachidonic acid-insensitive GH4 cells.