P327The KATP channel subunit, Kir6.2, is regulated by a Ca2+-dependent isoform of Protein Kinase C

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ATP-sensitive potassium (KATP) channels couple cell metabolism to membrane potential in many cell types to control vascular smooth muscle tone and excitability in neurons and muscle. Protein Kinases are involved in the modulation of channel activity and are particularly important in regulating smooth muscle tone. In previous work, Kir6.1/SUR2B channels have been shown to be inhibited by Protein Kinase C (PKC) whilst Kir6.2/SUR2B channels are activated or have no response. In this study we show that Kir6.2/SUR2B channels can be inhibited by PKC in a Ca2+-dependent manner and identify a specific site for potential PKC phosphorylation.


Human embryonic kidney cells (HEK293) stably transfected with SUR2B and Kir6.1 or Kir6.2 were subjected to Rubidium flux and whole cell patch-clamp electrophysiology. An in vitro phosphorylation assay and phosphopeptide mapping were used to determine potential PKC phosphorylation sites.


Rubidium flux experiments with Kir6.1/SUR2B and Kir6.2/SUR2B showed that both were inhibited by the activation of PKC. In the presence of the Ca2+ chelator, BAPTA-AM, inhibition by PKC was observed only in Kir6.1/SUR2B cells suggesting that the PKC effect on Kir6.2/SUR2B is Ca2+ dependent. This was confirmed using whole cell patch clamp recordings, where KATP current inhibition was only seen when free intracellular Ca2+ was 100 nM or higher. A serine residue at position 372 was identified by phosphopeptide mapping to be a potential PKC phosphorylation site. In vitro phosphorylation was reduced when Serine 372 was mutated to an alanine residue compared to wild type. Currents evoked from cells expressing Kir6.2-S372A/SUR2B were not inhibited by PKC activation in the presence of 300nM free intracellular Ca2+.


The results in this study show that Kir6.2/SUR2B is regulated by PKC and that this is Ca2+-dependent. In addition, Serine 372 in the Kir6.2 subunit has been identified as a novel site for PKC phosphorylation.

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