TASK2 is a member of the two-pore domain potassium (K2P) channel family that plays a role in acid-base homeostasis; TASK2 knock-out animals have plasma electrolyte patterns typical of the human clinical condition of renal tubular acidosis (Warth et al., 2004). It is expressed preferentially in epithelia, including the proximal tubules of the kidney. In common with the other TASK channels, TASK2 is sensitive to changes in extracellular pH, although the molecular mechanism of such pH-sensing is not understood (Morton et al., 2003). We have examined the role of charged residues in the extracellular domains in pH-sensing in murine TASK2 using a mutational approach. Mutant channels were transiently expressed in CHO cells and studied by whole-cell and single channel patch clamp. Results are given as mean ± SEM with n, the number of observations. Statistical significance was assumed at P<0.05. Neutralisation of no single amino acid in isolation gave substantial loss of pH-sensitivity. However, the combined removal of five charged amino acids (E28, K32, K35, K42 & K47) in the large extracellular loop linking the first transmembrane and pore domains, the M1-P1 loop, resulted in a marked reduction in pH-sensitivity (WT = 84.6±0.02% inhibition at pH 5.8 v pH 8.8, n = 14; E28 = 21.1±0.05% inhibition at pH 5.8 v pH 8.8, n=8). This result was confirmed in single channel studies, where channels containing the five point mutations were stabilised in the open state. Wild-type channels contain two M1-P1 loops, but a concatemeric construct, comprised of one wild-type subunit and one containing the five mutations, was fully pH sensitive, indicating that only one M1-P1 loop is required to yield a fully pH sensitive channel. This is the first demonstration of a regulatory role of this distinctive structure in K2P channels. Thus pH-sensing in TASK2 channels is conferred by the combined action of several charged residues in the large extracellular M1-P1 loop, and a single functional loop is sufficient for pH-sensing.