TASK-2 is a two-pore domain K⁺ channel (K_2P) sensitive to extracellular pH; currents are maximal at alkaline pH but inhibited as pH decreases (Reyes et al., 1998). TASK-2 is found principally in epithelial tissues and in the kidney is located in the basolateral membrane of proximal tubule cells where it is coupled to HCO₃^– transport (Warth et al., 2004). Mouse TASK-2 possesses one putative PKA concensus site (S266, PROSITE). Our aim was to investigate whether (de)phosphorylation of this residue regulates channel activity. Wild-type (WT) mTASK-2 cDNA was subcloned into the bicistronic vector pIRES-CD8 (Invitrogen). Mutations were generated by PCR and confirmed by sequencing (Lark). Chinese hamster ovary (CHO) cells were transfected using Fugene (Roche). After 24 to 72 hours CHO cells expressing the CD8 antigen were identified with anti-CD8 magnetic beads (Dynal). Whole-cell currents were recorded in mammalian Ringer containing (in mM); NaCl 145, KCl 5, MgCl₂ 1, CaCl₂ 2, HEPES 5, PIPES 5, pH 7.8. Pipette solution contained (in mM); K-gluconate 135, KCl 10, MgCl₂ 5, CaCl₂ 611 μM, EGTA 5, HEPES 10 and K₂-ATP 5. Free Ca²⁺ activity was 20 nM (React program, Godfrey Smith, Glasgow University). Fluoride was added by replacing 20 mM K-gluconate with KF. Channel rundown was measured as the channel activity remaining after 2 mins. Results are given as means ± SEM, compared using the unpaired Students t-test. mTASK-2 currents were outwardly rectifying and pH sensitive. With time channel activity decreased such that after 2 mins of whole-cell recording only 56.7±0.03% (n=7) of channel activity remained. In the presence of 20 mM F, a non-specific inhibitor of phosphatase activity, channel rundown was abolished; 98.8±0.03% channel activity remained after 2 mins. The PKA inhibitor, H89 (5 μM, in the presence of F) caused a reversible decrease in channel activity; 75.9±0.03% WT channel activity remained after 2 mins (n=8). S266A and S266D mutants expressed pH sensitive currents not different from WT. After 2 mins, 72.2±0.02% (n=11) and 74.7±0.12% (n=4) channel activity remained, respectively. The C-terminus of mTASK-2 is 252 amino acids long and encodes one concensus PKA site at residue S266. The inhibition of rundown by F and rundown caused by H89 suggest that PKA has a role in the regulation of TASK-2; normal channel activity is maintained by PKA-mediated phosphorylation. Given that both S266 mutants express as WT, PKA must be acting either at a site other than S266 or on an, as yet unidentified, accessory subunit.