Separate gating mechanisms mediate the regulation of K2P potassium channel TASK-2 by intra- and extracellular pH

Abstract

TASK-2 (KCNK5 or K(2P)5.1) is a background K(+) channel that is opened by extracellular alkalinization and plays a role in renal bicarbonate reabsorption and central chemoreception. Here, we demonstrate that in addition to its regulation by extracellular protons (pH(o)) TASK-2 is gated open by intracellular alkalinization. The following pieces of evidence suggest that the gating process controlled by intracellular pH (pH(i)) is independent from that under the command of pH(o). It was not possible to overcome closure by extracellular acidification by means of intracellular alkalinization. The mutant TASK-2-R224A that lacks sensitivity to pH(o) had normal pH(i)-dependent gating. Increasing extracellular K(+) concentration acid shifts pH(o) activity curve of TASK-2 yet did not affect pH(i) gating of TASK-2. pH(o) modulation of TASK-2 is voltage-dependent, whereas pH(i) gating was not altered by membrane potential. These results suggest that pH(o), which controls a selectivity filter external gate, and pH(i) act at different gating processes to open and close TASK-2 channels. We speculate that pH(i) regulates an inner gate. We demonstrate that neutralization of a lysine residue (Lys(245)) located at the C-terminal end of transmembrane domain 4 by mutation to alanine abolishes gating by pH(i). We postulate that this lysine acts as an intracellular pH sensor as its mutation to histidine acid-shifts the pH(i)-dependence curve of TASK-2 as expected from its lower pK(a). We conclude that intracellular pH, together with pH(o), is a critical determinant of TASK-2 activity and therefore of its physiological function.

FIGURE 1.

TALK-type K⁺ channels are sensitive to intracellular pH. A, K⁺ current recorded at 0 mV in HEK-293 cells expressing K_2P channels TASK-2, TALK-2, or TASK-3. During the time indicated the solution bathing the cells was switched to one containing 10 mm NH₄Cl. Results are means ± S.E. of six, three, and three experiments, respectively for TASK-2, TALK-2, and TASK-3. B, a similar experiment as in A but using only TASK-2 and a solution saturated with 5% CO₂ and containing 33 mm HCO₃⁻. Means ± S.E. of eight experiments.

FIGURE 2.

Lack of effect of disabling the extracellular pH sensor and of voltage on TASK-2-mediated currents. A, time course of TASK-2-mediated current recorded at 0 mV at various indicated intracellular pH values. These were achieved in cells containing 50 mm acetate and extracellular acetate concentrations ranging from 1.26 to 126 mm to yield pH_i values ranging from 7.0 to 9.0 (as described under “Experimental Procedures”). On the right, average ± S.E. values at pH_i 9.0 and 7.0 (n = 7) are reported. Extracellular pH was 7.4 throughout. B, an experiment performed as in A but using TASK-2-R224A pH_o-insensitive mutant. Average values shown on the right are from six experiments. C, pH_i dependence of TASK-2 at different voltages (means ± S.E., n = 4–8). Extracellular and intracellular K⁺ concentrations were 5 and 140 mm, respectively, in A–C. The line shows a fit of a Hill function to the data at 0 mV.

FIGURE 3.

TASK-2 gating by intracellular pH is independent on extracellular K⁺ concentration and voltage. A, pH_i dependence of TASK-2 first measured at a [K⁺]_o of 5 mm at 0 mV (triangles) and then at 140 mm [K⁺]_o at voltages from −60 to 60 mV as indicated. Values, normalized to measurements at pH_i 7.5, are means ± S.E. (n = 3–8). Extracellular pH was 7.4 throughout. B, effect of [K⁺]_o on the pH_o dependence of TASK-2. To obtain approximately similar driving forces, measurements were taken at −20 and 60 mV at [K⁺]_o 5 and 140 mm, respectively (means ± S.E., of n = 6 and 8, respectively). C, effect of voltage on the pH_o dependence of TASK-2. Measurements (means ± S.E., n = 11) were taken at −100 and 60 mV using [K⁺]_o 5 mm. Intracellular K⁺ was 140 mm in A–C.

FIGURE 4.

Closure of TASK-2 channels by extracellular acidification cannot be overcome by intracellular alkalinization. A, continuous recording of TASK-2-mediated current using an intracellular acetate concentration of 50 mm and changing extracellular acetate concentration to simultaneously change pH_o and pH_i as indicated. B, bar chart summarizing the normalized activation of TASK-2 channel by an increase in pH_i from 7.5 to 8.5 at extracellular pH of 6.0, 7.4, and 7.9. Means ± S.E., n = 3–9. Extracellular and intracellular K⁺ concentrations were 5 and 140 mm.

FIGURE 5.

Search for a pH_i sensor controlling the gating of TASK-2. A, chimaeras constructed from TASK-2 and TASK-3. The borders between segments of the two origins are depicted. Segments of TASK-2 origin are shaded in gray. B, portion of TASK-sequence containing TM4 and the putative glycine hinge (highlighted in gray). The lines below illustrate the functional result of mutagenesis done on this segment followed by (−), which indicates that the construct retained pH_i sensitivity, or (+) to indicate removal of intracellular pH dependence. C, portion of TASK-2 sequence containing TM4 and TM2 with their putative glycine hinges (highlighted in gray). The lines below illustrate the functional results of mutagenesis as in B.

FIGURE 6.

Lysine 245 is a sensor that commands TASK-2 gating by intracellular pH. A and B, time course of currents mediated by K245A and K245H mutants of TASK-2 recorded at 0 mV at various, indicated intracellular pH values. These experiments were performed exactly as described in the legend to Fig. 2A. B, pH_i dependence of TASK-2-K245A and TASK-2-K245C measured at 0 mV (means ± S.E., n = 6 and 3) is compared with that of WT TASK-2 that had been taken from Fig. 2C without error bars. D, pH_i dependence of TASK-2-K245H measured at 0 mV (means ± S.E., n = 6). Extracellular and intracellular K⁺ concentrations were 5 and 140 mm, respectively in A–D.

FIGURE 7.

Effect of mutation K245A on single channel activity of TASK-2 channels. The pH_i sensitivity of WT TASK-2 (left) and the mutant R245A (right) are shown at three different pH values of the bathing medium. The recording configuration was inside out, and the short lines at the left of the recordings show the closed channel levels. Openings are downward deflections and the patches illustrated contained at least three channels. The holding potential was −60 mV, and both pipette and bath solutions contained 140 mm K⁺.