Extracellular acidification exerts opposite actions on TREK1 and TREK2 potassium channels via a single conserved histidine residue

Abstract

Mechanosensitive K(+) channels TREK1 and TREK2 form a subclass of two P-domain K(+) channels. They are potently activated by polyunsaturated fatty acids and are involved in neuroprotection, anesthesia, and pain perception. Here, we show that acidification of the extracellular medium strongly inhibits TREK1 with an apparent pK near to 7.4 corresponding to the physiological pH. The all-or-none effect of pH variation is steep and is observed within one pH unit. TREK2 is not inhibited but activated by acidification within the same range of pH, despite its close homology with TREK1. A single conserved residue, H126 in TREK1 and H151 in TREK2, is involved in proton sensing. This histidine is located in the M1P1 extracellular loop preceding the first P domain. The differential effect of acidification, that is, activation for TREK2 and inhibition for TREK1, involves other residues located in the P2M4 loop, linking the second P domain and the fourth membrane-spanning segment. Structural modeling of TREK1 and TREK2 and site-directed mutagenesis strongly suggest that attraction or repulsion between the protonated side chain of histidine and closely located negatively or positively charged residues in P2M4 control outer gating of these channels. The differential sensitivity of TREK1 and TREK2 to external pH variations discriminates between these two K(+) channels that otherwise share the same regulations by physical and chemical stimuli, and by hormones and neurotransmitters.

Fig. 1.

TREK1 is inhibited by extracellular acidification. (A) Effect of pH on TREK1 expressed in Xenopus oocytes. Currents were elicited by voltage-ramps (from −150 to + 50 mV, 1 s in duration). (B) pH dependence of the TREK1 channel activity at 0 mV. (C) pH dependence of TREK1E306A channel activity at 0 mV. Error bars, SEM. At higher levels of expression, TREK1E306 becomes resistant to inhibition. (D) Effect of DEPC preincubation (2 mM) on pH-sensitivity of TREK1. (Inset) the histograms represent the TREK1 current increase induced by a pH shift from 8 to 7.2 at 0 mV, in the absence or in the presence of DEPC. The number of cells tested is indicated in parenthesis.

Fig. 2.

pH sensitivity of mutated TREK1 channels. (A) Membrane topology of TREK1. Extracellular cysteine and histidine residues are indicated. (B) Current increase induced by a pH shift from 7.2 to 8 at 0 mV for TREK1 and point mutants. (C) Loss of pH effect on currents produced by TREK1H126A and TREK1H126K. Currents were elicited by voltage-ramps (from −150 to + 50 mV, 1 s in duration). (Inset) TREK1H126A and TREK1H126K current amplitude changes relative to TREK1 current at 0 mV at pH 7.4. Error bars, SEM. The number of cells tested is indicated in parenthesis.

Fig. 3.

pH sensitivity of TREK2. (A) Effect of pH on TREK2. Currents were elicited by voltage-ramps (from −150 to + 50 mV, 1 s in duration). (B) pH-dependence of TREK2 channel activities at 0 mV. (C) The histograms represent the current increase or decrease induced by a pH shift from 7.2 to 8 at 0 mV in the absence or in the presence of DEPC. Error bars, SEM. The number of cells tested is indicated in parenthesis.

Fig. 4.

A conserved histidine residue is the extracellular pH sensor. (A) Sequence alignment of the pore regions from TREK1 and TREK2. The pH-sensing histidine residue is in bold. Residues in TREK2 predicted to interact with the histidine pH sensor are boxed. (B) Percentage of inhibition at pH 8 relative to pH 7.2 at 0 mV for TREK2 and TREK2H151 mutants. (C) Effect of pH on representative TREK2H151A and TREK2H151K currents in Xenopus oocyte in ND96 solution at pH 7.2 and 8. Currents were elicited by voltage-ramps (from −150 to + 50 mV, 1 s in duration). (Inset) TREK2H151A and TREK2H151K current amplitude changes relative to TREK2 current at 0 mV.

Fig. 5.

Mapping of the region involved in the differential regulation of TREK1 and TREK2. pH dose-response curves of the variable proportion calculated from the normalized current of the current TREK2-TREK1 (A), TREK2-P2TREK1 (B), TREK2-P2Nt-TREK1 (C), and TREK2-P2Ct-TREK1 (D) chimeras, respectively. Dotted curves are the pH dose–response curve of TREK2, shown for comparison.

Fig. 6.

Effect of single and combined mutations on pH-sensitivity to TREK2. Top view of the extracellular side of homodimeric TREK1 (A) and TREK2 (B) channels. Cytoplasmic Nter and Cter domains are not displayed. Studied residues are colored in only one monomer. pH-sensing histidines are indicated. Positively charged residues are in blue and negatively charged residues in red. Neutral residues are depicted in green and aromatics F282 and Y272 are in yellow along with the oxygen atom in red in Y272. (C) The histograms represent the percentage of inhibition or activation at pH 8 relative to pH 7.2 at 0 mV for TREK2 and TREK2 mutants. (D) pH dose-response curves of the variable proportion calculated from the normalized current of TREK2 mutants. Error bars, SEM. The number of cells tested is indicated in parenthesis.