Acid-sensitive TWIK and TASK two-pore domain potassium channels change ion selectivity and become permeable to sodium in extracellular acidification

Abstract

Two-pore domain K(+) channels (K2P) mediate background K(+) conductance and play a key role in a variety of cellular functions. Among the 15 mammalian K2P isoforms, TWIK-1, TASK-1, and TASK-3 K(+) channels are sensitive to extracellular acidification. Lowered or acidic extracellular pH (pH(o)) strongly inhibits outward currents through these K2P channels. However, the mechanism of how low pH(o) affects these acid-sensitive K2P channels is not well understood. Here we show that in Na(+)-based bath solutions with physiological K(+) gradients, lowered pH(o) largely shifts the reversal potential of TWIK-1, TASK-1, and TASK-3 K(+) channels, which are heterologously expressed in Chinese hamster ovary cells, into the depolarizing direction and significantly increases their Na(+) to K(+) relative permeability. Low pH(o)-induced inhibitions in these acid-sensitive K2P channels are more profound in Na(+)-based bath solutions than in channel-impermeable N-methyl-D-glucamine-based bath solutions, consistent with increases in the Na(+) to K(+) relative permeability and decreases in electrochemical driving forces of outward K(+) currents of the channels. These findings indicate that TWIK-1, TASK-1, and TASK-3 K(+) channels change ion selectivity in response to lowered pH(o), provide insights on the understanding of how extracellular acidification modulates acid-sensitive K2P channels, and imply that these acid-sensitive K2P channels may regulate cellular function with dynamic changes in their ion selectivity.

FIGURE 1.

Lowered pH_o shifted the reversal potential and inhibited outward currents in TWIK-1 K⁺ channels. A, topology of a TWIK-1·K274E subunit. B, alignment of the selectivity sequences of P1-loops in five rat, human, and mouse K2P channels. C, the crystal structure of the selectivity filter (P1-loop) of TWIK-1 K⁺ channels (24). D and E, whole-cell currents of TWIK-1·K274E K⁺ channels are shown before (black or blue line) and after (red lines) change of pH from 7.4 to 6.0 in Na⁺-based (D) or NMDG⁺-based (E) bath solutions with 5 mm K⁺. Quinine blockade confirmed TWIK-1 currents in pH_o 6.0 in D (pink line). F, comparison of low pH_o-induced inhibitions of outward TWIK-1 K⁺ currents in Na⁺-based and NMDG⁺-based bath solutions. Currents at 60 mV obtained in Na⁺-based or NMDG⁺-based bath solutions with pH 6.0 were normalized (Norm.) by those recorded in pH 7.4 from a group of experiments in D or E. *, p < 0.001. G, reversal potentials (E_rev) of the channels were plotted as a function of [K⁺]_o in Na⁺-based solutions with pH 7.4 (black circles) or 6.0 (red circles) (n = 4–11). The continuous curves are fits with the Goldman-Hodgkin-Katz equation: E_rev = RT/zF × ln((ρ[Na⁺]_o + [K⁺]_o)/(ρ[Na⁺]_i + [K⁺]_i)), yielding a Na⁺ to K⁺ relative permeability ρ of 0.005 at pH 7.4 and 0.09 at pH 6.0, respectively. Whole-cell currents in all figures were obtained in transfected CHO cells. The currents measured in lowered pH_o represent those at equilibrium in all figures, unless indicated specifically.

FIGURE 2.

Kinetics of low pH_o-induced changes in the reversal potential and current amplitude of TWIK-1 K⁺ channels. A and C, two-phase effects of lowered pH of Na⁺-based (A) or NMDG⁺-based (C) bath solutions on whole-cell currents of TWIK-1·K274E K⁺ channels. Whole-cell currents are each shown in 30 and 60 s in the left and right panels, respectively. B and D, kinetics of the changes in the reversal potential (filled circles and squares) and TWIK-1 currents at 60 mV (open circles and squares) after pH_o was lowered in A or C. The superimposed single-exponential fit yields a time constant of 284 ± 25 s (orange line, n = 9), 94 ± 8 s (pink line, n = 9), and 148 ± 11 s (purple line, n = 6), respectively.

FIGURE 3.

Effects of lowered pH_o on the reversal potential and outward currents of TASK-3 wild type and TASK-3·H98D mutant K⁺ channels. A–D, whole-cell currents are shown for TASK-3 (A and B) and TASK-3·H98D (C and D) K⁺ channels before (black or blue line) and after (red lines) change of pH from 7.4 to 6.0 in Na⁺-based (A and C) or NMDG⁺-based (B and D) bath solutions. The dashed pink line in A represents the current after pH_o was switched backed to 7.4 from 6.0. Insets: current traces are shown in shorter voltage ranges and narrow current amplitudes (−100 to +100 pA) so that the reversal potentials are clearly visible. E, comparison of low pH_o-induced inhibitions of outward K⁺ currents in Na⁺-based and NMDG⁺-based bath solutions for the channels. Whole-cell currents at 60 mV obtained in Na⁺-based or NMDG⁺-based bath solutions with pH 6.0 were normalized by those recorded in pH 7.4. *, p < 0.001. F, reversal potentials of TASK-3 K⁺ channels were plotted as a function of [K⁺]_o in Na⁺-based solutions with pH 7.4 (black triangles) or 6.0 (red triangles) (n = 11–29). The continuous curves are fits with the Goldman-Hodgkin-Katz equation, yielding a Na⁺ to K⁺ relative permeability of 0.006 at pH 7.4 and 0.13 at pH 6.0, respectively.

FIGURE 4.

Negative controls for low pH_o-induced changes in ion selectivity of TWIK-1, TASK-1, and TASK-3 K⁺ channels. A and B, whole-cell currents of TRESK-2 K⁺ channels are shown before (black or blue line) and after (red lines) change of pH from 7.4 to 6.0 in Na⁺-based (A) or NMDG⁺-based (B) bath solutions with 5 mm K⁺. C, comparison of low pH_o-induced inhibitions of outward TRESK-2 K⁺ currents in Na⁺-based and NMDG⁺-based bath solutions. Currents at 60 mV obtained in Na⁺-based or NMDG⁺-based bath solutions with pH 6.0 were normalized (Norm.) by those recorded in pH 7.4 from a group of experiments in A or B (n = 4–5). D–F, whole-cell currents of TREK-1, TASK-3, and TWIK-1 K⁺ channels are shown before (black or purple lines) and after (red lines) change of pH from 7.4 to 6.0 in Na⁺-based bath solutions with 5 mm K⁺ (D) or Mg²⁺-based bath solutions with 0 mm K⁺ (E and F) (n = 5–8). Insets in A and B and D–F: current traces are shown in shorter voltage ranges and narrow current amplitudes (−100 to +100 pA) so that the reversal potential or no inward Mg²⁺ current is clearly visible.

FIGURE 5.

Effects of lowered pH_o on the reversal potential and outward currents of TASK-1 K⁺ channels. A and B, whole-cell currents of TASK-1 K⁺ channels are shown before (black or blue line) and after (pink lines) change of pH from 7.4 to 6.8 in Na⁺-based (A) or NMDG⁺-based (B) bath solutions. Insets: current traces were shown in shorter voltage ranges and narrow current amplitudes (−100 to +100 pA) so that the reversal potentials are clearly visible. The green line in A represents the current after pH_o was switched back to 7.4 from 6.8. C, comparison of low pH_o-induced inhibitions of outward TASK-1 currents in Na⁺-based and NMDG⁺-based bath solutions. Whole-cell currents at 60 mV obtained in Na⁺-based or NMDG⁺-based bath solutions with pH of 6.8 were normalized (Norm.) by those recorded in pH of 7.4. *, p < 0.001. D, reversal potentials of TASK-1 K⁺ channels were plotted as a function of [K⁺]_o in Na⁺-based solutions with pH 7.4 (black squares) or 6.8 (pink squares) (n = 3–11). The continuous curves are fits with the Goldman-Hodgkin-Katz equation, yielding a Na⁺ to K⁺ relative permeability of 0.005 at pH 7.4 and 0.06 at pH 6.8, respectively.