We have previously cloned and characterised the human potassium channel, heag2, a member of the ether-a-go-go ion channel family (Ju & Wray, 2002). Besides the usual six membrane-spanning domains, this voltage-activated channel has long intracellular N and C termini. The N terminal region contains a ‘PAS’ domain, thought to be involved in protein-protein interactions, while the C terminal region is even longer and contains a cyclic nucleotide binding domain (cNBD) as well as a number of other domains. The heag2 channel is predominantly expressed in the brain but is also found in skeletal muscle and the heart. In this study, we have looked for binding partners for intracellular regions of the channel. For this, we have made GST fusion proteins of parts of the intracellular regions, and used these to pull down binding partners which we have identified by mass spectrometry. The GST fusion protein constructs were made by PCR amplification of fragments of intracellular regions, and ligated into pGEX 4T-3 vector with suitable restriction enzymes. The GST fusion proteins were grown in BL21 cells and protein expression induced using IPTG; expression was confirmed using Western blotting with anti-GST antibody. After binding to glutathione beads and washing, the product was mixed with a human brain protein medley (BD Biosciences). Samples were eluted from the beads and run on SDS-PAGE gels; bands were extracted and trypsin digested after staining with Coomassie blue. MALDI-TOF mass spectrometry was then performed to identify the masses of the peptides in the tryptic digests, and then to identify the proteins using Mascot software and protein databases. A number of binding partners were identified for the C terminal regions of heag2. For the region between the end of S6 to the beginning of the cNBD (475-549), human protein binding partners that were identified were both α and β tubulin as well as a heat shock cognate 71 kDa protein HSP7C. For the region corresponding to the cNBD of heag2 (550-650), binding partners were again α and β tubulin, as well as myelin basic protein. For residues 717-826 of the C terminus, again binding proteins that were identified were α and β tubulin. Finally for the distal part of the C terminus (residues 909-988), myelin basic protein was also identified as a binding partner. Work is in progress to investigate protein binding partners at the N terminus. In summary, binding partners that were identified fell into several classes, including those concerned with cytoskeletal structure. However the roles of these proteins in the context of the heag2 channel need to be identified. M. Ju & D. Wray (2002) FEBS Lett, 524, 204-210.