Yang Z et al. (2000), Biophysical and molecular mechanisms underlying...

XB-ART-10762

J Gen Physiol 2000 Jul 01;1161:33-45.

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Biophysical and molecular mechanisms underlying the modulation of heteromeric Kir4.1-Kir5.1 channels by CO2 and pH.

Yang Z , Xu H , Cui N , Qu Z , Chanchevalap S , Shen W , Jiang C .

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CO2 chemoreception may be related to modulation of inward rectifier K+ channels (Kir channels) in brainstem neurons. Kir4.1 is expressed predominantly in the brainstem and inhibited during hypercapnia. Although the homomeric Kir4.1 only responds to severe intracellular acidification, coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivities to CO2 and pH. To understand the biophysical and molecular mechanisms underlying the modulation of these currents by CO2 and pH, heteromeric Kir4. 1-Kir5.1 were studied in inside-out patches. These Kir4.1-Kir5.1 currents showed a single channel conductance of 59 pS with open-state probability (P(open)) approximately 0.4 at pH 7.4. Channel activity reached the maximum at pH 8.5 and was completely suppressed at pH 6.5 with pKa 7.45. The effect of low pH on these currents was due to selective suppression of P(open) without evident effects on single channel conductance, leading to a decrease in the channel mean open time and an increase in the mean closed time. At pH 8.5, single-channel currents showed two sublevels of conductance at approximately 1/4 and 3/4 of the maximal openings. None of them was affected by lowering pH. The Kir4.1-Kir5.1 currents were modulated by phosphatidylinositol-4,5-bisphosphate (PIP2) that enhanced baseline P(open) and reduced channel sensitivity to intracellular protons. In the presence of 10 microM PIP2, the Kir4.1-Kir5.1 showed a pKa value of 7.22. The effect of PIP2, however, was not seen in homomeric Kir4.1 currents. The CO2/pH sensitivities were related to a lysine residue in the NH2 terminus of Kir4.1. Mutation of this residue (K67M, K67Q) completely eliminated the CO2 sensitivity of both homomeric Kir4.1 and heteromeric Kir4.1-Kir5.1. In excised patches, interestingly, the Kir4.1-Kir5.1 carrying K67M mutation remained sensitive to low pHi. Such pH sensitivity, however, disappeared in the presence of PIP2. The effect of PIP2 on shifting the titration curve of wild-type and mutant channels was totally abolished when Arg178 in Kir5.1 was mutated. Thus, these studies demonstrate a heteromeric Kir channel that can be modulated by both acidic and alkaline pH, show the modulation of pH sensitivity of Kir channels by PIP2, and provide information of the biophysical and molecular mechanisms underlying the Kir modulation by intracellular protons.

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Species referenced: Xenopus laevis
Genes referenced: kcnj10 kcnj16 pkm

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	Figure 1. Single-channel conductance of Kir4.1–Kir5.1. (A) Single-channel current was recorded from an inside-out patch using symmetric concentrations of K+ (150 mM) on both sides of the patch membrane at various membrane potentials (Vm). An active channel is seen with a clear inward rectification at hyperpolarizing Vm. Solid line, opening; dotted line, closure. (B) Single-channel conductance calculated from the channel in A is linear at Vm = −40 to −120 mV. The straight line represents a slope conductance of 59 pS.
	Figure 3. Dwell-time histograms of single channel Kir4.1–Kir5.1. (A and B) At pH 8.5, one active channel was recorded from an inside-out patch with equal concentrations of K+ on both sides of patch membrane and Vm of −80 mV. (A) Open dwell-time histogram of the Kir4.1–Kir5.1 current. Data fitting was done using four stretches of data collected from the same patch with an interval of 2 s and a total recording time of 80 s. The channel open dwell-time histogram can be fitted with two exponentials with time constants τO1 1.2 and τO2 59.9 ms. (B) Closed dwell-time histogram of this channel can also be fitted with three exponentials with time constants τC1 0.2, τC2 3.5, and τC3 224.6 ms. (C and D) Activity of the same channel (Popen) decreased by 50% at pH 7.5. (C) The open dwell-time histogram obtained from 10 stretches of data totaling 200 s is fitted using two exponentials with τO1 1.1 and τO2 41.2 ms. (D) Closed dwell-time histogram is expressed with three exponentials with τC1 0.8, τC2 3.2, and τC3 438.7 ms.
	Figure 2. Single-channel activity of the Kir4.1–Kir5.1 current. (A) Single-channel activity was recorded from an inside-out patch with equal concentrations of K+ on both sides of the patch membrane. An active channel was seen at Vm = −60 mV. At pH 8.5, this channel showed a high channel activity with Popen 0.915. In its long-lasting openings, brief closures can be seen that are better illustrated in extended time scales (a–c). a, b, and c are obtained from positions a, b, and c in the top trace, respectively. Also in a–c, sublevels of conductance can bee seen. Calibration: 2 s for the top trace and 200 ms for a–c; 2 pA for all. C, closure; S1, the first level of substate conductance; S2 the second level of substate conductance; S3, full opening. (B) All-point histogram of the same Kir4.1–Kir5.1 current shows channel openings at 3.78 pA at a membrane potential of −60 mV. Two sublevels of conductance are found between the closures and full openings with their peaks at ∼1/4 and 3/4 of the amplitude of full openings. Data are obtained from A as a stretch recording of 20 s, displayed as a logarithmic scale in the y axis and fitted using the Gaussian distribution with each peak at 0.0, 1.14, 3.11, and 3.78 pA.
	Figure 5. Concentration-dependent inhibition of single-channel activity by low pH. Single-channel currents were recorded from an inside-out patch using symmetric concentrations of K+ (150 mM) on both sides of the patch. At Vm of −80 mV, one active channel was seen at pHi 8.5, which had a Popen 0.934. No detectable change in channel activity was found at pH 8.0 (Popen 0.935). Channel activity decreased at pH 7.5 (Popen 0.548) and was almost completely inhibited at pHi 7.0 (Popen 0.001). Channel activity resumed to the baseline level (Popen = 0.899) after washout. C, closure; O, opening.
	Figure 4. Effects of pHi on macroscopic Kir4.1–Kir5.1 currents. (A) Kir4.1–Kir5.1 currents were recorded from a giant patch with equal concentrations of K+ (150 mM) on either side of the membrane. Inward-rectifying currents were seen at pH 8.5 using command potentials from −140 to 120 mV in 20-mV increments. When pH in the internal solution was reduced to 7.5, these currents were inhibited by ∼60%. Further decreases in pHi caused strong inhibitions of these currents. Inward-rectifying currents were almost totally suppressed at pH 6.5. Washout led to a recovery of these currents to the baseline level. (B) The relationship of Kir4.1–Kir5.1 currents with pHi. These currents are inhibited at pH 7.5, augmented at pH 8.0, and completely shut off at pH 6.5. The relationship of currents (I) to pHi can be expressed using the Hill equation (solid line): I = 1/[1 + (pKa/pHi)h], where pKa is the midpoint pH value for channel inhibition and h is the Hill coefficient. The pKa and h here are pH 7.45 and 2.3, respectively. In comparison, the Kir4.1 was inhibited at much lower pH levels, with pKa 5.99 and h 2.0. The pH sensitivity of Kir4.1–Kir5.1 but not homomeric Kir4.1 was modulated by PIP2. In the presence of 10 μM PIP2, its titration curve was shifted leftward by 0.22 pH U.
	Figure 6. Effects of intracellular pH on Popen and single-channel conductance. (A) At pHi 7.5, the channel Popen is only about a half of its value at pHi 8.5 (average Popen 0.846). The Popen reaches almost its maximum level at pHi 8.0, and becomes nearly zero at pHi 6.5. The relationship of Popen versus pHi can be expressed with the Hill equation (solid line). The pKa (the midpoint of the channel inhibition) and h (the Hill coefficient) are pH 7.48 and 2.3 (n = 6), respectively. (B) In contrast to Popen, single-channel conductance retains at ∼59 pS and does not change with pH levels. Data are presented as means ± SEM.
	Figure 7. Lack of effect of protons on substates of conductance. (A) Single-channel Kir4.1–Kir5.1 current was recorded from the same inside-out patch as Fig. 2 at pH 7.5. Two substate conductances were still seen with peaks at ∼1/4 and 3/4 of full openings, even though Popen was reduced 0.488 at such a pH level. C, closure; S1, substate 1; S2, substate 2. (B) All-point histogram of the current shows opening, closure, and two sublevels of conductance with each peak at 0.0, 1.18, 3.10, and 3.81 pA, respectively.
	Figure 8. Effects of Lys67 mutation on CO2 sensitivity of Kir4.1 and Kir4.1–Kir5.1 currents. (A) Whole-cell currents were recorded from oocytes. Inward-rectifier K+ currents were seen in an oocyte that had received an injection of K67M mutant Kir4.1. (B) Although the K67M mutation did not affect the baseline currents, channel sensitivity to 15% CO2 (5 min) was abolished. (C) The K67M Kir4.1 was created on a tandem dimer of Kir4.1–Kir5.1, the Kir4.1–Kir5.1 currents were observed 3 d after the injection. (D) These Kir4.1–Kir5.1 currents lost their hypercapnic sensitivity, as no evident change in the current amplitude was seen after 7 min exposure to 15% CO2. (E) Titration curves of Lys67 mutations on Kir4.1 and on a tandem dimer of Kir4.1–Kir5.1. While the Lys67 mutations abolished pHi sensitivity of Kir4.1, the Kir4.1–Kir5.1 with this mutation remained pHi sensitive (pKa 6.95, h 2.2). In the presence of 10 μM PIP2, however, Kir4.1–Kir5.1 carrying the K67M mutation lost its pHi sensitivity.
	Figure 9. Mutation of Arg178 in Kir5.1 eliminated the effect of PIP2 on channel sensitivity to pHi. In the tandem dimer of Kir4.1–Kir5.1, site-specific mutations were made at Lys67 in Kir4.1 and Arg178 in Kir5.1. Inward-rectifying currents were then studied in inside-out patches in the absence or presence of PIP2 (10 μM). The R178Q mutant showed pHi sensitivity similar to the wild-type Kir4.1–Kir5.1 with pK 7.50. Its pHi sensitivity was identical with or without PIP2 in the internal solution. The double mutant (K67M/R178Q) showed the pHi sensitivity (pK 7.15) similar to the Kir4.1–Kir5.1 with a single K67M mutation. Such pH sensitivity was not affected by PIP2.

References [+] :

Baukrowitz, PIP2 and PIP as determinants for ATP inhibition of KATP channels. 1998, Pubmed, Xenbase