Direct Activation of Cloned KATP Channels by Intracellular Acidosis*

Haoxing XuDagger, Ningren CuiDagger, Zhenjiang Yang, Jianping Wu, Lande R. Giwa, Latifat Abdulkadir, Puja Sharma, and Chun Jiang§

From the Department of Biology, Georgia State University, Atlanta, Georgia 30302-4010

Received for publication, October 23, 2000, and in revised form, January 22, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ATP-sensitive K+ (KATP) channels may be regulated by protons in addition to ATP, phospholipids, and other nucleotides. Such regulation allows a control of cellular excitability in conditions when pH is low but ATP concentration is normal. However, whether the KATP changes its activity with pH alterations remains uncertain. In this study we showed that the reconstituted KATP was strongly activated during hypercapnia and intracellular acidosis using whole-cell recordings. Further characterizations in excised patches indicated that channel activity increased with a moderate drop in intracellular pH and decreased with strong acidification. The channel activation was produced by a direct action of protons on the Kir6 subunit and relied on a histidine residue that is conserved in all KATP. The inhibition appeared to be a result of channel rundown and was not seen in whole-cell recordings. The biphasic response may explain the contradictory pH sensitivity observed in cell-endogenous KATP in excised patches. Site-specific mutations of two residues showed that pH and ATP sensitivities were independent of each other. Thus, these results demonstrate that the proton is a potent activator of the KATP. The pH-dependent activation may enable the KATP to control vascular tones, insulin secretion, and neuronal excitability in several pathophysiologic conditions.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hypercapnia and acidosis affect vascular tone, skeletal muscle contractility, insulin secretion, epithelial transport, and neuronal excitability, which may be mediated by KATP1 (1-5). However, previous studies on the pH sensitivity of these K+ channels were controversial and even contradictory. In the absence of ATP, acidic pH was shown to stimulate cell-endogenous KATP (6, 7), inhibit it (8, 9), and have little or no effect (10, 11). This inconsistency is further complicated by the indirect effect of ATP or Mg2+ and tissue-specific KATP species (8-12). Consequently, it is unclear whether KATP is modulated during hypercapnia and acidosis and what molecular mechanisms are underlying the modulations. The cloned KATP channels are ideal for addressing these questions because they allow for fine dissection of the modulatory mechanisms and elaborate manipulation of PCO2 and pH in an expression system (13, 14). Therefore, we studied the modulation of the cloned KATP (Kir6 with SUR, Ref. 15) by CO2 and acidic pH. To locate the pH sensors, we also studied Kir6.2 with a truncation of 36 amino acids at the C terminus (Kir6.2Delta C36) because it expresses functional channel without the SUR subunit and retains fair ATP sensitivity (16).

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Oocytes from 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). Then, the surgical incision was closed and the frogs were allowed to recover from the anesthesia. Xenopus oocytes were treated with 2 mg/ml collagenase (Type I, Sigma) in an OR2 solution consisting of (in mM) NaCl 82, KCl 2, MgCl2 1, and HEPES 5 (pH 7.4) for 90 min at room temperature. After three washes (10 min each) of the oocytes with the OR2 solution, cDNAs (25-50 ng in 50 nl of water) were injected into the oocytes. For coexpression, Kir6.x and SUR1 were injected in a 1:2 ratio. The oocytes were then incubated at 18 °C in the ND-96 solution containing (in mM) NaCl 96, KCl 2, MgCl2 1, CaCl2 1.8, HEPES 5, and sodium pyruvate 2.5 with 100 mg/liter geneticin added (pH 7.4).

Rat Kir6.1 (uKATP, GenBankTM/EBI accession number D42145) and mouse Kir6.2 (mBIR, GenBankTM/EBI accession number D50581) cDNAs were generously provided by Dr. S. Seino. Hamster SUR1 (GenBankTM accession number L40623) was a gift from Dr. L. Bryan. The cDNAs were subcloned to a eukaryotic expression vector (pcDNA3.1, Invitrogen Inc., Carlsbad, CA) and used for Xenopus oocyte expression without cRNA synthesis. Site-specific mutations were produced using a site-directed mutagenesis kit (Stratagene, La Jolla, CA). The orientation of the constructs and correct mutations were confirmed with DNA sequencing.

Whole-cell currents were studied on the oocytes 2-4 days after injection using a two-electrode voltage clamp with an amplifier (Geneclamp 500, Axon Instruments Inc., Foster City, CA) at room temperature (~24 °C). The extracellular solution contained (in mM) KCl 90, MgCl2 3, and HEPES 5 (pH 7.4). Extracellular acidification was done by titrating the extracellular solution to desired pH levels. The HEPES buffer was chosen because of its buffering range and membrane impermeability, as shown in our previous studies (17, 18). In intracellular acidification experiments, 90 mM KCl was replaced with the same concentration of KHCO3 (pH titrated to 7.4), so that the K+ concentration remained the same in these experiments (19). When oocytes were exposed to these perfusates, intracellular pH (pHi) and extracellular pH (pHo) were measured using ion-selective microelectrodes as described previously (14). Whole-cell currents were also studied with nigericin (10 µM) at various bath pH levels. This protonophore forms cation channels permeable primarily to protons (20, 21). Using 90 mM K+ in the bath solution as permeable cation, pHi becomes the same as pHo in the presence of nigericin (20). Exposure to nigericin increased oocyte-endogenous currents at each pH point. The current changes in these pH points were measured in oocytes without any injection, averaged (n = 4), and subtracted from current records of the Kir6-expressing cells because the alterations were due to nigericin rather than changes of pHi in these cells.

Experiments were performed in a semiclosed recording chamber (BSC-HT, Medical Systems Corp., Greenvale, NY) in which oocytes were placed on a supporting nylon mesh; 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 × 15-mm gap on the top cover of the chamber, which served as the gas outlet and an access to the oocytes for recording microelectrodes. 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 at various concentrations balanced with 21% O2 and N2 and superfused with the same gas (14, 17, 18). The high dissolvability of CO2 resulted in a detectable change in intra- or extracellular acidification as fast as 10 s in these oocytes.

Macroscopic and single-channel currents were recorded in excised patches at room temperature (~24 °C) as described previously (22, 23). In brief, the oocyte vitelline membranes were mechanically removed after being exposed to hypertonic solution (400 mosmol) for 5 min. Recordings were performed on the stripped oocytes using the same solution applied to bath and recording pipettes. The solution contained (in mM) KCl 10, potassium gluconate 130, potassium fluoride 5, EGTA 1, and HEPES 10 (pH 7.4). A parallel perfusion system was used to deliver low pH perfusates at a rate of ~1 ml/min with no dead space (22, 23). Macroscopic and single-channel currents were analyzed using the pClamp 6 software as detailed previously (22, 23).

Data are presented as means ± S.E. analysis of variance or Student's t test was used. Differences of CO2 and pH effects before versus during exposures were considered to be statistically significant if p <=  0.05.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expressions of Kir6.2 together with SUR1 (Kir6.2+SUR1) in Xenopus oocytes produced K+ currents with clear inward rectification in whole-cell recordings and ATP-dependent inhibition in inside-out patches. When oocytes expressing Kir6.2+SUR1 were exposed to 15% CO2 (14, 18), massive activation of the whole-cell inward rectifying currents occurred (129 ± 31%, n = 5). Similar channel activation was observed in Kir6.2Delta C36 (143 ± 15%, n = 11), whose CO2 sensitivity did not show any significant difference from the Kir6.2+SUR1 and Kir6.2Delta C36+SUR1 (p > 0.05), indicating that the CO2-sensing mechanism is located on the Kir6.2 subunit (Fig. 1, A and B). The effect was reversible and dependent on CO2 concentrations. An apparent increase in the current amplitude was seen with PCO2 as low as 7.6 torr (1%), and higher PCO2 resulted in stronger activation (Fig. 1C). Interestingly, the Kir6.1+SUR1 showed a similar CO2 sensitivity (141 ± 28%, n = 6; Fig. 1B), suggesting that various KATP channels consisting of Kir6.1 or Kir6.2 are likely to be activated by CO2. In contrast, Kir2.1 had no response to 15% CO2, whereas Kir1.1, Kir2.3, and Kir4.1 were inhibited (Fig. 1B).


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 1.   Augmentation of cloned KATP currents during hypercapnia and intracellular acidification. A, whole-cell K+ currents were studied with a series of voltage commands (from -160 mV to 140 mV with 20-mV increments at a holding potential of 0 mV) using a bath solution containing 90 mM K+. The Kir6.2Delta C36 currents show a clear inward rectification of ~4 µA at -160 mV. When the oocyte was exposed to 15% CO2, the inward rectifying currents were markedly enhanced. Washout led to a complete recovery. B, similar channel activation was observed in Kir6.2+SUR1, Kir6.2Delta C36+SUR1, and Kir6.1+SUR1, all of which showed CO2 (15%) sensitivity indistinguishable from Kir6.2Delta C36 (p > 0.05). In contrast, the same CO2 exposure had no effect on Kir2.1 and caused inhibition of Kir1.1, Kir2.3, and Kir4.1. Data are presented as means ± S.E. C, activation of Kir6.2Delta C36 currents with various concentrations of CO2. Significant increase in the current amplitude was seen with 1% CO2 (7.6 torr). Higher PCO2 resulted in much stronger activation. Asterisk, exposure to the atmospheric air showing that all changes are statistically significant (p < 0.05). pHi was measured as described previously (14). The pHi values were obtained from four different oocytes at each CO2 level, averaged and shown in parentheses following the CO2 concentrations. D, In another cell, intracellular acidification was produced using 90 mM KHCO3 (pHo 7.4). Such a manipulation reduced pHi to 6.6, and reversibly enhanced the Kir6.2Delta C36 currents. E, Intracellular acidification to pHa 6.6 using high concentration bicarbonate or nigericin (10 µM in the KD90 solution) caused an activation of the Kir6.2Delta C36 that was almost identical to that produced by 15% CO2. Extracellular acidification to the pH level seen during 15% CO2 exposure (pHo 6.2) using membrane-impermeable HEPES buffer had little effect, however, which is significantly different from channel activation by CO2 and acidic pHi (asterisk, p < 0.001). I, current; WS, washout.

To understand whether the channel activation was produced by pH changes, currents were studied with a selective decrease in intracellular pH (pHi) to 6.6 or extracellular pH (pHo) to 6.2 as we have previously measured during CO2 (15%) exposure (14). Selective intracellular acidification using bicarbonate (14, 18, 19) without changing pHo activated the Kir6.2Delta C36 currents by 162 ± 31% (n = 4), which showed no significant difference from the hypercapnic effect (p > 0.05, Fig. 1, D and E). Lowering pHo to 6.2 without changing pHi, however, increased the currents only modestly (8 ± 2%, n = 4; Fig. 1E), suggesting that the channels are stimulated predominantly by intracellular protons. The pH sensitivity of whole-cell Kir6.2Delta C36 currents was examined by permeabilization of the plasma membranes using protonophore nigericin (10 µM) at various pHo levels (20, 21). Graded activation of Kir6.2Delta C36 currents was seen with graded acidification (Fig. 2B). When the maximal activation was reached at pH ~6.6, the current amplitude increased by 150 ± 11% (n = 6), which should mainly be produced by a drop in pH because acidic pHo had little effect on the currents. Thus, a consistent enhancement of the channel activity was demonstrated at pHi 6.6 in the presence or absence of CO2.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2.   The pH-dependent activation of Kir6.2 currents. A, macroscopic currents were recorded in an inside-out patch obtained from a Kir6.2Delta C36-expressing oocyte. Symmetric concentrations of K+ (145 mM) were applied to both sides of the patch membrane. Ramp command potentials from 100 to -100 mV were applied to the patch from a holding potential of 0 mV. When the internal surface of the patch membrane was exposed to a perfusate with various pH levels, a clear increase in the inward rectifying currents was seen. The maximal activation occurred at pH 6.6. A further decrease in pHi caused a rapid suppression in the current amplitude. This effect is likely to be the channel rundown because the currents did not recover after ~5 min of washout. Note that eight superimposed traces are shown in each panel. B, the current/pHi relationship can be described using a sum of two Hill equations (solid line): y = (1.3 (1 + (pH/pK1)h1)) + (1.3 (1+(pK2/pH)h2)), where pK1 = 7.15, the midpoint channel activation; h1 = 2.0, the Hill coefficient for channel activation; pK2 = 6.48, the midpoint channel rundown; and h2 = 6.0, the Hill coefficient for channel rundown. Such a description can also be applied to the macroscopic Kir6.2+SUR1 currents and to the NPo of single-channel (SC) Kir6.2Delta C36 currents (SC Kir6.2Delta C36). Whole-cell (WC) Kir6.2Delta C36 currents are plotted against pHi produced by CO2 at various concentrations as shown in Fig. 1C (WC Kir6.2Delta C36 CO2) and protonophore nigericin (10 µM) at 6 pHo levels (WC Kir6.2Delta C36 nigericin). The pH-current relationship can be expressed using the regular Hill equation (broken line): y = (1/(1 + (pH/pK)h)), where pK = 7.08 and h = 2.0. I, current; WS, washout; Popen, the open state probability.

To determine whether the channel activation is carried out by the inherent mechanism of Kir6.2 or mediated by cytosolic factors, Kir6.2Delta C36 and Kir6.2+SUR1 currents were studied in excised inside-out patches in the absence of ATP, ADP, and other cytosolic soluble factors (22, 23). Exposure of the internal surface of patch membranes to acidic pH augmented the macroscopic inward rectifying currents (Fig. 2A). The peak activation occurred at pHi 6.6-6.8 (Fig. 2B). A further decrease in pHi caused rapid inhibition of the Kir currents (Fig. 2, A and B). The inhibition appeared to be channel rundown because it was not seen in whole-cell recordings and channel activity showed little or no recovery during washout at pH 7.4, particularly after a long period of exposure in patches (>30 s). Thus, extremely acidic pH may accelerate KATP rundown as described previously (7, 11-13). A similar bell-shaped current-pHi relationship was observed in Kir6.2+SUR1 (Fig. 2B), further supporting the observation that pHi sensitivity is independent of the SUR subunit. Single-channel recordings showed that the current stimulation was mainly caused by augmentation of the open state probability (NPo) with concurrent moderate suppression (by 10.7 ± 0.2% measured at pH 6.2, n = 4) of the single-channel conductance. When the NPo was plotted as a function of pHi a bell-shaped NPo-pH relationship was also obtained, which was almost identical to the response of macroscopic currents to acidic pHi (Figs. 2B and 3). The relationship of channel activity (i.e. macroscopic currents and NPo) versus pHi can be described using a sum of two Hill equations with one for channel activation (pKa 7.15, h 2.0) and the other for channel rundown (pKa 6.48, h 6.0) (Fig. 2B). The pHi-dependent channel activation was virtually superimposed with that of whole-cell currents, consistent with the idea that the pH sensing mechanism exists inherently in the channel protein.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 3.   Single channel recordings from Kir6.2Delta C36 in an inside-out patch. Three active channels were seen at pHi 7.4 at -60 mV. A graded decrease in pHi produced graded augmentation of channel activity with the number of active channels increased to 5 at pH 6.8. Further acidification to pHi 6.5 and 6.2 caused inhibition of these channels. Only a partial recovery was seen after washout. Labels on the left indicate numbers of openings with C as closure. The pHi level is shown on top of each panel, and NPo on the bottom. The two lower traces are taken from the first and third panel with magnification in time scales. WS, washout.

If the pHi sensing is indeed an intrinsic property of the channel protein, there should be specific protein domains or amino acid residues responsible for the proton detection. To test this hypothesis, we performed site-directed mutagenesis on potentially titratable residues of histidine, an amino acid with its side chain pK (6.04) closest to the channel activation pKa (7.15). Among nine histidine residues studied in the intracellular N- and C-terminal domains, we found that His-175 was critical for the pHi sensitivity of Kir6.2Delta C36. Mutation of this residue to lysine (residue found in other Kir channels, H175K) or alanine (H175A) completely eliminated the proton-induced channel activation (Fig. 4). These mutants were even inhibited during hypercapnia in whole-cell recordings (Fig. 4, A and C) and by acidic pHi in excised patches (Fig. 4B), which were also observed in mutants expressed with SUR1. Single-channel conductance of the H175K was significantly smaller than its wild-type counterpart (64.4 ± 0.9 picosiemens, n = 4; p < 0.05). Interestingly, a relief of channel rundown was seen in some of the His-175 mutations. In the H175K, NPo remained above 0.1 after 200 s of perfusion with a solution of pH 6.2 (n = 3). In contrast, NPo dropped to below 0.01 within 40 s in the wild-type channel (n = 12) and H175A mutant under the same condition (n = 3). Mutations of other histidine residues had no significant effect on the CO2 sensitivity (p > 0.05, Fig. 4C).


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 4.   Dependence of the pH sensitivity on histidine residues. A, whole-cell currents were recorded from an oocyte expressing the H175A mutant Kir6.2Delta C36 under the same conditions as described in Fig. 1A. The currents were no longer stimulated by hypercapnia, although current inhibition was seen. B, in an inside-out patch, the H175A mutation completely abolished channel activation by low pHi, whereas the currents were suppressed at acidic pHi. Moderate recovery occurred after washout. C, comparison of the CO2 sensitivity of Kir6.2Delta C36 currents with histidine mutations studied in the same experimental condition. Channel stimulation disappeared in the H175A and H175K, whereas other mutants showed no significant changes. D, alignment of amino acid sequences of several members of the Kir family around the His-175 in Kir6.2 (bold). This histidine residue is found exclusively in the Kir6 subfamily. WS, washout.

Finally, we examined if ATP and pH sensitivities depended on each other. It is known that the K185E mutation greatly reduces the ATP sensitivity of Kir6.2Delta C36 (24). We found that the K185E had identical pH sensitivity to the wild-type Kir6.2Delta C36, whereas the H175K mutation did not affect the ATP sensitivity (Fig. 5).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5.   ATP and pH sensitivities in Lys-185 and His-175 mutant Kir6.2Delta C36. A, current/pH relationship shows pH sensitivity of K185E identical to Kir6.2Delta C36 but clearly different from H175A. B, the ATP sensitivity of H175K remains the same to Kir6.2Delta C36, whereas the K185E is insensitive to ATP up to 10 mM (24). I, current.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

KATP channels are regulated by ATP, ADP, and phospholipids (11, 24-28). Such modulations allow them to control cellular activity during energy insufficiency. In the present studies, we have demonstrated the proton as another KATP regulator. We have found that hypercapnia and acidic pH at near physiological levels augment KATP activity in striking contrast to other members of the Kir family that are either inhibited by acidic pH or lack response (Fig. 1B) (29).

The effect of hypercapnia is likely to be mediated by a decrease in intracellular pH, inasmuch as similar channel activation occurs with a drop in pHi but not pHo. In cell-free excised patches we have found a moderate decrease in pHi augments KATP activity consistent with our whole-cell recordings in which pHi drops to 6.6 with 15% CO2 (14). A further decrease in pHi causes channel rundown in excised patches consistent with previous observations (7, 11-13). Such channel stimulation followed by inhibition may explain by and large the contradictory results of the pH sensitivity obtained previously in the cell-endogenous KATP using excised patches, i.e. when the KATP is studied with moderate acidification in a short period of exposure, channel activity increases. On the other hand, lower pH levels with a longer period of exposure produces channel inhibition with poor reversibility (8, 9). In the presence of ATP, however, channel rundown is largely diminished so that only activation remains (6-12). Because the biphasic response to acidic pH is not seen in our whole-cell recordings with severe hypercapnia (15% CO2) and acidification (pH 5.8), the channel inhibition or rundown may not occur in intact cells.

Our current studies have also begun to shed insight into the molecular mechanisms underlying KATP activation during acidosis. We have shown that the pH-sensing mechanisms are located on the Kir6 subunit rather than in the SUR. Indeed, we have demonstrated that mutation of a single histidine residue (H175A or H175K) is sufficient to eliminate completely the acid-induced channel activation. Instead of stimulation, the His-175 mutants show a significant inhibition by hypercapnic acidosis. The inhibition appears to suggest that the pH-dependent channel rundown may include a proportion of channel inhibition that is not seen in the wild-type channels under whole-cell recordings when the channel activation is dominant. The channel inhibition manifests itself when the channel stimulation is eliminated, as for His-175 mutations. When this inhibition is considered, the activation phase of the pH-current relationship curve shown in Figs. 2B and 5A can be even steeper. The inhibition, however, cannot totally account for the pH-dependent rundown, because rundown, though relieved in the H175K, is still seen in the H175A mutation. The histidine-dependent pH sensitivity thus is consistent with the proton-mediated regulation of a large number of proteins. Because the histidine is conserved in both Kir6.1 and Kir6.2 in all known species (Fig. 4D), it is very likely that the pH-sensing mechanism exists in KATP channels in various tissues. This unique site may offer an approach to control the KATP selectively without interference of other Kir channels.

Our data indicate that pH sensitivity is independent of ATP for the following reasons: 1) acid-induced channel activation is seen in the absence of ATP; 2) channel pH sensitivity in excised patches resembles that in whole-cell recordings; 3) the K185E mutation greatly reduces ATP sensitivity without affecting the pH sensitivity; and 4) the H175K mutation does not compromise the ATP sensitivity. Therefore, proton sensing in the KATP is very unlikely to be mediated by ATP, although it may be modulated by ATP.

Because a drop in pH often accompanies metabolic stresses and is more frequently seen than sole energy depletion, pH sensitivity enables the KATP channels to play a role in a wide variety of pathophysiological conditions. Pharmacological manipulation of the KATP in coronary arteries and cerebral vasculature has been shown to affect vascular tones during hypercapnia and acidosis (5, 30). KATP may be activated by lactoacidosis during skeletal muscle fatigue (31), contributing to the decrease in tetanic force and the protection against injury (3). Excessive neuronal activity also reduces pHi (32), which may activate the KATP, leading to a suppression of hyperexcitability and a cessation of seizure activity (2). Therefore, the demonstration of KATP modulation by pHi has a profound impact on understanding cellular functions during metabolic stress and offers a potential intervention to control the cellular activity by manipulating the inherent pH sensing mechanism of the KATP channels in the treatment and prevention of stroke, epilepsy, diabetes mellitus, and coronary heart diseases.

    ACKNOWLEDGEMENTS

We thank Dr. S. Seino for providing Kir6.1 and Kir6.2 cNDAs and Dr. L. Bryan for the SUR1 cDNA.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL58410, American Diabetes Association Grant 01039, and American Heart Association Grant 9950528N.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These two authors contributed equally to this work.

§ To whom correspondence should be addressed: Dept. of Biology, Georgia State University, 24 Peachtree Central Ave., Atlanta, GA 30303-4010. Tel.: 404-651-0913; Fax: 404-651-2509; E-mail: cjiang@gsu.edu.

Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M009631200

    ABBREVIATIONS

The abbreviations used are: KATP, ATP-sensitive K+; SUR, sulfonylurea receptor; pHi, intracellular pH; pHo, extracellular pH; NPo, open state probability.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Aspinwall, C. A., Brooks, S. A., Kennedy, R. T., and Lakey, J. R. (1997) J. Biol. Chem. 272, 31308-31314[Abstract/Free Full Text]
2. Dost, R., and Rundfeldt, C. (2000) Epilepsy Res. 38, 53-66[CrossRef][Medline] [Order article via Infotrieve]
3. Gramolini, A., and Renaud, J. M. (1997) Am. J. Physiol. 272, C1936-C1946[Abstract/Free Full Text]
4. Wang, W., Hebert, S. C., and Giebisch, G. (1997) Annu. Rev. Physiol. 59, 413-436[CrossRef][Medline] [Order article via Infotrieve]
5. Wei, E. P., and Kontos, H. A. (1999) Stroke 30, 851-853[Abstract/Free Full Text]
6. Koyano, T., Kakei, M., Nakashima, H., Yoshinaga, M., Matsuoka, T., and Tanaka, H. (1993) J. Physiol. (Lond.) 463, 747-766[Abstract]
7. Vivaudou, M., and Forestier, C. (1995) J. Physiol. (Lond.) 486, 629-645[Abstract]
8. Allard, B., Lazdunski, M., and Rougier, O. (1995) J. Physiol. (Lond.) 485, 283-296[Abstract]
9. Proks, P., Takano, M., and Ashcroft, F. M. (1994) J. Physiol. (Lond.) 475, 33-44[Abstract]
10. Cook, D. L., and Hales, C. N. (1984) Nature 311, 271-273[Medline] [Order article via Infotrieve]
11. Davies, N. W., Standen, N. B., and Stanfield, P. R. (1992) J. Physiol. (Lond.) 445, 549-568[Abstract]
12. Fan, Z., Tokuyama, Y., and Makielski, J. C. (1994) Am. J. Physiol. 267, C1036-C1044[Abstract/Free Full Text]
13. Baukrowitz, T., Tucker, S. J., Schulte, U., Benndorf, K., Ruppersberg, J. P., and Fakler, B. (1999) EMBO J. 18, 847-853[Abstract/Free Full Text]
14. Zhu, G. Y., Chanchevalap, S., Liu, C., Xu, H., and Jiang, C. (2000) J. Cell. Physiol. 183, 53-64[CrossRef][Medline] [Order article via Infotrieve]
15. Ashcroft, F. M., and Gribble, F. M. (1998) Trends Neurosci. 21, 288-294[CrossRef][Medline] [Order article via Infotrieve]
16. Tucker, S. J., Gribble, F. M., Zhao, C., Trapp, S., and Ashcroft, F. M. (1997) Nature 387, 179-183[CrossRef][Medline] [Order article via Infotrieve]
17. Qu, Z., Zhu, G. Y., Yang, Z., Cui, N., Li, Y., Chanchevalap, S., Sulaiman, S., Haynie, H., and Jiang, C. (1999) J. Biol. Chem. 274, 13783-13789[Abstract/Free Full Text]
18. Xu, H., Cui, N., Yang, Z., Qu, Z., and Jiang, C. (2000) J. Physiol. (Lond.) 524, 725-735[Abstract/Free Full Text]
19. Fakler, B., Schultz, J. H., Yang, J., Schulte, U., Brandle, U., Zenner, H. P., Jan, L. Y., and Ruppersberg, J. P. (1996) EMBO J. 15, 4093-4099[Abstract]
20. Grinstein, S., Cohen, S., and Rothstein, A. (1984) J. Gen. Physiol. 83, 341-369[Abstract]
21. Wilding, T. J., Cheng, B., and Roos, A. (1992) J. Gen. Physiol. 100, 593-608[Abstract]
22. Zhu, G. Y., Chanchevalap, S., Cui, N., and Jiang, C. (1999) J. Physiol. (Lond.) 516, 699-710[Abstract/Free Full Text]
23. Yang, Z., Xu, H., Cui, N., Qu, Z., Chanchevalap, S., Shen, W., and Jiang, C. (2000) J. Gen. Physiol. 116, 33-46[Abstract/Free Full Text]
24. Reimann, F., Ryder, T. J., Tucker, S. J., and Ashcroft, F. M. (1999) J. Physiol. (Lond.) 520, 661-669[Abstract/Free Full Text]
25. Larsson, O., Ammala, C., Bokvist, K., Fredholm, B., and Rorsman, P. (1993) J. Physiol. (Lond.) 463, 349-365[Abstract]
26. Fan, Z., and Makielski, J. C. (1997) J. Biol. Chem. 272, 5388-5395[Abstract/Free Full Text]
27. Baukrowitz, T., Schulte, U., Oliver, D., Herlitze, S., Krauter, T., Tucker, S. J., Ruppersberg, J. P., and Fakler, B. (1998) Science 282, 1141-1144[Abstract/Free Full Text]
28. Shyng, S. L., and Nichols, C. G. (1998) Science 282, 1138-1141[Abstract/Free Full Text]
29. Nichols, C. G., and Lopatin, A. N. (1997) Annu. Rev. Physiol. 59, 171-191[CrossRef][Medline] [Order article via Infotrieve]
30. Ishizaka, H., Gudi, S. R., Frangos, J. A., and Kuo, L. (1999) Circulation 99, 558-563[Abstract/Free Full Text]
31. Light, P. E., Comtois, A. S., and Renaud, J. M. (1994) J. Physiol. (Lond.) 475, 495-507[Abstract]
32. Trapp, S., Luckermann, M., Brooks, P. A., and Ballanyi, K. (1996) J. Physiol. (Lond.) 496, 695-710[Abstract]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.