Protein-protein interaction between cPLA2 and splice variants of alpha-subunit of BK channels.
Altering the splice variant composition of large-conductance Ca(2+)-activated potassium (BK) channels can alter their activity and apparent sensitivity to Ca(2+) 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 alpha-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 A(2) (cPLA(2)). Here we used the Mammalian Matchmaker two-hybrid assay and two BK alpha-subunit constructs with [rSlo(27)] and without [rSlo(0)] the 27-amino acid motif to determine whether cPLA(2) associates with one construct [rSlo(27)] and not the other. We hypothesized that differential association of cPLA(2) might explain the differing responsiveness of the two constructs and GH3 and GH4 cells to arachidonic acid. We found that cPLA(2) 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 cPLA(2) to the COOH terminus of the rSlo(0) alpha-subunit, which is very similar to the splice variant found in the arachidonic acid-insensitive GH4 cells.
Li
Juan
J
Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Al-Khalili
Otor
O
Ramosevac
Semra
S
Eaton
Douglas C
DC
Denson
Donald D
DD
D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D054513:Group IV Phospholipases A2 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D051381:Rats; D014162:Transfection; D020798:Two-Hybrid System Techniques
D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D020033:Protein Isoforms; D016718:Arachidonic Acid; D054513:Group IV Phospholipases A2