Site of action of fatty acids and other charged lipids on BKCa channels from arterial smooth muscle cells

Fattyacids and other negatively charged single-chain lipids increaselarge-conductance Ca²⁺-activated K⁺(BK_Ca) channel activity, whereas sphingosine and otherpositively charged single-chain lipids suppress activity. Because thesemolecules are effective on both inside-out and outside-out patches andbecause they can flip across the bilayer, the location of their site of action is unclear. To identify the site of action of charged lipids onthis channel, we used two compounds that are unlikely to flip acrossthe lipid bilayer. Palmitoyl coenzyme A (PCoA) was used to identify thesite of action of negatively charged lipids, and a positively chargedmyristoylated pentapeptide (myr-KPRPK) was used to investigate the siteof action of positively charged lipids. The effect of these compoundson channel activity was studied in excised patches using patch-clamptechniques. In "normal" ionic strength solutions and in experimentswhere high-ionic strength solutions were used to shield membranesurface charge, PCoA increased channel activity only when applied tooutside-out patches, suggesting that the site of action of negativelycharged lipids is located on the outer surface of the membrane. Adecrease in activity, similar to that of other positively chargedlipids, was observed only when myr-KPRPK was applied to outside-outpatches, suggesting that positively charged lipids suppress activity byalso acting on the outer membrane surface. Some channel blockadeeffects of myr-KPRPK and KPRPK are also described. The sidedness ofaction suggests that modulation of channel activity by single-chainlipids can occur by their interaction with the channel protein.