Dahlmann A et al. (2004), Regulation of Kir channels by intracellular pH ...

XB-ART-3767

J Gen Physiol 2004 Apr 01;1234:441-54. doi: 10.1085/jgp.200308989.

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Regulation of Kir channels by intracellular pH and extracellular K(+): mechanisms of coupling.

Dahlmann A , Li M , Gao Z , McGarrigle D , Sackin H , Palmer LG .

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ROMK channels are regulated by internal pH (pH(i)) and extracellular K(+) (K(+)(o)). The mechanisms underlying this regulation were studied in these channels after expression in Xenopus oocytes. Replacement of the COOH-terminal portion of ROMK2 (Kir1.1b) with the corresponding region of the pH-insensitive channel IRK1 (Kir 2.1) produced a chimeric channel (termed C13) with enhanced sensitivity to inhibition by intracellular H(+), increasing the apparent pKa for inhibition by approximately 0.9 pH units. Three amino acid substitutions at the COOH-terminal end of the second transmembrane helix (I159V, L160M, and I163M) accounted for these effects. These substitutions also made the channels more sensitive to reduction in K(+)(o), consistent with coupling between the responses to pH(i) and K(+)(o). The ion selectivity sequence of the activation of the channel by cations was K(+) congruent with Rb(+) > NH(4)(+) > Na(+), similar to that for ion permeability, suggesting an interaction with the selectivity filter. We tested a model of coupling in which a pH-sensitive gate can close the pore from the inside, preventing access of K(+) from the cytoplasm and increasing sensitivity of the selectivity filter to removal of K(+)(o). We mimicked closure of this gate using positive membrane potentials to elicit block by intracellular cations. With K(+)(o) between 10 and 110 mM, this resulted in a slow, reversible decrease in conductance. However, additional channel constructs, in which inward rectification was maintained but the pH sensor was abolished, failed to respond to voltage under the same conditions. This indicates that blocking access of intracellular K(+) to the selectivity filter cannot account for coupling. The C13 chimera was 10 times more sensitive to extracellular Ba(2+) block than was ROMK2, indicating that changes in the COOH terminus affect ion binding to the outer part of the pore. This effect correlated with the sensitivity to inactivation by H(+). We conclude that decreasing pH(I) increases the sensitivity of ROMK2 channels to K(+)(o) by altering the properties of the selectivity filter.

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Species referenced: Xenopus laevis
Genes referenced: kcnj1 kcnj2

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	Figure 1. . ROMK2/IRK1 chimeras. (A) Each protein is divided into the cytoplasmic NH2 and COOH terminus, the two transmembrane domains M1 and M2, and the extracellular loop (ECL), which includes the pore region (P) containing the selectivity filter. Light segments represent components from ROMK2; shaded segments represent components from IRK1. Arrows indicate the positions of the three point mutations K61M, I159V, and I163M. (B) Sequence comparison of ROMK2 and C127.
	Figure 2. . pH dependence of ROMK2 and C13. (A and C) Current traces for ROMK2 and C13 at different extracellular/intracellular pH. External solutions contained 55 mM KCl and 55 mM K acetate adjusted to the indicated pH values. Holding potential was 0 mV. Currents were measured at test potentials of −60 to +60 mV. Reducing pHo from 8.2 to 7.4 had little effect on ROMK2 currents (A) but markedly reduced the inward currents through the C13 chimera (C). (B and D) Steady-state I-V relationships for ROMK2 (B) and C13 (D) at different pH.
	Figure 3. . pH dependence of inward conductance through ROMK2, C13, and ROMK2 K61M. (A) Conductance was measured in experiments similar to those shown in Fig. 2. Values were normalized to those at pH 8.2 and plotted as a function of external pH. Data represent means ± SEM for 4–6 oocytes. (B) Conductance measured in cut-open oocytes, expressing either ROMK2 or C13. Data were normalized to values obtained at pHi = 9. ROMK2: open squares, solid line, pKa = 6.8 ± .03, Hill coefficient 2.2 ± 0.3 (n = 4). C13: solid circles, dashed line, pKa = 7.7 ± .04, Hill coefficient 2.3 ± 0.3 (n = 7).
	Figure 4. . pH dependence of inward conductance through the ROMK2 mutants I163M, L160M, and I159V and the C13 mutant “C13-3M” (C13 V159I/M160L/M163I). Conductance was measured and plotted as in Fig. 3 A. Data represent means ± SEM for 4–5 oocytes. Dashed and dotted lines show data for ROMK2 and C13, respectively, from Fig. 3 A.
	Figure 5. . Effect of external K+o on inward conductance through C13. (Left) Current traces for a C13-expressing oocyte in 10 mM K+ (A), 1 min after increasing K+ to 25 mM (B) and 40 min after the solution change (C). (Bottom right) Time course of inward conductance. (Top right) I-V curves at times A–C.
	Figure 6. . Steady-state concentration-conductance relationships for ROMK2, C13, C13–3M (A), and for I163M and I159V (B). Oocytes were perfused with solutions containing KCl concentrations of 1–110 mM, substituting for NaCl. Data represent means ± SEM for 5–11 oocytes.
	Figure 7. . Effect of pH and Ko+ on inward conductance through C107 and C107 I163M. (A) pH dependence. (B) Ko+ dependence. Data represent means ± SEM for five oocytes.
	Figure 8. . Activation of C13 and ROMK2 channels by extracellular K+, Rb+, and NH+4. Oocytes were incubated in medium containing 1 mM permeant ion + 109 mM NaCl until a steady-state conductance was achieved. Permeant ion concentrations were then increased in steps up to 110 mM, substituting for Na+. Conductance was normalized to maximal values measured at the 110 mM concentration. (A and B) Current-voltage relationships for C13 with increasing concentrations of K+ (A) or NH4+ (B). (C) Conductance-concentration relationships for C13. (D) Conductance-concentration relationships for ROMK2. Smooth curves are drawn through the data points and have no theoretical meaning. Data represent the means ± SEM for 4–6 oocytes.
	Figure 9. . Schematic representation of two possible mechanisms of coupling between inner and outer channel gates, and an experimental test of coupling through restricted K+ access.
	Figure 10. . Effect of a depolarizing holding potential on the inward conductance of strongly rectifying channels. (A) Time course of inward conductance changes with C13. Oocytes were superfused with solution containing 25 mM K. Inward conductances were measured between −60 and −80 mV with a holding potential of −40 mV, close to EK and the resting membrane potential. After a steady-state was achieved, the holding potential was changed to +10 mV, and the conductance continued measured at the same test potentials. After 20 min the holding potential was returned to −40 mV. Data represent means ± SEM for five oocytes. (B) Summary of experiments similar to those shown in A using C13 under different initial conditions, including initial holding potential and K+o concentration. (C) Summary of experiments similar to those shown in A using different pH-insensitive channels. The plots in B and C show fractional changes in conductance in response to a 50-mV depolarization. Data represent means ± SEM for 4–6 oocytes.
	Figure 11. . Ba2+ block of K+ channels. Oocytes were superfused with solution containing 110 mM KCl. BaCl2 was then added to the solution at concentrations of 0.01, 0.1, 1, and 10 mM. Currents were measured at voltages between −20 and −160 mV under each condition and were normalized to values obtained in the absence of Ba2+. (A and B) Typical traces for ROMK2 (A) and C13 (B) -expressing oocytes with zero, submaximal (0.1 mM), and near-maximal (10 mM) Ba2+. (C) Dose–response relationships of different channels at a voltage of −160 mV. Data represent means ± SEM (shown for representative data points) of 4–7 oocytes. (D) KBa values as a function of voltage. Mean values obtained from best-fits to the data points to the equation: KBa (V) = KBa(0)exp(2FδBaV/RT). Values of KBa(0) (mM) and δBa were: ROMK2 (13.8; 0.27) C13 (1.4; 0.21) C25 (7.8; 0.21) C13K61M (10.0; 0.19) ROMK2I163M (2.8; 0.23).
	Figure 12. . Effect of pH on Ba2+ block of ROMK2 channels. Data were obtained in experiments similar to those shown in Fig. 5 using pHo 7.8 (A) and 7.0 (B). Best fits to the data using the equation described in Fig. 6 gave values of KBa(0) = 10.4 mM (pH 7.8) and 1.5 mM (pH 7.0). (C and D) Voltage dependence of block at pH 7.8 (C) and 7.0 (D). Values of δBa were 0.23 (pH 7.8) and 0.24 (pH 7.0), respectively.
	Figure 13. . Possible interactions between I163 at the end of the second transmembrane helix of subunit A with residues (e.g., W50) on the slide helix of subunit D. The relevant residues for Kir 1.1 pH gating (space-filled) are superimposed on the crystal structure of a KirBac1.1. Only 2 of 4 KirBac1.1 subunits are shown.

References [+] :

Armstrong, Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons. 1971, Pubmed