Molecular mechanisms of large-conductance ca (2+) -activated potassium channel activation by ginseng gintonin

Gintonin is a unique lysophosphatidic acid (LPA) receptor ligand found in Panax ginseng. Gintonin induces transient [Ca(2+)]i through G protein-coupled LPA receptors. Large-conductance Ca(2+)-activated K(+) (BKCa) channels are expressed in blood vessels and neurons and play important roles in blood vessel relaxation and attenuation of neuronal excitability. BKCa channels are activated by transient [Ca(2+)]i and are regulated by various Ca(2+)-dependent kinases. We investigated the molecular mechanisms of BKCa channel activation by gintonin. BKCa channels are heterologously expressed in Xenopus oocytes. Gintonin treatment induced BKCa channel activation in oocytes expressing the BKCa channel α subunit in a concentration-dependent manner (EC50 = 0.71 ± 0.08 µg/mL). Gintonin-mediated BKCa channel activation was blocked by a PKC inhibitor, calphostin, and by the calmodulin inhibitor, calmidazolium. Site-directed mutations in BKCa channels targeting CaM kinase II or PKC phosphorylation sites but not PKA phosphorylation sites attenuated gintonin action. Mutations in the Ca(2+) bowl and the regulator of K(+) conductance (RCK) site also blocked gintonin action. These results indicate that gintonin-mediated BKCa channel activations are achieved through LPA1 receptor-phospholipase C-IP3-Ca(2+)-PKC-calmodulin-CaM kinase II pathways and calcium binding to the Ca(2+) bowl and RCK domain. Gintonin could be a novel contributor against blood vessel constriction and over-excitation of neurons.