EFFECTS OF ISOFLURANE ON THE EXPRESSED Ca_v2.2 CURRENTS IN XENOPUS OOCYTES DEPEND ON THE ACTIVATION OF PROTEIN KINASE C 8 AND ITS PHOSPHORYLATION SITES IN THE Ca_v2.2alpha-1 SUBUNITS

摘要

The effects of isoflurane on the modulation of two neuronal voltage-gated calcium channels (Ca_v; Ca_v2.1 and 2.2) by protein kinase C (PKC) isozymes J3II, e or 8 and their combination were examined. Ca_v2.1o:1 or Ca_v2.2apha 1 with 01b and a2S subunits were expressed in Xenopus oocytes and the currents (l_(Ba)) were recorded by two-electrode voltage clamp. Isoflurane (0.70 mM) decreased both Ca_v2.1 and 2.2 currents by 20-35% and also caused translocation of PKCS to the membrane. Compared to the wild type (WT), isoflurane caused greater inhibition of Cav2.2 currents in the absence of stimulatory PKC sites (Thr-422, Ser-1757, Ser-2108, Ser-2132) and in the presence of inhibitory PKC site (Ser-425). In contrast, isoflurane caused less inhibition of l_(Ba) in the oocytes expressing S425A, the inhibitory site mutant, compared to WT. PKCS by itself did not modulate Ca_v2.2 currents, but potentiated these currents in the presence of isoflurane. PKCe increased Ca_v2.2 currents either alone or in combination with isoflurane. Ca_v2.1 currents were not modulated by phorbol-12-myristate, 13-acetate (PMA) or acetyl-beta-methyl-choline (MCh), activators of PKC. Yet the presence of isoflurane caused PMA (but not MCh) to enhance Ca_v2.1 currents. PKCjSII and PKCe isozymes activated by PMA, did not alter Ca_v2.1 currents. However, in the presence of isoflurane, these two isozymes together potentiated Cav2.1 currents. The variable responses of Cav2.1 currents to PKC/3II and PKCe and Cav2.2 currents to PKCS in the presence of isoflurane may be due to increased affinity or accessibility of these isozymes to their Ser/Thr PKC sites of Ca_va1 subunits.