UP-MODULATION AND DOWN-MODULATION OF A CLONED APLYSIA K-KINASE-C( CHANNEL (AKV1.1A) BY THE ACTIVATORS OF PROTEIN)

Modulation of a cloned Aplysia K+ channel, AKv1.1a, by protein kinase C (PKC) activators was examined in Xenopus oocytes expression system. Following the application of phorbol esters (phorbol 12-myristate 13-a cetate, PMA; phorbol 12,13-dibutyrate, PDBu), or a diacylgrycerol anal ogue (1-oleoyl-2-acetyl-sn-glycerol, GAG), the fast inactivation of th e AKv1.1a became slower and the peak current increased (up-modulation) . However, the effect was transient. The expressed current was decreas ed even below control level about 15 to 20 min after the treatment (do wn-modulation). Both effects by PMA was blocked by the kinase inhibito r, H7, suggesting that phosphorylation by PKC is involved. The amino a cid sequence of AKv1.1a contains three putative phosphorylation sites by PKC (Ser(24), Thr(345), Se-349). We tested their contributions to t he PMA-induced modulation by site-directed mutagenesis. The results su ggest that the up-modulation by PKC activators is due to the inhibitio n of the fast inactivation by the amino-terminal domain (N-type inacti vation), thereby increase the time the channels are conductive. Phosph orylation of Ser(24) may enhance the PKC-induced down-modulation, whil e phosphorylation of Thr(345) may inhibit the downmodulation. By contr ast, mutation of Ser(349) did not affect the modulation. The N-type in activation were not indispensable for the down-modulation because the amino-terminal deletion mutant also showed some down-modulation althou gh its onset was quite slow. Thus, the down-modulation of AKv1.1a may be heterogeneous. Because some modulation was still observed even in a mutant which lacks all putative phosphorylation sites mentioned above , additional mechanisms such as the regulation by other phosphorylated protein(s) exist endogenously in oocytes and/or recruitment of other kinases by PKC-activation may also be involved in the observed modulat ion of the AKv1.1a.