Suppression of Kir2.3 activity by protein kinase C phosphorylation of the channel protein at threonine 53

Kir2.3 plays an important part in the maintenance of membrane potential in neurons and myocardium. Identification of intracellular signaling molecules controlling this channel thus may lead to an understanding of the regulati on of membrane excitability. To determine whether Kir2.3 is modulated by di rect phosphorylation of its channel protein and identify the phosphorylatio n site of protein kinase C (PKC), we performed experiments using several re combinant and mutant Kir2.3 channels, Whole-cell Kir2.3 currents were inhib ited by phorbol la-myristate 13-acetate (PMA) in Xenopus oocytes. When the N-terminal region of Kir2.3 was replaced with that of Kir2.1, another membe r in the Kir2 family that is insensitive to PMA, the chimerical channel los t its PMA sensitivity. However, substitution of the C terminus was ineffect ive. Four potential PKC phosphorylation sites in the N terminus were studie d by comparing mutations of serine or threonine with their counterpart resi dues in Kir2.1. Whereas substitutions of serine residues at positions 5, 36 , and 39 had no effect on the channel sensitivity to PMA, mutation of threo nine 53 completely eliminated the channel response to PMA. Interestingly, c reation of this threonine residue at the corresponding position (I79T) in K ir2.1 lent the mutant channel a PMA sensitivity almost identical to the wil dtype Kir2.3. These results therefore indicate that Kir2.3 is directly modu lated by PKC phosphorylation of its channel protein and threonine 53 is the PKC phosphorylation site in Kir2.3.