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• W1987866631 abstract "ATP-sensitive K+ channels (KATP) are regulated by pH in addition to ATP, ADP, and phospholipids. In the study we found evidence for the molecular basis of gating the cloned KATP by intracellular protons. Systematic constructions of chimerical Kir6.2-Kir1.1 channels indicated that full pH sensitivity required the N terminus, C terminus, and M2 region. Three amino acid residues were identified in these protein domains, which are Thr-71 in the N terminus, Cys-166 in the M2 region, and His-175 in the C terminus. Mutation of any of them to their counterpart residues in Kir1.1 was sufficient to completely eliminate the pH sensitivity. Creation of these residues rendered the mutant channels clear pH-dependent activation. Thus, critical players in gating KATP by protons are demonstrated. The pH sensitivity enables the KATP to regulate cell excitability in a number of physiological and pathophysiological conditions when pH is low but ATP concentration is normal. ATP-sensitive K+ channels (KATP) are regulated by pH in addition to ATP, ADP, and phospholipids. In the study we found evidence for the molecular basis of gating the cloned KATP by intracellular protons. Systematic constructions of chimerical Kir6.2-Kir1.1 channels indicated that full pH sensitivity required the N terminus, C terminus, and M2 region. Three amino acid residues were identified in these protein domains, which are Thr-71 in the N terminus, Cys-166 in the M2 region, and His-175 in the C terminus. Mutation of any of them to their counterpart residues in Kir1.1 was sufficient to completely eliminate the pH sensitivity. Creation of these residues rendered the mutant channels clear pH-dependent activation. Thus, critical players in gating KATP by protons are demonstrated. The pH sensitivity enables the KATP to regulate cell excitability in a number of physiological and pathophysiological conditions when pH is low but ATP concentration is normal. ATP-sensitive K+ channels Kir6.2ΔC36 wild type sulfonylurea receptor ATP-sensitive K+ channel (KATP)1is a unique member in the K+ channel family, which directly couples the intermediary metabolism to cellular excitability (1Ashcroft F.M. Gribble F.M. Trends Neurosci. 1998; 21: 288-294Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 2Quayle J.M. Nelson M.T. Standen N.B. Physiol. Rev. 1997; 77: 1165-1232Crossref PubMed Scopus (718) Google Scholar). Such a property enables the KATP to play an important role in regulating vascular tone, skeletal muscle contractility, insulin secretion, epithelial transport, and neuronal excitability under a variety of physiological and pathophysiological conditions (3Dost R. Rundfeldt C. Epilepsy Res. 2000; 38: 53-66Crossref PubMed Scopus (55) Google Scholar, 4Gramolini A. Renaud J.M. Am. J. Physiol. 1997; 272: C1936-C1946Crossref PubMed Google Scholar, 5Light P.E. Comtois A.S. Renaud J.M. J. Physiol. (Lond.). 1994; 475: 495-507Crossref Scopus (54) Google Scholar, 6Wang W. Hebert S.C. Giebisch G. Annu. Rev. Physiol. 1997; 59: 413-436Crossref PubMed Scopus (176) Google Scholar, 7Wei E.P. Kontos H.A. Beckman J.S. Stroke. 1998; 29: 817-822Crossref PubMed Scopus (31) Google Scholar). Although ATP is the primary regulator of theKATP, several other cytosolic factors are also involved in the control of channel activity, including ADP, phospholipids, and hydrogen ion (8–20). Our recent studies have shown that the cloned KATP also responds to acidic pH (21Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). These channels are strongly stimulated by hypercapnia and intracellular acidosis. The pH sensitivity is independent of the SUR subunit and other cytosolic factors, suggesting that the pH sensing mechanisms are located in the Kir (inward rectifier K+ channel) subunit (21Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). If the pH sensitivity is an inherent property of Kir6 proteins, there should be special structures responsible for channel gating by protons. These structures are likely to be located in the Kir6 subunit, since the SUR subunit is not required for the pH sensitivity. To identify these structures, we performed these experiments in which we used the Kir6.2 with a truncation of 36 amino acids at the C-terminal end,i.e. Kir6.2ΔC36 that expresses functional channels without the SUR subunit and retains fair ATP sensitivity (22Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (680) Google Scholar). Several chimerical channels were generated based on peptide sequences of Kir6.2 and Kir1.1, a Kir channel that is inhibited by intracellular acidosis (23Chanchevalap S. Yang Z. Cui N. Qu Z. Zhu G. Liu C. Giwa L.R. Abdulkadir L. Jiang C. J. Biol. Chem. 2000; 275: 7811-7817Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 24Fakler B. Schultz J.H. Yang J. Schulte U. Brandle U. Zenner H.P. Jan L.Y. Ruppersberg J.P. EMBO J. 1996; 15: 4093-4099Crossref PubMed Scopus (163) Google Scholar, 25Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar) and has been shown to express functional channels in its chimeras with Kir6.2 (26Drain P. Li L. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13953-13958Crossref PubMed Scopus (174) Google Scholar). These chimeras were studied in whole-cell recording using hypercapnia, a condition that does not cause channel rundown (21Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Our results indicate that there are three separate protein domains in the Kir6.2 protein that are crucial for the pH sensitivity. We have identified critical amino acid residues within each of these protein domains. Replacement of any of them with their counterpart residues in Kir1.1 interrupts the channel sensitivity to CO2/pH. Frog oocytes were obtained from Xenopus laevis. The 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 OR2 solution (NaCl 82 mm, KCl 2 mm, MgCl2 1 mm, and HEPES 5 mm, pH 7.4) for 90 min at room temperature. After 3 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. The oocytes were then incubated at 18 °C in the ND-96 solution containing 96 mm NaCl, 2 mm KCl, 1 mmMgCl2, 1.8 mm CaCl2, 5 mm HEPES, and 2.5 mm sodium pyruvate with 100 mg/liter Geneticin added, pH 7.4. Rat Kir1.1 (ROMK1, GenBankTM accession number X72341) and mouse Kir6.2 (mBIR, GenBankTM accession number D50581) cDNAs were generously provided by Dr. S. Hebert at Yale University and Dr. S. Seino at Chiba University in Japan, respectively. The cDNAs were subcloned to a eukaryotic expression vector (pcDNA3.1, Invitrogen Inc., Carlsbad, CA) and used forXenopus oocyte expression without cRNA synthesis. Chimerical constructs were prepared by the overlap extension at the junction of the interested domains using the polymerase chain reaction (Pfu DNA polymerase, Stratagene, La Jolla, CA). Site-specific mutations were made using a site-directed mutagenesis kit (Stratagene). 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. Two-electrode voltage clamp was performed using an amplifier (Geneclamp 500, Axon Instruments Inc., Foster City, CA) at room temperature (∼24 °C). The microelectrodes were filled with 3m KCl. One of the electrodes (1.0–2.0 megaohms) served for voltage measurements, and the other electrode (0.3–0.6 megaohms) was used for current recording. Current records were low-pass filtered (Bessel, 4-pole filter, 3 dB at 5 kHz), digitized at 5 kHz (12-bit resolution), and stored on computer disc for later analysis (pClamp 6, Axon Instruments) (21Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 27Xu H. Yang Z. Cui N. Giwa L.R. Abdulkadir L. Patel M. Sharma P. Shan G. Shen W. Jiang C. Am. J. Physiol. Cell Physiol. 2000; 279: C1464-C1471Crossref PubMed Google Scholar, 28Yang Z. Jiang C. J. Physiol. (Lond.). 1999; 520: 921-927Crossref Scopus (48) Google Scholar, 29Yang Z. Xu H. Cui N. Qu Z. Chanchevalap S. Shen W. Jiang C. J. Gen. Physiol. 2000; 116: 33-45Crossref PubMed Scopus (96) Google Scholar, 30Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar). The extracellular solution contained 90 mm KCl, 3 mm MgCl2, and 5 mm HEPES, pH 7.4. Under this condition, cells showed a membrane potential of ∼0 mV, so that we studied the currents with a holding potential of 0 mV. Expressions of functional Kir channels were confirmed using one or two of the following methods. 1) The amplitude of the inward rectifying currents was significantly larger than that recorded from the pcDNA3-injected oocytes, 2) the currents were strongly activated with the exposure to 3 mm azide, and 3) 100 μm Ba2+ inhibited the currents. When a mutant failed to express functional channels in ∼60 oocytes tested, another two injections of the same mutant from different colonies were followed in ∼60 cells each. If there was still a lack of expression, we believed that the mutation was too severe to produce any functional channels, and further experimentation was not attempted. Experiments were performed in a semi-closed recording chamber (BSC-HT, Medical System, Greenvale, NY) 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 × 15-mm gap on the top cover of the chamber that served as the gas outlet and an access to the oocytes for recording microelectrodes. At base line, the chamber was ventilated with atmospheric air. Exposure of the oocytes to CO2 was carried out by switching to 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 (21Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 27Xu H. Yang Z. Cui N. Giwa L.R. Abdulkadir L. Patel M. Sharma P. Shan G. Shen W. Jiang C. Am. J. Physiol. Cell Physiol. 2000; 279: C1464-C1471Crossref PubMed Google Scholar, 28Yang Z. Jiang C. J. Physiol. (Lond.). 1999; 520: 921-927Crossref Scopus (48) Google Scholar, 29Yang Z. Xu H. Cui N. Qu Z. Chanchevalap S. Shen W. Jiang C. J. Gen. Physiol. 2000; 116: 33-45Crossref PubMed Scopus (96) Google Scholar, 30Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar). The high solubility of CO2 resulted in a detectable change in intra- or extracellular acidification as fast as 10 s in these oocytes. The following nomenclatures were used in the present study. Kir6.2 and Kir1.1 were divided into three segments in their peptide chains,i.e. 1) the N terminus, 2) C terminus, and 3) all the rest M1 through M2 regions. A chimera with both N and C termini from Kir6.2 and the rest from Kir1.1 was named SOS. If only the N terminus was from Kir6.2, it was called SOO. If the SOS carried the M1 region from Kir6.2, it was referred to SOSm1. To facilitate the comparison of amino acid residues between the SO chimeras and the wt channels, they were numbered by their original locations in the wt Kir1.1 or Kir6.2 protein rather than by their new positions in the sequences of the chimeras. Data are presented as means ± S.E. Analysis of variance or Student's t test was used. Differences of CO2and pH effects before versus during exposures were considered to be statistically significant if p ≤ 0.05. The Kir6.2ΔC36 was expressed in Xenopus oocytes. As reported previously (21Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 22Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (680) Google Scholar), inward rectifying currents were recorded in the whole-cell configuration after Kir6.2ΔC36 cDNA injection. Exposure to CO2 produced reversible and concentration-dependent activation of the inward rectifying currents (Fig. 1A). The effect was mediated by pH rather than molecular CO2, as intracellular, but not extracellular, acidification to the same levels seen during CO2 exposures produced the same degrees of channel activation (22Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (680) Google Scholar). In excised inside-out patches, the Kir6.2ΔC36 currents were also stimulated by modest acidification on the cytosolic side of membranes in the absence of ATP and other cytosolic soluble factors. With strong acidification, however, the channel activation was followed by marked inhibition that appeared to be a result of channel rundown as reported previously (8Baukrowitz T. Schulte U. Oliver D. Herlitze S. Krauter T. Tucker S.J. Ruppersberg J.P. Fakler B. Science. 1998; 282: 1141-1144Crossref PubMed Scopus (441) Google Scholar, 11Davies N.W. Nature. 1990; 343: 375-377Crossref PubMed Scopus (189) Google Scholar, 13Koyano T. Kakei M. Nakashima H. Yoshinaga M. Matsuoka T. Tanaka H. J. Physiol. (Lond.). 1993; 463: 747-766Crossref Scopus (55) Google Scholar, 20Vivaudou M. Forestier C. J. Physiol. (Lond.). 1995; 486: 629-645Crossref Scopus (35) Google Scholar,31Fan Z. Tokuyama Y. Makielski J.C. Am. J. Physiol. 1994; 267: C1036-C1044Crossref PubMed Google Scholar). Similar effects were observed in the presence of 1 mmATP in the internal solution, 2J. Wu and C. Jiang, unpublished observations. indicating that the pH sensitivity is independent of ATP. Since the channel rundown occurs in excised patches, further studies were done using whole-cell recording and hypercapnic acidosis with 15% CO2, an experimental condition that we have well documented previously (21Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar,27Xu H. Yang Z. Cui N. Giwa L.R. Abdulkadir L. Patel M. Sharma P. Shan G. Shen W. Jiang C. Am. J. Physiol. Cell Physiol. 2000; 279: C1464-C1471Crossref PubMed Google Scholar, 28Yang Z. Jiang C. J. Physiol. (Lond.). 1999; 520: 921-927Crossref Scopus (48) Google Scholar, 29Yang Z. Xu H. Cui N. Qu Z. Chanchevalap S. Shen W. Jiang C. J. Gen. Physiol. 2000; 116: 33-45Crossref PubMed Scopus (96) Google Scholar, 30Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar). In contrast to the Kir6.2, Kir1.1 was inhibited during hypercapnia and intracellular acidification (Fig. 1B), as demonstrated previously (23Chanchevalap S. Yang Z. Cui N. Qu Z. Zhu G. Liu C. Giwa L.R. Abdulkadir L. Jiang C. J. Biol. Chem. 2000; 275: 7811-7817Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 24Fakler B. Schultz J.H. Yang J. Schulte U. Brandle U. Zenner H.P. Jan L.Y. Ruppersberg J.P. EMBO J. 1996; 15: 4093-4099Crossref PubMed Scopus (163) Google Scholar, 25Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar, 27Xu H. Yang Z. Cui N. Giwa L.R. Abdulkadir L. Patel M. Sharma P. Shan G. Shen W. Jiang C. Am. J. Physiol. Cell Physiol. 2000; 279: C1464-C1471Crossref PubMed Google Scholar, 32Doi T. Fakler B. Schultz J.H. Schulte U. Brandle U. Weidemann S. Zenner H.P. Lang F. Ruppersberg J.P. J. Biol. Chem. 1996; 271: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Our results suggest that the pH sensitivity in the Kir6.2 is an inherent property of the channel protein, similar to those described in several other Kir channels (23Chanchevalap S. Yang Z. Cui N. Qu Z. Zhu G. Liu C. Giwa L.R. Abdulkadir L. Jiang C. J. Biol. Chem. 2000; 275: 7811-7817Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 24Fakler B. Schultz J.H. Yang J. Schulte U. Brandle U. Zenner H.P. Jan L.Y. Ruppersberg J.P. EMBO J. 1996; 15: 4093-4099Crossref PubMed Scopus (163) Google Scholar, 25Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar, 27Xu H. Yang Z. Cui N. Giwa L.R. Abdulkadir L. Patel M. Sharma P. Shan G. Shen W. Jiang C. Am. J. Physiol. Cell Physiol. 2000; 279: C1464-C1471Crossref PubMed Google Scholar, 30Zhu G. Chanchevalap S. Cui N. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar, 32Doi T. Fakler B. Schultz J.H. Schulte U. Brandle U. Weidemann S. Zenner H.P. Lang F. Ruppersberg J.P. J. Biol. Chem. 1996; 271: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 33Coulter K.L. Perier F. Radeke C.M. Vandenberg C.A. Neuron. 1995; 15: 1157-1168Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 34Qu Z. Yang Z. Cui N. Zhu G. Liu C. Xu H. Chanchevalap S. Shen W. Wu J. Li Y. Jiang C. J. Biol. Chem. 2000; 275: 31573-31580Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). To identify the specific structures in the Kir6.2 protein that enable the channel to respond to acidic pH, chimerical Kir channels were constructed by recombination of protein domains of Kir6.2 and Kir1.1. We reasoned that the pH sensitivity relied on the integrity of the proton-sensing and channel-gating mechanisms in the Kir6.2 protein. Interruption of the integrity will cause a loss of the pH sensitivity. Thus, we divided Kir6.2 and Kir1.1 into three segments in their peptide chains, i.e. 1) the N terminus, 2) C terminus, and 3) all the rest M1 through M2 regions. The chimera with both N and C termini from Kir6.2 and the rest from Kir1.1 is named SOS (S refers to six, and O to one). If the N terminus is the only part from Kir6.2, it is called SOO. Accordingly, the wild-type (wt) Kir6.2ΔC36 refers to SSS, and the wt Kir1.1 to OOO (Fig.2A). Six chimerical channels were systematically constructed using these Kir6.2 and Kir1.1 protein domains (Fig. 2A). Most of the recombinant channels showed inward rectifying currents of 1–3 μA, which were significantly larger than those recorded from the vector-injected cells (0.5 ± 0.1 μA, n = 9). The SSO and OSO were the only two that did not show evident channel expression. Azide (3 mm) treatment of the cells injected with SSO or OSO did not reveal any additional increase in the current amplitude. Thus, how SSO and OSO respond to acidosis is unclear. Although all other chimeras expressed detectable inward rectifying currents, none of them displayed a hypercapnic response as large as the wt Kir6.2ΔC36 (Fig. 2A, TableI). Clearly, these chimeras had caused interruptions of the pH-dependent gating mechanisms.Table IKir6.2, Kir6.2ΔC36. See Fig. 2 for nomenclature of chimeras. Data are presented as means ± S.E.NameBase-line current% CO2effectnExpressionμA Kir6.2 (SSS)2.1 ± 0.4130.3 ± 12.214Yes Kir1.1 (OOO)19.3 ± 5.5−69.0 ± 5.412YesChimeras OOS0.8 ± 0.345.7 ± 11.29Yes OSONo OSS1.0 ± 0.423.5 ± 10.57Yes SOO2.4 ± 0.517.8 ± 6.85Yes SOS6.8 ± 1.143.8 ± 7.94Yes SSONo OOSm2No SOOm2No SOSm11.9 ± 0.519.2 ± 5.84Yes SOSm24.9 ± 3.0123.9 ± 15.17YesN terminus Kir6.2-T71DNo Kir6.2-T71K2.4 ± 0.5−11.7 ± 6.48Yes Kir6.2-T71M1.6 ± 0.447.1 ± 14.94Yes Kir6.2-T71N4.6 ± 1.1160.3 ± 19.45Yes Kir6.2-T71S4.2 ± 1.1112.7 ± 7.65Yes OOSm2-K80TNo OSS-K80T1.9 ± 0.472.0 ± 18.25Yes SOSm2-T71K3.3 ± 0.6−1.9 ± 1.55YesM2 region Kir1.1-F173I5.2 ± 1.0−27.2 ± 3.84Yes Kir1.1-A177C1.7 ± 0.9−29.2 ± 9.014Yes Kir1.1-C175L/A177CNo Kir1.1-L179FNo Kir6.2-C166A12.5 ± 4.6−1.9 ± 1.74Yes Kir6.2-C166E19.6 ± 5.85.1 ± 1.54Yes Kir6.2-C166H5.6 ± 0.563.4 ± 2.74Yes Kir6.2-C166KNo Kir6.2-C166S18.7 ± 5.0−0.5 ± 1.86Yes Kir6.2-C166V1.5 ± 0.3114.7 ± 26.15Yes Kir6.2-F168LNo Kir6.2-I162FNo SOS-A177C6.7 ± 2.5−4.5 ± 4.54Yes SOS-F173I31.2 ± 9.5−3.9 ± 1.85Yes SOSm2-C166A1.7 ± 2.0−13.4 ± 8.08Yes SOSm2-C166E19.5 ± 0.81.5 ± 1.84Yes SOSm2-C166KNo SOSm2-C166S1.7 ± 0.64.7 ± 0.54Yes SOSm2-F168LNo SOSm2-I162F1.1 ± 0.274.4 ± 14.68YesC terminus Kir6.2-H175K5.6 ± 1.2−18.6 ± 2.75Yes SOSm2-H175K11.6 ± 3.16.5 ± 0.74Yes SOOm2-K186HNo Kir1.1-K186H12.0 ± 3.4−63.1 ± 4.15Yes SOO-K186H3.4 ± 0.544.4 ± 12.96YesMultiple domains Kir1.1-K80T/C175L/A177C/K186HNo OOS-K80T/C175L/A177C2.1 ± 0.555.1 ± 12.84Yes SOO-C175L/A177C/K186HNo SOS-C175L/A177C4.7 ± 1.7−20.5 ± 3.54Yes SOS-ALC1.1 ± 0.2142.5 ± 15.05Yes SOS-ILC3.9 ± 0.77.0 ± 6.56Yes SOS-AILC1.3 ± 0.2118.3 ± 23.05Yes Open table in a new tab Because none of the chimeras had a full CO2/pH sensitivity as did the wt Kir6.2ΔC36, it is possible that the essential structures for the pH sensitivity are not contained in these chimeras. To include them, we extended the C terminus to the entire M2 region of Kir6.2. This chimera SOSm2 expressed large inward rectifying currents (4.9 ± 3.0 μA, n = 7), with its inward rectification more like Kir6.2 than Kir1.1 (Fig. 2B). Exposure of the SOSm2 to 15% CO2 enhanced the inward rectifying currents by 123.9 ± 15.1% (n = 7), which were slightly smaller but not significantly different from the wt Kir6.2ΔC36 (p > 0.05) (Figs. 2B). Inclusion of the M1 sequence to the N terminus (SOSm1), however, did not produce any significant additional effect on the CO2sensitivity over the SOS (Fig. 2A). To determine if two of the three protein domains are sufficient for the pH sensitivity, chimeras containing two of the M2, N terminus and C terminus from Kir6.2 and the remaining sequences from Kir1.1 were constructed. The OOSm2 and SOOm2 failed to yield any functional channels, whereas the SOS had pH sensitivity of only about one-third that of the SOSm2 (Fig. 2A). These results thus indicate that protein domains necessary for the full CO2/pH sensitivity consist of at least the N terminus, C terminus, and M2 region in Kir6.2. The N terminus has been previously shown to play an important part in pH sensitivity in several Kir channels. A lysine residue at near the M1 region (Lys-80 in Kir1.1, Lys67 in Kir4.1) is a critical player (24Fakler B. Schultz J.H. Yang J. Schulte U. Brandle U. Zenner H.P. Jan L.Y. Ruppersberg J.P. EMBO J. 1996; 15: 4093-4099Crossref PubMed Scopus (163) Google Scholar, 27Xu H. Yang Z. Cui N. Giwa L.R. Abdulkadir L. Patel M. Sharma P. Shan G. Shen W. Jiang C. Am. J. Physiol. Cell Physiol. 2000; 279: C1464-C1471Crossref PubMed Google Scholar). This lysine residue is not found in Kir6.2. At the same location, the Kir6.2 has a threonine instead (Thr-71). Since Kir1.1 and Kir4.1 channels with this lysine residue are inhibited by acidic pH (25Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar, 28Yang Z. Jiang C. J. Physiol. (Lond.). 1999; 520: 921-927Crossref Scopus (48) Google Scholar), it is possible that the lack of the positively charged residues renders the Kir6.2 an opposite pH sensitivity. To test this hypothesis, we performed site-directed mutagenesis experiments at this site. Mutation of the Thr-71 to lysine yielded fair inward rectifying currents in whole-cell recordings. When the T71K was exposed to 15% CO2, we found that the mutant channels became insensitive to hypercapnia (Fig.3A). The same result was obtained in the SOSm2 carrying the T71K mutation (Fig. 3D). Systematic mutagenesis was subsequently carried out on the Thr-71 by replacing it with acidic, nonpolar, and other polar-neutral residues. There is a methionine at this site in Kir2.1, Kir2.3, Kir5.1, and Kir7.1. Thus we constructed the T71M mutant. The substitution of the Thr-71 with such a nonpolar residue greatly reduced the pH sensitivity (Fig. 3D). Replacement of the Thr-71 with an aspartate did not produce a functional channel. When it was mutated to serine, the mutant T71S showed an increase in the current amplitude by >100% with hypercapnia (Fig. 3D). Since both threonine and serine are the potential substrate of phosphorylation, we mutated the Thr-71 to another polar-neutral residue, asparagine. Currents of the T71N mutant were enhanced by 160.3 ± 19.4% (n = 5) by 15% CO2 (Fig. 3, B and D), suggesting that a polar-neutral residue at this site is necessary for maintaining the pH sensitivity in Kir6.2. Creation of the threonine residue in the OSS produced clear inward rectifying currents that increased reversibly by 72.0 ± 18.2% (n = 5) in response to 15% CO2 (Fig. 3,C and D), an increase that was significantly larger than the OSS (p < 0.01). Construction of this threonine in OOSm2 failed to produce functional expression. These results therefore suggest that the Thr-71 is the determinant residue in the N terminus for the pH sensitivity of Kir6.2. The M2 region of several Kir channels was aligned in Fig.4A. Amino acid residues in this region are highly homologous between Kir1.1 and Kir6.2. It is known that the area on the cytosolic side of the M2 domain is involved in lining the conductive pore. There are five residues in this area that are clearly different between Kir6.2 and Kir1.1. Among them are two phenylalanines with one found in Kir1.1 (Phe-173) and the other in Kir6.2 (Phe-168). Phenylalanine has a side chain much larger than that of leucine and valine, seen at its counterpart positions. To elucidate whether the size of residues controls the pH sensitivity, we site-specifically mutated these residues in Kir6.2 to those found in Kir1.1. The F168L and I162F mutants were constructed using both Kir6.2ΔC36 and SOSm2 as the template. Of the four mutants studied, the SOSm2-I162F was the only one expressing inward rectifying currents (Table I), which remained, although smaller, to be stimulated during hypercapnia by 74.4 ± 14.6% (n = 8) (Fig.4B). Subsequently, we studied two other sites in which a cysteine residue exists in each of Kir6.2 and Kir1.1. Mutations of Cys-175 to leucine in Kir1.1 and SOS did not produce channels that were stimulated by CO2. The other cysteine mutation, however, was remarkable. The Kir6.2-C166A mutant showed large base-line currents (12.5 ± 4.6 μA, n = 4) and a much weaker inward rectification than the wt Kir6.2ΔC36 (Fig. 4C). At highly negative membrane potentials, the inward rectifying currents also became weaker, suggesting that this site contributes to the voltage-dependent rectification. More interestingly, substitution of this single residue with alanine (Kir6.2-C166A) completely abolished the CO2 sensitivity of Kir6.2 (−1.9 ± 1.1%, n = 4) (Fig. 4, B andC). The same effect was also observed in the SOSm2-based mutant SOSm2-C166A (−13.4 ± 8.0%, n = 8). Systematic mutations of the Cys-166 were thereafter performed using Kir6.2ΔC36 and SOSm2. The mutant channels became CO2-insensitive when this residue was replaced with a negative or a polar-neutral residue, i.e. Kir6.2-C166E, Kir6.2-C166S, SOSm2-C166E, and SOSm2-C166S (Fig. 4B). Mutation to a positive residue (Kir6.2-C166K and SOSm2-C166K) did not yield functional expression. In contrast, the mutant channels remained stimulated by hypercapnic acidosis, when a valine or histidine was the replacement (Table I). Indeed, the Kir6.2-C166V showed CO2sensitivity almost identical to the SOSm2 (Fig. 4B). Reversal mutations of this cysteine residue, however, did not show any dramatic effect. The Kir1.1-A177C was still inhibited by CO2, and the SOS-A177C remained pH-insensitive (Fig.4B). Therefore, it is possible that other adjacent residues within the M2 are also involved. To identify these residues, we performed further mutagenesis studies. We found that the formation of disulfide bond with Cys-175 was not a reason, because combined mutations of these two residues still showed an inhibited phenotype in SOS (SOS-C175L/A177C, -20.5 ± 3.5%, n = 4). Subsequently, we created mutants to include Ser-172 and/or Phe-173. The SOS-based mutants S172A/F173I/C175L/A177C (SOS-AILC) and S172A/C175L/A177C (SOS-ALC) expressed functional currents with clear inward rectification. Both of them were strongly stimulated by 15% CO2 to a degree that statistically was not different from the SOSm2 and the wt Kir6.2ΔC36 channels (Fig. 4D). In contrast, the F173I/C175L/A177C (SOS-ILC) had no significant effect. Thus, these results suggest that the molecular determinant for the pH-dependent activation is likely to be the short motif (∼ 6 amino acids) at the intracellular end of the M2 centered by the Cys-166. Our previous studies show that His-175 in the C terminus is critical for the pH sensitivity of Kir6.2 (21Xu H. Cui N. Yang Z. Wu J. Giwa L.R. Abdulkadir L. Sharma P. Jiang C. J. Biol. Chem. 2001; 276: 12898-12902Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This was confirmed in our current studies (Table I). In addition, we found that mutation of this histidine residue in SOSm2 greatly reduced the current response to 15% CO2 (6.5 ± 0.7%, n = 4) (Fig.5A). Although construction of a histidine residue at the same site in SOOm2 did not produce functional expression, creation of such a residue in the SOO (SOO-K186H) gave rise to funct" @default.
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• W1987866631 title "Requirement of Multiple Protein Domains and Residues for GatingKATP Channels by Intracellular pH" @default.
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