Co-localization of the alpha-subunit of BK-channels and c-PLA2 in GH3 cells.
Large conductance, calcium-activated potassium channels (maxi K- or BK-channels) can be regulated by arachidonic acid produced by c-Phospholipase A2 (c-PLA2). Since in every excised patch from GH3 cells where there was BK-channel activity, treatment with either a stimulator or inhibitor of c-PLA2 resulted in a corresponding increase or decrease in BK-channel activity, we hypothesized that there must be a close association between BK-channel proteins and c-PLA2 in the cell membrane. To test this hypothesis, we first determined whether the two proteins would co-immunoprecipitate. We then used confocal imaging of fluorescently tagged proteins to determine where in the cells BK-channel proteins and c-PLA2 co-localize. The alpha-subunit of the BK-channel was strongly co-immunoprecipitated by c-PLA2 antibodies, suggesting that most of the BK channel alpha-subunits are associated with c-PLA2. This interaction was not affected by pharmacologically inhibiting c-PLA2 suggesting that the association does not require functionally active c-PLA2. Following dual immunohistochemical labeling and confocal microscopy, image analysis revealed that in the cytosol there was some co-localization, but most of the c-PLA2 was separate from BK-channel proteins. On the other hand, the c-PLA2 and BK-channel proteins at the plasma membrane were strongly co-localized. Immunoprecipitation experiments conducted with plasma membrane proteins support these findings. We conclude that c-PLA2 is likely physically associated with BK-channel proteins at the cell surface.
Denson
D D
DD
Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA 30322, USA. Don_Denson@emoryhealthcare.org
Li
Juan
J
Eaton
D C
DC
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D007150:Immunohistochemistry; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport; D051381:Rats
D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D021122:Protein Subunits; D000001:Calcimycin; D010741:Phospholipases A; D054467:Phospholipases A2