Abstract
Vascular ATP-sensitive K(+) channels are inhibited by multiple vasoconstricting hormones via the protein kinase C (PKC) pathway. However, the molecular substrates for PKC phosphorylation remain unknown. To identify the PKC sites, Kir6.1/SUR2B and Kir6.2/SUR2B were expressed in HEK293 cells. Following channel activation by pinacidil, the catalytic fragment of PKC inhibited the Kir6.1/SUR2B currents but not the Kir6.2/SUR2B currents. Phorbol 12-myristate 13-acetate (a PKC activator) had similar effects. Using Kir6.1-Kir6.2 chimeras, two critical protein domains for the PKC-dependent channel inhibition were identified. The proximal N terminus of Kir6.1 was necessary for channel inhibition. Because there was no PKC phosphorylation site in the N-terminal region, our results suggest its potential involvement in channel gating. The distal C terminus of Kir6.1 was crucial where there are several consensus PKC sites. Mutation of Ser-354, Ser-379, Ser-385, Ser-391, or Ser-397 to nonphosphorylatable alanine reduced PKC inhibition moderately but significantly. Combined mutations of these residues had greater effects. The channel inhibition was almost completely abolished when 5 of them were jointly mutated. In vitro phosphorylation assay showed that 4 of the serine residues were necessary for the PKC-dependent (32)P incorporation into the distal C-terminal peptides. Thus, a motif containing four phosphorylation repeats is identified in the Kir6.1 subunit underlying the PKC-dependent inhibition of the Kir6.1/SUR2B channel. The presence of the phosphorylation motif in Kir6.1, but not in its close relative Kir6.2, suggests that the vascular K(ATP) channel may have undergone evolutionary optimization, allowing it to be regulated by a variety of vasoconstricting hormones and neurotransmitters.
Highlights
Experimental evidence suggests that vasoconstrictors act on the vascular KATP channel through the PKC2 signaling system
Our previous studies have shown that the Kir6.1/SUR2B channel and its counterpart in vascular smooth muscle cells are inhibited by vasopressin and that channel inhibition can be abolished by specific protein kinase C (PKC) blockers [6]
These results indicate that the Kir6.1/SUR2B channel is inhibited by PKC independently of cytosolic soluble components, consistent with previous reports [20]
Summary
Experimental evidence suggests that vasoconstrictors act on the vascular KATP channel through the PKC2 signaling system. By comparing the effects of the acetylcholine M3 receptor on Kir6.1/ SUR2B and Kir6.2/SUR2B channels, Quinn et al [16] suggest that Kir6.1/SUR2B channel inhibition is mediated via a direct effect of PKC rather than a change in phosphatidyl 4,5-bisphosphate concentrations They show evidence for Kir6.1 phosphorylation using in vitro biochemical assay [16]. Purified PKC inhibits the cloned Kir6.1/SUR2B and vascular smooth muscle endogenous KATP channels in inside-out patches where cytosolic soluble components are absent [4, 20]. These previous studies have significantly improved our understanding of vascular KATP channel regulation by vasoconstrictors, the molecular substrate of PKC remains unknown.
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