Correspondence to: Chun Jiang, Associate Professor, Department of Biology, Georgia State University, 24 Peachtree Central Avenue, Atlanta, GA 30303-4010. Fax:404-651-2509 E-mail:cjiang{at}gsu.edu.
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Abstract |
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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 Kir4.1Kir5.1 were studied in inside-out patches. These Kir4.1Kir5.1 currents showed a single channel conductance of 59 pS with open-state probability (Popen) 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 Popen 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
1/4 and 3/4 of the maximal openings. None of them was affected by lowering pH. The Kir4.1Kir5.1 currents were modulated by phosphatidylinositol-4,5-bisphosphate (PIP2) that enhanced baseline Popen and reduced channel sensitivity to intracellular protons. In the presence of 10 µM PIP2, the Kir4.1Kir5.1 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 Kir4.1Kir5.1. In excised patches, interestingly, the Kir4.1Kir5.1 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.
Key Words: CO2 chemoreception, pH, phosphatidylinositol-4,5-bisphosphate, excised patch, brainstem
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INTRODUCTION |
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Inward rectifier K+ channels (Kir channels)1 are the primary regulators of membrane excitability and are themselves also regulated by several intra- and extracellular factors (
Several cloned Kir channels respond to CO2 and pH similarly to the K+ currents seen in brainstem neurons. Kir1.1 and Kir1.2 are inhibited by a decrease in intracellular pH (
In contrast, Kir4.1 is expressed predominantly in the brainstem ( 6.0), coexpression of Kir4.1 with the brain Kir5.1 greatly enhances CO2 and pH sensitivities of the heteromeric Kir4.1Kir5.1 channels (
Several mechanisms can underlie the modulation of the heteromeric Kir4.1Kir5.1 by CO2. Our previous studies have shown that Kir channel inhibition during hypercapnia is mediated by protons rather than molecular CO2 (
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METHODS |
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Oocytes from female frogs (Xenopus laevis) were used in the present studies. Frogs were anesthetized by bathing them in 0.3% 3-aminobenzoic acid ethyl ester. A few lobes of ovaries were removed after a small abdominal incision (5 mm). The surgical incision was closed and the frogs were allowed to recover from the anesthesia. Xenopus oocytes were treated with 2 mg/ml of collagenase (Type I; Sigma-Aldrich) in OR2 solution (mM): 82 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES, pH 7.4, for 90 min at room temperature. After three washes (10-min each) of the oocytes with OR2 solution, cDNAs (2550 ng in 50 nl double-distilled water) were injected into the oocytes. The oocytes were then incubated at 18°C in ND-96 solution containing (mM): 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES, and 2.5 sodium pyruvate with 100 mg/liter geneticin added (pH 7.4).
Brain Kir4.1 (BIRK10) and brain Kir5.1 (BIRK9) cDNAs were generously provided by Dr. John Adelman (Organ Health Science University) (
Whole-cell currents were studied on the oocytes 24 d after injection. Two-electrode voltage clamp was performed using an amplifier (Geneclamp 500; Axon Instruments Inc.) at room temperature (2326°C). The extracellular solution contained (mM): 90 KCl, 3 MgCl2, and 5 HEPES, pH 7.4. Cells were impaled using electrodes filled with 3 M KCl. The potential leakage of KCl from the recording electrodes was not corrected because of the large volume of oocytes. One of the electrodes (1.02.0 M) served as voltage recording, which was connected to an HS-2 x 1L headstage (input resistance = 1011
), and the other electrode (0.30.6 M
) was used for current recording connected to an HS-2 x 10MG headstage (maximum current = 130 µA). Oocytes were accepted for further experiments only if their leak currents, measured as a difference before and after leak subtractions, were <10% of the peak currents. The leak subtraction was applied to oocytes if their leak currents were 510%, which was done by subtracting a sum of currents produced by five small depolarizing prepulses in 1/5 of command potentials. Current records were low-pass filtered (Bessel, four-pole filter, 3 dB at 5 kHz), digitized at 5 kHz (12-bit resolution), and stored on computer disk for later analysis (PCLAMP 6; Axon Instruments, Inc.) (
Patch-clamp experiments were performed at room temperature (25°C) as described previously (
) were made from 1.2-mm borosilicate capillary glass (Sutter Instrument Co.). Single channel currents were recorded from inside-out, outside-out, and cell-attached patches (
1.0 M
. Current records were low-pass filtered (2,000 Hz, Bessel, four-pole filter, -3 dB), digitized (10 kHz, 12-bit resolution), and stored on computer disk for later analysis (PCLAMP 6; Axon Instruments, Inc.). Junction potentials between bath and pipette solutions were appropriately nulled before seal formation.
For single channel recordings, the oocyte vitelline membranes were mechanically removed after being exposed to hypertonic solution (400 mOsm) for 15 min. The stripped oocytes were placed in a petri dish containing regular bath solution (see below). Recordings were performed using solutions containing equal concentrations of K+ applied to the bath and recording pipettes. The bath solution contained (mM): 40 KCl, 75 potassium gluconate, 5 potassium fluoride, 0.1 sodium vanadate, and 10 potassium pyrophosphate, 1 EGTA, 0.2 ADP, 10 PIPES, 10 glucose, and 0.1 spermine (FVPP solution, pH 7.4). The pipette was filled with the same FVPP solution used in the bath or a solution containing (mM): 40 KCl, 110 potassium gluconate, 0.2 ADP, 1 EGTA, 10 HEPES, 10 glucose, 2 MgCl2, pH 7.4. This bath solution was chosen after several others had been tested regarding channel rundown in excised patches. In a control experiment, we found that macroscopic currents recorded from giant inside-out patches were very well maintained, showing <10% reduction over a 17-min period of recordings in such a bath solution. This period is sufficient for all our single-channel recording protocols that were designed to be completed generally within 10 min.
CO2 exposures were performed in a semi-closed recording chamber (BSC-HT; Medical System), in which oocytes were placed on a supporting nylon mesh, and the perfusion solution bathed both the top and bottom surface of the oocytes. The perfusate and the superfusion gas entered the chamber from two inlets at one end and flowed out at the other end. There was a 3 x 15-mm gap on the top cover of the chamber, which served as the gas outlet and the access to the oocytes for recording microelectrodes. The perfusate contained (mM): 90 KCl, 3 MgCl2, and 5 HEPES, pH 7.4. At baseline, the chamber was ventilated with atmospheric air. Exposure of the oocytes to CO2 was carried out by switching a perfusate that had been bubbled for at least 30 min with a gas mixture containing CO2 (5, 10, or 15%) balanced with 21% O2 and N2, and superfused with the same gas. The high solubility of CO2 resulted in a detectable change in intra- or extracellular acidification as fast as 10 s in these oocytes. Thus, in most experiments, only the superfusion air was switched to CO2, in which similar results were produced.
A parallel perfusion system was used to administer agents to patches or cells at a rate of 1 ml/min with no dead space (
For single-channel analysis, data were further filtered (01,000 Hz) with a Gaussian filter. This filtering causes events shorter than 100 µs to be ignored. No correction was attempted for the missed events. Single channel conductance was measured as a slope conductance with at least two voltage points. Popen was calculated by first measuring the time, tj, spent at current levels corresponding to j = 0,1,2,...n channels open (nj = 1 tj j)/tn, where n is the number of channels active in the patch and t is the duration of recordings. Popen values were calculated from stretches of data having a total duration of 20200 s. Open and closed times were measured from records in which only a single channel was active. The open- and closed-time distributions were fitted using the method of maximum likelihood (
Data are presented as means ± SEM (n = number of patches). Differences in means were tested with the Student's t test and were accepted as significant if P 0.05.
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RESULTS |
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Inhibition of Kir Currents by CO2
Effects of CO2 on K+ currents were studied using a high K+ (90 mM) bath solution. Membrane potential was held at 0 mV and stepped from -160 to 100 mV in 20-mV increments. Under such a condition, clear inward rectifying currents were observed in the oocytes receiving an injection of Kir4.1 or a tandem-linked Kir4.1 and Kir5.1 (Kir4.1Kir5.1) cDNA 23 d earlier. Exposure of these oocytes to 15% CO2 for 46 min produced an inhibition of Kir4.1 and Kir4.1Kir5.1 currents. The degree of the inhibition was different between these two currents. At the maximal inhibition (measured at -120 mV), the Kir4.1Kir5.1 currents were suppressed by 59.4 ± 3.7% (n = 9) and the Kir4.1 by only 23.3 ± 4.8% (n = 8). The effect of CO2 on these currents was reversible and depended on CO2 concentrations. Evident inhibition of these Kir currents was seen with 5% CO2. Higher concentrations of CO2 (1015%) induced a much stronger inhibition of these currents. This inhibition was likely to be mediated by intracellular acidification, since lowering intra- but not extracellular pH to levels measured during 15% CO2 produced a similar inhibition to 15% CO2. These results are therefore consistent with our previous observations (
Baseline Single Channel Properties of Kir4.1Kir5.1
To understand channel biophysical properties underlying the effect of protons, single-channel activity was studied in inside-out patches, after the expression of Kir currents was identified in the two-electrode voltage-clamp mode. These patches were exposed to symmetric concentrations of K+ (150 mM) on both sides of plasma membranes; command potentials from -120 to 100 mV were applied to these patches. When inward-rectifying currents were seen, the slope conductance was first measured. Fig 1 A shows a single-channel current recorded from a Kir4.1Kir5.1-injected oocyte. The current showed a clear inward rectification with the slope conductance of 59 pS in the inside-out patch configuration (Fig 1 B). In comparison with the homomeric Kir4.1, the single channel conductance of the Kir4.1Kir5.1 was much larger, which averaged 59.2 ± 1.4 pS (n = 17) vs. 22.2 ± 0.5 pS (n = 22) in the Kir4.1 currents. Although the Kir4.1 showed a high baseline channel activity (Popen = 0.890 ± 0.021, n = 5), the heteromeric Kir4.1Kir5.1 channels had a mean Popen of only 0.416 ± 0.095 (n = 6) at pH 7.4. When the pH level in the internal solution increased to pH 8.5, a large increase in channel activity was seen (Fig 2 A), suggesting that channel activity was partially inhibited at physiological pH level. The Popen was not affected by a change in membrane potential from -100 to -40 mV in both Kir4.1 and Kir4.1Kir5.1. At pH 8.0 and 8.5, single channel Kir4.1Kir5.1 currents showed two sublevels of conductance, which were 1/4 and 3/4 of the conductance of the full open state (Fig 2A and Fig B). No bursting activity was observed at pH 8.0 and 8.5.
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At pH 8.5, single-channel Kir4.1Kir5.1 currents showed long periods of openings and short periods of closures (Fig 2). The mean open time of these currents was 49.4 ± 3.6 ms (n = 4), and the mean closed time was 12.6 ± 4.5 ms (n = 4). Fig 3A and Fig B, illustrates dwell-time histograms of the Kir4.1Kir5.1 currents, in which two components of time constant are revealed for the channel open state and three for the closed state (O1 = 0.9 ± 0.2 ms;
O2 = 57.0 ± 3.8 ms;
C1 = 0.3 ± 0.1 ms;
C2 = 4.0 ± 0.3 ms;
C3 = 135.5 ± 53.0 ms; n = 5).
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It is known that activity of Kir6, Kir3, and Kir1 members is also subject to the modulation of phosphatidylinositol-4,5-bisphosphate (PIP2) (
Modulation of Macroscopic Currents by Intracellular Protons
The effect of pHi on macroscopic Kir currents was studied using giant patches under conditions similar to that described above. At pH 8.5, inward-rectifying currents as large as 2 nA were seen in most patches obtained from Kir4.1Kir5.1-injected oocytes at a membrane potential of -100 mV. Fig 4 illustrates modulations of the Kir4.1Kir5.1 currents by exposures of the internal patch membrane to solutions of various pH levels. The current amplitude remained roughly the same at pH 8.0 and 8.5, started decreasing at pH 7.5, and reached almost zero at pH 7.06.5 (Fig 4 A). This effect was fast, reversible, and dependent on pH levels. The relationship of the current amplitude to pHi was described using the Hill equation with pKa (pH level at a half of the maximal currents) 7.45 and the Hill coefficient (h) 2.3 (n = 9; Fig 4 B). When the affected currents obtained by subtraction of the remaining currents at pH 7.5 from those at pH 8.5 were scaled to the same amplitude of the baseline currents, slopes of these two current recordings were almost identical, suggesting that the pH effect is not a voltage-dependent process (not shown). In comparison, the Kir4.1 was only inhibited at much lower pH levels with pKa 5.99 and h 2.0 (Fig 4 B).
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The pHi sensitivity of the Kir4.1Kir5.1 was affected by PIP2. The titration curve left-shifted by 0.23 pH U in the presence of 10 µM PIP2 (Fig 4 B, Table 1). Similar exposure to PIP2, however, had no evident effect on the pHi sensitivity of Kir4.1 currents (Fig 4 B, Table 1).
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Effects of pHi on Single Channel Properties of Kir4.1Kir5.1
Fig 5 shows modulations of single channel activity in a conventional inside-out patch when the internal surface of the patch was exposed to solutions with different pH levels. The Kir4.1Kir5.1 current showed a high channel activity at pH 8.5 (Popen 0.934) and pH 8.0 (Popen 0.935). Channel activity started being inhibited at pH 7.5. This inhibition is due to the appearance of repetitive bursting activity with no detectable reduction in the current amplitude (Fig 5). The channel was shut off at pH 6.5. The Popen of the Kir4.1Kir5.1 currents also can be expressed as a function of pHi with pKa 7.48 and h 2.3 (n = 6) (Fig 6 A).
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The inhibition of Popen was due to a decrease in the channel mean open time and an increase in the mean closed time. At pHi 7.5, single-channel Kir4.1Kir5.1 currents had a mean open time of 25.0 ± 9.2 ms (n = 4), and the mean closed time 48.6 ± 7.4 ms (n = 4). These figures are significantly different from those obtained at pH 8.5 (see above, P < 0.05). At pHi 7.5, the dwell-time histograms still showed two components of time constant in the open state (O1 = 1.3 ± 0.4 ms;
O2 = 30.3 ± 8.4 ms; n = 4) and three in the closed state (
C1 = 0.4 ± 0.1 ms;
C2 = 7.5 ± 4.3 ms;
C3 = 471.9 ± 173.8 ms; n = 4) (Fig 3C and Fig D). Among these components, the
O2 was significantly reduced, and the
C3 increased at pH 7.5 in comparison with those at pH 8.5 (P < 0.05). The reduction in the long-lasting openings and the increase in long-lasting closures are consistent with the observed bursting activity emerging at pH 7.5.
The single-channel conductance was examined at these pH levels. Fig 6 B shows that the single channel conductance is fairly constant at a pH range of 7.08.5, indicating that protons selectively inhibit Popen without affecting the single-channel conductance. At pH 7.0 and 7.5, when channel activity was markedly inhibited, the substate conductances were still clearly seen (Fig 7), suggesting these sublevels of conductance are independent of intracellular protons.
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Critical Role of Lysine 67 in pHi Sensing
To understand the molecular mechanisms underlying the pH sensitivity of the Kir4.1Kir5.1 and Kir4.1 channels, we studied the Lys67 in Kir4.1 and Thr68 in Kir5.1 using site-directed mutagenesis. The reason for choosing these residues is that, at corresponding positions, a lysine residue (Lys80) in Kir1.1 and a threonine in Kir2.3 have been known to be critical in pH sensing of these channels (
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The effects of the Lys67 mutations on channel sensitivity to pHi were examined in inside-out patches. Consistent with our data obtained from the whole-cell voltage clamp, the K67M mutation completely eliminated pHi sensitivity of the homomeric Kir4.1 (Fig 8 E). Interestingly, the heteromeric Kir4.1Kir5.1 with the K67M mutation remained pHi sensitive. Such pHi sensitivity, however, was eliminated in the presence 10 µM PIP2 (Fig 8 E); i.e., this mutant was only modestly inhibited at extremely acidic pHi with pH sensitivity similar to the K67M-mutant Kir4.1. Since there are micromolar concentrations of PIP2 in oocytes (
To understand how PIP2 affects pHi sensitivity of the Kir4.1Kir5.1, we mutated the potential PIP2-binding site Arg178 in Kir5.1. The Arg178 is a conserved residue found in most Kir channels, which has been shown to be a potential PIP2-binding site in Kir6.2 (
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DISCUSSION |
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In our current studies, we have demonstrated the first K+ channel that responds to either an increase or a decrease in intracellular pH from its physiological level, shown the modulation of pH sensitivity of Kir channels by PIP2, and provided information about the biophysical and molecular mechanisms underlying the modulation of the heteromeric Kir4.1Kir5.1 channels by CO2 and pH.
Baseline Single-Channel Properties
Baseline single-channel properties of the homomeric Kir4.1 and heteromeric Kir4.1Kir5.1 have not been well studied previously. The only known single-channel property as yet is the conductance. In cell-attached patches, the Kir4.1 shows a single-channel conductance of 16 pS and the Kir4.1Kir5.1 has 40 pS (30% larger than those measured in cell-attached patches, which seems to result from different K+ concentrations used in these two experimental conditions (150 mM in the present study vs. 90 mM by
0.4) that increases with an increase in pHi and decreases with a drop in pHi, suggesting that these channels are potentially down or up regulated by either acidic or alkaline pH. At pH 8.08.5, when channel activity reaches its maximum, the Kir4.1Kir5.1 channels show two open and three closed states with a mean open time of
50 ms. Another interesting feature of these channels is the substate of conductance. These K+ channels show two sublevels of conductance a
1/4 and 3/4 of the conductance of the full openings at pH 8.08.5. The sublevels of conductance have also been observed in outward currents of Kir2.1 (
It is known that baseline channel activity of Kir channels can be modulated by PIP2 (
Detection of CO2 and Intracellular Protons
Several members of the Kir4 family have been demonstrated to be pH sensitive.
Mechanisms for the Proton Sensitivity of these Kir Channels
Our previous studies have indicated that a decrease in pH during CO2 exposure is the primary cause for the Kir channel inhibition (
Interestingly, we found that the pH sensitivity of the heteromeric Kir4.1Kir5.1 is modulated by PIP2. Channel sensitivity to intracellular protons is reduced in the presence of PIP2. Considering the presence of micromolar concentrations of PIP2 in an intact cell (
The inhibition of Kir channels by low pHi is unlikely to be caused by changes in concentrations of cytosolic soluble factors such as second messengers, polyamines, or Mg2+ since it is seen in cell-free excised patches. Also, our results do not support the idea that protein phosphorylation is responsible for the modulation of these K+ currents by intracellular protons. Several blockers of phosphatase and phosphodiesterase such as vanadate, fluoride, and pyrophosphate were used in the intracellular solution. These chemicals tend to inhibit protein dephosphorylation. In addition, there was no Mg2+ or ATP in this intracellular solution. Under such a condition, the turnover of protein phosphorylation and dephosphorylation should not occur, at least in the time frame of our low pH experiments (0.52.0 min). Therefore, the modulation of the Kir4.1Kir5.1 during low pH may not be a result of protein phosphorylation.
Our studies suggest that amino acid sequences and tertiary structures in Kir channel proteins are the molecular basis underlying the modulation of these K+ channels by protons. We have found that Lys67 in the Kir4.1 is critical in the modulation. Channel sensitivity to CO2 is completely eliminated when this lysine residue is mutated to methionine or glutamine. A lysine residue found at the same position of Kir1.1 and Kir1.2 channels has been demonstrated to play a crucial role in pH sensing of these channels (
We understand that this lysine residue may also be a part of the gating mechanisms in these Kir channels. With an interruption of the gating mechanism, the Kir4.1 and Kir4.1Kir5.1 may not be able to close appropriately during hypercapnia or intracellular acidification, and thus show a marked decrease in pH sensitivity. Since the binding versus gating has been a common problem in studies of all ligand-gated ion channels (
Functional Implications
Inward-rectifier K+ channels are important players in the maintenance of plasma membrane excitability and the control of intra- and extracellular K+ ionic homeostasis. In the brainstem where the Kir4.1 and Kir5.1 channels are expressed, the inhibition of heteromeric Kir4.1Kir5.1 channels by hypercapnia can have a major impact not only on cells expressing these channels, but also on local neuronal networks. The inhibition of these K+ channels produces depolarization and increases membrane excitability (
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Footnotes |
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1 Abbreviations used in this paper: Kir channel, inward rectifier K+ channel; PIP2, phosphatidylinositol-4,5-bisphosphate.
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Acknowledgements |
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We thank Dr. John Adelman for his generosity in sharing with us the Kir4.1 and Kir5.1 cDNAs.
This work was supported by the National Institutes of Health (RO1 HL58410-01) and a Grant-in-Aid award (9950528N) from the American Heart Association.
Submitted: 28 January 2000
Revised: 17 May 2000
Accepted: 18 May 2000
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References |
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