Protein kinase A (PKA) is an important, broad-specificity kinase that is activated by the diffusible second messenger cAMP. Since PKA is capable of phosphorylating a broad range of target proteins, specificity in PKA signalling is achieved via A-kinase anchoring proteins (AKAPs) that segregate different pools of PKA with their appropriate substrate. Ion channels represent a diverse group of kinase substrates, and kinase targeting through association with AKAPs facilitates PKA-mediated phosphorylation and regulation of AMPA/kainate receptors, L-type calcium channels and calcium-activated and inwardly rectifying potassium channels (Fraser & Scott, 1999).

We have shown recently that cAMP-dependent modulation of whole-cell currents of the inwardly rectifying potassium channel, Kir2.1, requires the presence of the multivalent anchoring protein, AKAP79, and that Kir2.1 and AKAP79 exist together in a complex within intact cells (Dart & Leyland, 2001). Here, we extend this study by identifying a binding site of AKAP79 on the intracellular C-domain of Kir2.1.

To determine possible sites of interaction, glutathione-S-transferase (GST) fusion proteins of various regions of the C-terminal domain of Kir2.1 were screened for their ability to isolate AKAP79 from lysates of HEK293 cells transfected with cDNA encoding AKAP79 (a gift from Dr John Scott, Vollum Institute, Portland, OR, USA). A GST fusion protein of residues 182-282, GST-C(182-282) was found to isolate AKAP79 from HEK293 lysates, while GST alone and GST fusion proteins of other regions of the C-terminus failed to interact with AKAP79 (n = 3). Additional smaller fusion proteins made from the interacting region showed that GST-C(182-232) retained its ability to isolate AKAP79, while GST-C(233-282) did not (n = 3).

These data suggest that AKAP79 targets a region between residues 182 and 232 on the intracellular C-domain of Kir2.1, presumably to anchor PKA in close proximity to channel phosphorylation sites. Interestingly, this region of Kir2.1 contains a modified leucine zipper-like motif (a periodic repetition of hydrophobic residues at every seventh position) that has been shown to mediate AKAP binding to L-type calcium channels and to the KvLQT1 ion channel (Hulme et al. 2002; Marx et al. 2002). This motif may represent a conserved mechanism by which AKAPs interact with ion channels.

This work was supported by the MRC and the Royal Society.