TASK-1 is a tandem-pore domain K⁺ channel sensitive to extracellular pH. This non-rectifying channel was cloned from a mouse brain cDNA library and is present in a number of tissues including kidney, heart and brain (Duprat et al. 1997). Previous work has shown that mutation of the pore-neighbouring residue His(H)98, to either asparagine (N) or aspartic acid (D), results in a lowered sensitivity to protons (Lopes et al. 2001; O’Connell et al. 2001), suggesting that this residue may also be involved in cation binding. In the present study we have investigated the Ba²⁺ sensitivity of these TASK-1 mutants.

cRNA encoding wild-type (WT, 50 ng), H98N or H98D (100 ng) was injected into stage V or VI Xenopus oocytes. Currents were measured using two-electrode voltage clamp. Bath solutions contained (mM): 100 KCl, 1 MgCl₂, 1 CaCl₂ and 10 Hepes, and varied concentrations of BaCl₂. Solutions were titrated to pH 7.8 with KOH. Cells were held at 0 mV and test potentials from +20 to -100 mV, in 20 mV steps, were applied. The interpulse interval was 2 s. Results are given as means ± S.E.M. and statistical analysis was by ANOVA, with significance assumed at the 5 % level.

WT, H98N and H98D channels all show concentration-and voltage-dependent block by Ba²⁺. However, mutant channels exhibit increased sensitivity to barium compared with WT. The dissociation constants for Ba²⁺ (mM) at 0 mV were: H98N, 7.58 ± 3.38 (n = 5); H98D, 0.41 ± 0.10 (n = 5); WT, 35.6 ± 10.0 (n = 6). Hill coefficients of all channels were close to 1. The fraction (δ) of the membrane electric field sensed by Ba²⁺ may be interpreted as the physical distance across the membrane at which an ion binds. H98N and WT have similar δ values of 0.58 ± 0.09 (n = 5) and 0.64 ± 0.16 (n = 6), respectively. However, H98D has a δ of 0.20 ± 0.05 (n = 5), which was significantly lower than WT or H98N.

Thus both mutants have a higher sensitivity to Ba²⁺ than WT, indicating either that Ba²⁺ has easier access, or binds more strongly, to residues within the pore. The lower δ of H98D suggests that, in this mutant, Ba²⁺ binds to a site closer to the extracellular mouth of the pore than in WT or H98N channels, perhaps by stabilisation of Ba²⁺ at the external K⁺ binding site within the selectivity filter. The enhanced sensitivity of the mutants could be due to the decreasing positivity of side-chain charge, but the altered δ values argue against direct binding of Ba²⁺ to these residues.

We are grateful to Dr Steve Goldstein (Yale, USA) for the TASK-1 clone. The financial support of the MRC is thankfully acknowledged.