Two pore domain potassium (K2P) channels are regulated by a wide variety of pharmacological and physiological mediators, and are responsible for leak K⁺ currents in many neurons. Little is known, however, about the regions of mammalian K2P channels that underlie their gating. In this study, we have considered the role of E30 in the K2P channel, TASK3. This conserved glutamate residue has been shown to be important for gating in drosophila KCNK0 K2P channels (Zilberberg et al. 2001) and for C-type inactivation of Shaker voltage-gated K⁺ channels (Larsson & Elinder, 2000). Whole-cell electrophysiological recordings were made from tsA-201 cells, transiently transfected with wild type (WT) and mutated TASK3 channels. TASK3 channel current density, at -40 mV, was significantly reduced from 67 ± 9 pA/pF (mean ± S.E.M., n = 18) to 22 ± 5 pA/pF (n = 13), following mutation E30C (p < 0.05, Student's t test). Current through E30C mutated channels could be recovered following treatment with 2.5 mM MTSES, to covalently restore a negative charge at this site (50 ± 5 pA/pF, n = 12). In contrast, MTSES had no effect on the amplitude of current through WT TASK-3 channels. In addition, the E30C mutation significantly increased the effectiveness of zinc block of TASK3 channels (Clarke et al. 2004), with 10 μM zinc producing 75 ± 3% (n = 7) inhibition of E30C channels compared to 29 ± 3% (n = 3) for WT TASK3 (p < 0.05). This was not due to the introduction of a novel inhibitory binding site for zinc (C at position 30) since the triple mutation E30C/E70K/H98A (like E70K/H98A, Veale et al. 2005) was not inhibited by zinc. The sensitivity of E30C channels to changes in pH_o was unaltered, and there was no significant change in the sensitivity of the channels to methanandamide (3 μM producing an inhibition of 78 ± 3% (n = 6) and 66 ± 8% (n = 4) for WT and E30C channels, respectively, p > 0.05). Thus mutation E30C alters the gating properties of TASK3 channels and, as a result, changes the degree of block produced by some (zinc) but not all (pH, methanandamide) regulators of activity. Our data supports the suggestion (Zilberberg et al. 2001) that this conserved glutamate residue is important for gating of K2P channels and that these channels gate in response to conformational changes in the external pore.