Two-pore-domain potassium (K_2P) channels underlie background currents that control the resting membrane potential and excitability of many cells. Although classed as “leak” channels, currents through K_2P channels are tightly regulated. For example, the pH sensitive K_2P (TASK) channels are inhibited by extracellular acidification with pK_as of 7.3 for TASK1 and 6.6 for TASK3. Whilst a pore neighbouring histidine is thought to be the pH sensor of TASK3 and TASK1 it is not present in TASK2 and does not account for the differing pK_as of TASK1 and TASK3. TASK2 was the second member of the TASK channel subfamily to be cloned; however it is now known to have closer amino acid homology to the alkaline sensitive TALK family channels. More like the TALK family, TASK2 is also activated at alkaline pH with a pK_a of around 8. Residues within the large extracellular M1-P1 loop have been implicated as forming the pH sensor of TASK2 (Morton et al., 2005), but this data has recently been refuted and an arginine within the second pore demonstrated to be the real pH sensor (Niemeyer et al., 2007)
In this study we show that the M1-P1 loop is a structured entity that, whilst not being the pH sensor per se, contributes to the differential pH sensitivity of TASK channels. Chimaeric TASK channels, constructed by swapping M1-P1 loops between family members, were expressed in Xenopus oocytes as previously described (Clarke et al., 2006). Channels were characterised using standard two-electrode voltage-clamp techniques. All data is shown as mean ± SEM. NMR spectroscopy studies were performed on a synthetic peptide of TASK2 M1-P1 (Auspep).
The pH sensitivity of TASK2 was essentially abolished with the replacement of its M1-P1 loop with that of TASK3 and vice versa (TASK2: pK_a = 8.0 ± 0.04, n=8; T2/T3 M1P1: pK_a = 5.3 ± 0.2, n=9; T3/T2 M1P1: pK_a = 4.9 ± 0.1, n=10).
Intriguingly, replacement of the TASK1 M1-P1 with that of TASK3 led to channels with TASK3 like pH sensitivity (TASK-1: pK_a = 7.3 ± 0.07, n=7; T1/T3 M1P1: pK_a = 6.7 ± 0.06, n=6, TASK-3, pK_a = 6.6 ± 0.04, n=7), however TASK3 pH sensitivity was not affected by the replacement of its M1-P1 loop with that of TASK1 (T3/T1 M1P1: pK_a = 6.6 ± 0.04, n=5).
Our work demonstrates that the M1-P1 is essential for the pH sensitivity of TASK2 and TASK1 channels and the mechanism of channel closure during extracellular acidification appears more complex than simple channel block by protonation of single pore lining residues.