Inwardly rectifying potassium channel, Kir 2.1, plays an important role in the control of membrane excitability in the vasculature (Quayle et al. 1997). Synapse-associated protein-97 (SAP97) belongs to a group of membrane-associated guanylate kinase proteins (MAGUKs) that interact with potassium channels through specialised PDZ domains. The inwardly rectifying potassium channel, Kir 2.2, carries a C-terminal PDZ binding motif and co-localises with SAP97 in ventricular myocytes (Leonoudakis et al. 2001). The present study examined the interaction of SAP97 with Kir 2.1 in the porcine coronary artery. Expression of inwardly rectifying channels belonging to the Kir 2.0 family and SAP97 in the porcine coronary artery was examined by RT-PCR. Whole hearts of adult pigs were obtained from a local abattoir, coronary arteries dissected and total RNA extracted. Sequence-specific oligonucleotide primers were designed against Kir 2.1, Kir 2.2, Kir 2.3, Kir 2.4 and SAP97. A one-tube RT-PCR reaction was performed and transcripts for Kir 2.1 and SAP97 detected. RT-PCR failed to detect transcripts for Kir 2.2, 2.3 and 2.4 (n = 3).
Association of SAP97 with the C-terminal PDZ binding motif of Kir 2.1 (amino acids 424-428) was examined using GST pull-down assays. A GST-tagged peptide representing C-terminal amino acids (421-428) of Kir 2.1 isolated SAP97 from porcine coronary artery lysates and HEK-293 cells transfected with cDNA for SAP97, a kind gift from Dr C. Garner (University of Birmingham, Alabama, USA). GST alone failed to bind SAP97 (n = 3).
SAP97 harbours three distinct PDZ domains (PDZ 1-3). Interaction of individual domains with Kir 2.1 was defined by overlay assay. A His-tagged construct representing C terminal amino acids of Kir 2.1 (307-428) was overlaid with GST-tagged fusion proteins representing single PDZ domains of SAP97. The C terminus of Kir 2.1 interacted specifically with PDZ domains 2 and 3 of SAP97. PDZ domain 1 failed to interact.
These data demonstrate that the inwardly rectifying potassium channel, Kir 2.1, and MAGUK protein, SAP97, are expressed in porcine coronary artery. Furthermore, Kir 2.1 interacts with PDZ domains 2 and 3 of SAP97, present in porcine coronary artery, via its C terminal PDZ binding motif.
This work was supported by the British Heart Foundation.