The amino acid residues situated in the pore domain and constituting the selectivity filter are highly conserved amongst potassium channels. Studies of Shaker (Larsson & Elinder 2000) and KcsA (Doyle 1998) have also shown a region at the extracellular end of S5 to contain a glutamate residue that both appears highly conserved within the voltage-gated potassium channel family and is thought to play a crucial role in C-type inactivation. This glutamate is also present at the equivalent position in M1 and M3 in the tandem pore K⁺ channel TASK-1 and is conserved throughout the family. We have mutated in turn both glutamate residues (E30 and E182) to cysteine in TASK-1 and observed the effects on ionic selectivity and pH sensitivity. Channels were expressed in oocytes taken from Xenopus frogs that had been anaesthetised by immersion in 0.3% w/v MS222 and killed by destruction of the brain and spinal cord. Two-electrode voltage clamp was used to measure the pH sensitivity, and the shift in reversal potential when K⁺ in the external medium was replaced by Rb⁺ or Na⁺. Surprisingly, these mutated residues, located outside the pore region of the channel, exhibited altered selectivity compared to wild type channels. P_Rb/P_K was significantly increased from 0.77 ± 0.02 (n=6) in wild type to 0.88 ± 0.03 (n=6) in E30C and 0.93 ± 0.02 in E182C (n=5; mean ± s.e.m; P<0.05, using ANOVA). Unlike wild type, both mutants were noticeably Na⁺-permeant, with an increase in P_Na/P_K from 0.02 ± 0.003 (n=6) to 0.20 ± 0.04 (n=6) and 0.45 ± 0.03 (n=5; P<0.001) for E30C and E182C respectively. We found that like wild type channels (pK_a of 6.02 at +40mV), E30C exhibited complete pH dependence, albeit with a reduced affinity for protonation, indicated by a pK_a of 6.60 at +40mV. However, E182C showed a reduced pH dependence, and incomplete channel closure at physiological pH. Fitting the residual pH-sensitive current gave a pK_a of 7.60 at +40mV. The change in pH dependence and selectivity exhibited with these non-pore mutations suggests that these residues may have a role in channel gating. The equivalent residue in KcsA and shaker is hypothesised to H-bond with local residues to stabilise the inactivation gate in its open conformation (Larsson & Elinder 2000). Our preliminary model of TASK-1 also indicates that E30 and E182 may form H-bonds with local residues T103 and Q209.