Zinc is essential for brain function. It is sequestered into and released from specialised ‘zinc-containing’ neurons. An increased zinc concentration in the extracellular solution has been shown to alter the activity of several membrane receptors and channels (e.g. Takeda, 2000). In the present study we have investigated the effects of extracellular zinc on TASK-1 and TASK-3 channels, acid-sensitive members of the ‘background’ two-pore domain potassium channel family.
Human TASK-1 (hTASK-1) and hTASK-3 were expressed in Xenopus oocytes as previously described (Clarke et al. 2000). Relatively large, outwardly rectifying currents were recorded in low external K+ (1 mM) when voltage ramps from -120 to +80 mV were applied. These currents were blocked by acidification of the external solution. Full pH response curves show that hTASK-1 is more pH sensitive that hTASK-3 with pH IC50 values of 7.3 ± 0.08 and 6.6 ± 0.04, respectively (means ± S.E.M.).
Application of 100 µM zinc had no effect on hTASK-1 currents (-4.4 ± 6.5 %, n = 10), a small inhibitory effect on rat TASK-1 currents (17.1 ± 3.9 %, n = 5), but blocked hTASK-3 currents, in a voltage-independent manner, by 68.9 ± 5.2 % (n = 9). The zinc concentration-response curve for hTASK-3 had an IC50 of 19.8 ± 2.7 µM (n = 6). Mutation of histidine (H) 98 of hTASK-3 to alanine (A) by site-directed mutagenesis, which led to a loss of pH sensitivity of the channel, also reduced the zinc sensitivity of the channel (with an IC50 of 95.8 ± 14.1 µM, n = 5).
As the equivalent histidine residue is also present in hTASK-1 and as zinc sensitivity is not completely lost in mutant H98A of TASK-3, it appeared that another residue might also be involved in the blocking action of zinc. Previous work by Derst et al. (2002) has identified glutamic acid (E) 70 of TASK-3 as an important determinant of sensitivity to extracellular divalent cations. TASK-1 does not have the equivalent residue in this position. Mutation of hTASK-3 E70 to lysine (K) by site-directed mutagenesis also led to a reduction of zinc block, with an IC50 of 104.8 ± 24.3 µM.
In conclusion, our data suggest that both residues H98, predicted to lie at the mouth of the first pore region and E70, which is within the large M1-P1 loop, are involved in the selective block by zinc of human TASK-3 channels.
This work was supported by the BBSRC and MRC.