Recently a new K+ channel superfamily, the two pore-domain K+ (K2P) channel family, was identified (Lesage & Lazdunski, 2000). hTASK-1 (KCNK3) is a member of this new gene family and generates characteristically, pH-sensitive currents. Evidence has accumulated for the involvement of TASK-like channels in O2-sensing by both airway (O’Kelly et al. 1999) and carotid body (Buckler et al. 2000) chemoreceptors. However, direct demonstration that acute hypoxia suppresses hTASK currents is lacking.
HEK293 cells were transfected with pcDNA3.1-hTASK-1 using SuperfectTM transfection reagent and selected with 1 mg ml-1 GENETICIN¿ (G418 Sulphate). The whole-cell patch-clamp technique was used to examine membrane currents of cells stably expressing hTASK-1. Extracellular solutions were made hypoxic by bubbling solutions with nitrogen gas for 30 min prior to perfusing the cells, and caused no alteration in pH. Data were taken at 0 mV and expressed as means ± S.E.M., and were analysed by Student’s paired t test with significance assumed at the 5 % level.
Current amplitude at all test potentials (-100 to +60 mV) was, as anticipated, extremely sensitive to extracellular pH, particularly within the physiological range. Raising pH from 7.4 to 8.4 elicited a significant augmentation of currents (35.0 ± 8.3 %, P < 0.05), whilst decreasing pH to 6.4 and 5.4 resulted in significant reductions of 62.1 ± 3.1 and 83.5 ± 3.0 %, respectively (P < 0.05); IC50 7.01 ± 0.09, Hill coefficient 2.17 (n = 6, for all pH levels). Importantly, all cells expressing this signature pH sensitivity were sensitive to changes in PO2. Reduction of perfusate PO2 levels from 150 to < 40 mmHg (pH 7.4) resulted in rapid and reversible suppression of pH-sensitive K+ currents by 19.6 ± 4.7 % (n = 8). Furthermore, these two regulatory signals clearly acted at the same channel, since the magnitude of the O2-sensitive current was dependent on the extracellular pH. Lowering pHo from 8.4 to 5.4 significantly reduced the hypoxic response from 20.9 ± 3.8 % (n = 4) to 7.6 ± 2.9 % (n = 3), respectively (P < 0.05).
These data represent the first direct verification that hTASK-1 is an O2-sensitive K+ channel, and reinforces the notion that this channel is key to O2 sensing in airway and also possibly carotid body chemoreceptors (Peers & Kemp, 2001).
This work was funded by The Wellcome Trust and British Heart Foundation.
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