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• W2100797127 abstract "Ion channels play an important role in cellular functions, and specific cellular activity can be produced by gating them. One important gating mechanism is produced by intra- or extracellular ligands. Although the ligand-mediated channel gating is an important cellular process, the relationship between ligand binding and channel gating is not well understood. It is possible that ligands are involved in the interactions of different protein domains of the channel leading to opening or closing. To test this hypothesis, we studied the gating of Kir2.3 (HIR) by intracellular protons. Our results showed that hypercapnia or intracellular acidification strongly inhibited these channels. This effect relied on both the N and C termini. The CO2/pH sensitivities were abolished or compromised when one of the intracellular termini was replaced. Using purified N- and C-terminal peptides, we found that the N and C termini bound to each other in vitro. Although their binding was weak at pH 7.4, stronger binding was seen at pH 6.6. Two short sequences in the N and C termini were found to be critical for the N/C-terminal interaction. Interestingly, there was no titratable residue in these motifs. To identify the potential protonation sites, we systematically mutated most histidine residues in the intracellular N and C termini. We found that mutations of several histidine residues in the C but not the N terminus had a major effect on channel sensitivities to CO2 and pHi. These results suggest that at acidic pH, protons appear to interact with the C-terminal histidine residues and present the C terminus to the N terminus. Consequentially, these two intracellular termini bound to each other through two short motifs and closed the channel. Thus, a novel mechanism for K+ channel gating is demonstrated, which involves the N- and C-terminal interaction with protons as the mediator. Ion channels play an important role in cellular functions, and specific cellular activity can be produced by gating them. One important gating mechanism is produced by intra- or extracellular ligands. Although the ligand-mediated channel gating is an important cellular process, the relationship between ligand binding and channel gating is not well understood. It is possible that ligands are involved in the interactions of different protein domains of the channel leading to opening or closing. To test this hypothesis, we studied the gating of Kir2.3 (HIR) by intracellular protons. Our results showed that hypercapnia or intracellular acidification strongly inhibited these channels. This effect relied on both the N and C termini. The CO2/pH sensitivities were abolished or compromised when one of the intracellular termini was replaced. Using purified N- and C-terminal peptides, we found that the N and C termini bound to each other in vitro. Although their binding was weak at pH 7.4, stronger binding was seen at pH 6.6. Two short sequences in the N and C termini were found to be critical for the N/C-terminal interaction. Interestingly, there was no titratable residue in these motifs. To identify the potential protonation sites, we systematically mutated most histidine residues in the intracellular N and C termini. We found that mutations of several histidine residues in the C but not the N terminus had a major effect on channel sensitivities to CO2 and pHi. These results suggest that at acidic pH, protons appear to interact with the C-terminal histidine residues and present the C terminus to the N terminus. Consequentially, these two intracellular termini bound to each other through two short motifs and closed the channel. Thus, a novel mechanism for K+ channel gating is demonstrated, which involves the N- and C-terminal interaction with protons as the mediator. inward rectifier K+ 1,4-piperazinediethanesulfonic acid glutathione S-transferase amino acid(s) Ion channels are a group of membrane proteins characterized by ion-selective permeation and event-specific gating. By controlling or gating the transition between their open and closed states, specific cellular functions can be produced (1Hille B. Ionic Channels of Excitable Membranes. Sinauer Associates, Sunderland, MA1992: 472-503Google Scholar). Several membrane and cytosolic mechanisms are involved in the channel gating. For instance, a voltage-gated ion channel can be activated by depolarization; intra- and extracellular ligands can retain a ligand-gated ion channel to the open or closed state. The gating of inward rectifier K+(Kir)1 channels is carried out by membrane-bound and cytosolic molecules including G proteins, nucleotides, and protons (2Nichols C.G. Lopatin A.N. Annu. Rev. Physiol. 1997; 59: 171-191Crossref PubMed Scopus (657) Google Scholar). The gating of Kir channels by protons may allow cells to produce corresponding responses to a change in intra- and extracellular pH seen under a number of pathophysiological conditions. Several members in the Kir family are regulated by intra- and extracellular protons (3Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar, 4Coulter K.L. Perier F. Radeke C.M. Vandenberg C.A. Neuron. 1995; 15: 1157-1168Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 5Fakler 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 (162) Google Scholar, 6Choe H. Zhou H. Palmer L.G. Sackin H. Am. J. Physiol. 1997; 273: F516-F529PubMed Google Scholar, 7Shuck M.E. Piser T.M. Bock J.H. Slightom J.L. Lee K.S. Bienkowski M.J. J. Biol. Chem. 1997; 272: 586-593Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 8Zhu G.Y. Chanchevalap S. Cui N.R. Jiang C. J. Physiol. (Lond. ). 1999; 516: 699-710Crossref Scopus (69) Google Scholar). Channels activity is completely shut off by acidification, whereas full channel openings are achieved with an increase in the pH level. In Kir1.1, Lys80 is a critical player in channel gating by intracellular protons, although several other residues are also involved (5Fakler 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 (162) Google Scholar, 6Choe H. Zhou H. Palmer L.G. Sackin H. Am. J. Physiol. 1997; 273: F516-F529PubMed Google Scholar, 9Schulte U. Hahn H. Konrad M. Jeck N. Derst C. Wild K. Weidemann S. Ruppersberg J.P. Fakler B. Ludwig J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15298-15303Crossref PubMed Scopus (132) Google Scholar, 10Chanchevalap, S., Yang, Z. J., Cui, N. R., Qu, Z., Zhu, G. Y., Liu, C. X., Giwa, L. R., Abdulkadir, L., and Jiang, C. J. Biol. Chem. 275, 7811–7817.Google Scholar). Mutation of this lysine residue totally abolishes the pH sensitivity (5Fakler 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 (162) Google Scholar). The Lys80 may become protonated at acidic pH by its interactions with other protein domains, leading to a closure of the channel (9Schulte U. Hahn H. Konrad M. Jeck N. Derst C. Wild K. Weidemann S. Ruppersberg J.P. Fakler B. Ludwig J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15298-15303Crossref PubMed Scopus (132) Google Scholar). In Kir2.3 (HIR), we have previously identified a short motif in the N terminus that is crucial in the gating of this channel by intracellular protons (11Qu Z.Q. Zhu G.Y. Yang J. Cui N.R. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Unlike Kir1.1, there is no proton-binding site within or nearby to this motif in HIR. Thus, it is possible that protons interact with other intermediate sites that subsequently act on this short motif and close the channel. Recent studies have shown that a large number of residues in the channel proteins are involved in the gating process by pH (5Fakler 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 (162) Google Scholar, 6Choe H. Zhou H. Palmer L.G. Sackin H. Am. J. Physiol. 1997; 273: F516-F529PubMed Google Scholar, 9Schulte U. Hahn H. Konrad M. Jeck N. Derst C. Wild K. Weidemann S. Ruppersberg J.P. Fakler B. Ludwig J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15298-15303Crossref PubMed Scopus (132) Google Scholar,10Chanchevalap, S., Yang, Z. J., Cui, N. R., Qu, Z., Zhu, G. Y., Liu, C. X., Giwa, L. R., Abdulkadir, L., and Jiang, C. J. Biol. Chem. 275, 7811–7817.Google Scholar). However, it is unknown how these multiple sites are harmonized in opening or closing a channel. A potential mechanism is that proton binding initiates a cascade of events involving consequential interactions of the proton-binding sites with other protein domains, leading to a rearrangement of protein conformation and a switch of channel activity. To test this hypothesis, we studied the gating of Kir by intracellular protons (8Zhu G.Y. Chanchevalap S. Cui N.R. Jiang C. J. Physiol. (Lond. ). 1999; 516: 699-710Crossref Scopus (69) Google Scholar, 11Qu Z.Q. Zhu G.Y. Yang J. Cui N.R. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 12Zhu G Chanchevalap S Liu C Xu H. Jiang C. J. Cell. Physiol. 2000; 183: 53-64Crossref PubMed Scopus (38) Google Scholar). We chose to use HIR because there is no titratable residue in the identified N-terminal motif. Thus, the gating site seems to be separate from the proton-sensing sites in this channel. Our results indicate that interactions of different parts of channel proteins occur during the gating process, demonstrating a novel channel-gating mechanism of K+ channels by intracellular protons. Kir2.3 (HIR) and Kir2.1 (IRK1) cDNAs were generously provided by Carol A. Vandenberg and Lily L. Jan, respectively. These cDNAs were inserted into pcDNA3.1, a eukaryotic expression vector that allows expression of its insert gene in eukaryotic cells without injecting it into the nucleus (Invitrogen, Carlsbad, NM). Chimerical constructs were prepared by the overlap extension at the junction of the interested domains using polymerase chain reaction (Pfu DNA polymerase, Stratagene, La Jolla, CA). Junction sites of all chimeras were chosen where there were conserved amino acid residues. Thus, none of the chimeras had missing or additional residues at the junction sites. Site-specific mutations were made using a site-directed mutagenesis kit (Stratagene). Orientation of the constructs and correct mutations were confirmed with DNA sequencing. Oocytes were surgically removed from adult frog (Xenopus laevis) and treated with 2 mg/ml collagenase (Type I, Sigma) in the OR2 solution (82 mm NaCl, 2 mm KCl, 1 mm MgCl2, and HEPES, pH 7.4) for 90 min at room temperature (∼25 °C). After three washes with OR2 solution, cDNA in the pcDNA3.1 vector (40–50 ng in 50 nl of double-distilled water) was injected into the oocytes. The oocytes were then incubated at 18 °C in ND-96 solution containing (in mm) NaCl 96, KCl 2, MgCl2 1, CaCl21.8, HEPES 5, and sodium pyruvate 2.5, plus 100 mg/liter geneticin (pH 7.4). Xenopus oocytes were placed in a semi-closed recording chamber (Medical System, Greenvale, NY), where perfusion solution bathed both the top and bottom surfaces of the oocytes. The perfusate and the superfused gas entered the chamber from the inlet 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 as access to the oocytes for recording microelectrodes. The perfusate (KD 90) contained 90 mm KCl, 3 mm MgCl2, and 5 mm HEPES (pH 7.4). At baseline, the chamber was ventilated with atmospheric air. Exposure of the oocytes to CO2 was carried out by switching the superfused air to a gas mixture containing CO2 (15%) balanced with 21% O2 and N2. The high dissolvability of CO2 resulted in a detectable change in intra- or extracellular acidification in as fast as 10 s in these oocytes (11Qu Z.Q. Zhu G.Y. Yang J. Cui N.R. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). 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 (∼25 °C). The extracellular solution contained 90 mm KCl, 3 mmMgCl2, and 5 mm HEPES (pH 7.4). Cells were impaled using electrodes filled with 3 m KCl. One of the electrodes (1.0–2.0 megaohms) served as voltage 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). Patch clamp experiments were performed at room temperature as described (8Zhu G.Y. Chanchevalap S. Cui N.R. Jiang C. J. Physiol. (Lond. ). 1999; 516: 699-710Crossref Scopus (69) Google Scholar, 13Yang Z. Jiang C. J. Physiol. (Lond. ). 1999; 520: 921-927Crossref Scopus (48) Google Scholar). In brief, fire-polished patch pipettes (0.5–2.0 megaohms) were made from 1.2-mm borosilicate capillary glass. The oocyte vitelline membranes were mechanically removed after exposing hypertonic solution (400 mosm) for 5 min. The stripped oocytes were placed in FVPP solution (in mm: 40 KCl, 75 potassium gluconate, 5 potassium fluoride, 0.1 sodium vanadate, 10 potassium pyrophosphate, 1 EGTA, 0.2 adenosine diphosphate, 10 PIPES, pH 7.4, 10 glucose, and 0.1 spermine) for giant inside-out patch preparation. The same solution was applied to the pipette. Macroscopic current recordings were performed using FVPP solutions containing equal concentrations of K+applied to both sides of the patch membranes. In a control experiment, we found that macroscopic currents recorded from giant inside-out patches showed less than 10% reduction over a 17-min period of recordings in FVPP solution. Current records were low-pass filtered (2000 Hz, Bessel 4-pole filter, −3 dB), digitized (10 kHz, 12-bit resolution) and stored on computer disc for later analysis (PCLAMP 6, Axon Instruments). Junction potentials between bath and pipette solutions were appropriately nulled before seal formation. A parallel perfusion system was used to administer agents to patches at a rate of ∼1 ml/min with no dead space (8Zhu G.Y. Chanchevalap S. Cui N.R. Jiang C. J. Physiol. (Lond. ). 1999; 516: 699-710Crossref Scopus (69) Google Scholar, 13Yang Z. Jiang C. J. Physiol. (Lond. ). 1999; 520: 921-927Crossref Scopus (48) Google Scholar). Low pH exposures were carried out using FVPP solutions that had been titrated to various pH levels as required. DNA fragments encoding the sequences of N- or C-terminal regions of HIR (HIRn, AA 1–50; HIRc, AA 171–445) were synthesized using polymerase chain reaction. The N-terminal region was fused to glutathionineS-transferase (GST) in pGEX-4T-3 (Amersham Pharmacia Biotech) by insertion of a polymerase chain reaction fragment of the HIRn at BamHI and XhoI restriction sites created with primers. Immediately following the BamHI site was the first methionine residue of the HIRn, so that there was no additional residue introduced. At the C-terminal end of the GST-HIRn, however, there were seven additional residues (LERPHRD) coming along with the vector before the stop codon in the plasmid. The HIRn was expressed inEscherichia coli BL21 cells (Amersham Pharmacia Biotech) by transformation using 0.25 mmisopropyl-β-d-thiogalactoside (IPTG, Amersham Pharmacia Biotech) at 25–30 °C for 3–5 h. Bacteria were lysed with a French pressure cell in lysis buffer (50 mm Tris-HCl, pH 7.4, or PIPES, pH 6.6, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1 mm dithiothreitol, 0.1 mm EDTA) containing protease inhibitors. Soluble proteins were isolated by centrifugation at 17,000 × g. The fusion proteins were purified with glutathionine-Sepharose beads (Amersham Pharmacia Biotech). The FLAG epitope was fused to HIRc using polymerase chain reaction. At the N-terminal end, a methonine residue was added with the Kozak sequence, followed by Ile171 of HIR. The FLAG sequence was introduced to the C-terminal end by removing the stop codon. Thus, there was no other residue missing in the HIRc peptide. The construct was then cloned into pcDNA3.1 and expressed in the human embryonic kidney cell line (HEK-293, ATCC, Manassas, VA). 48 h after transfection, proteins were extracted with lysis buffer (pH 7.4 or 6.6). Glutathionine-Sepharose beads that had been pre-linked to GST fusion proteins were mixed with HEK cell extracts in lysis buffer (pH 7.4 or 6.6) rotated at 4 °C for 2 h. Similar amounts of GST fusion proteins were used for each assay. Beads were centrifuged at 2000 × g for 2 min and washed three times with 0.7 ml of lysis buffer (pH 7.5 or 6.6). After a final wash, beads were heated in sample buffers for SDS-polyacrylamide gel electrophoresis, and bound proteins were separated with 15% SDS-polyacrylamide gel (Bio-Rad). After transferring the protein to a polyvinylidene difluoride membrane, binding to GST fusion proteins were detected by an anti-FLAG antibody (Sigma) followed by a secondary antibody conjugated with alkaline phosphatase (Bio-Rad). Color development was completed with an alkaline phosphatase conjugate substrate kit (Bio-Rad). Interaction assays were repeated two to four times to determine their reproducibility. Mutation analyses were performed on HIR and IRK1. Chimerical recombinations of intracellular N and C termini were presented using three letters, with the first and last representing the originations of N and C termini and the middle letter for the source of the rest sequence, e.g. HIH, C and N termini of the mutant were from HIR and the rest from IRK1; HII, the N terminus was from HIR and rest from IRK1. Chimerical mutants within the C or N terminus were named with two pieces of information: 1) the channel in which mutations were constructed, such XXXHIR, XXXIRK, and HIHXXX (mutants were made based on HIR, IRK1, and HIH, respectively); 2) the location of the mutations, such as N1–50HIH, amino acids from 1 to 50 in HIH were mutated to a corresponding sequence in IRK1; IRK179–238C, residues from 179 to 238 were mutated to those in HIR. Site-specific mutations were described using single-letter amino acids symbols, e.g. H191A, histidine at position 191 was mutated to alanine. Data are presented as means ± S.E. (n ≥ 4). Differences in means were examined with the Student's ttest or an analysis-of-variance test and were accepted as significant if p ≤ 0.05. Whole-cell currents were studied inXenopus oocytes that had received an injection of HIR cDNA or one of its mutants. In the two-electrode voltage clamp mode, inward rectifying currents as large as 20 μA were seen in most oocytes. These currents were sensitive to micromolar concentrations of Ba2+ and Cs+ (8Zhu G.Y. Chanchevalap S. Cui N.R. Jiang C. J. Physiol. (Lond. ). 1999; 516: 699-710Crossref Scopus (69) Google Scholar, 11Qu Z.Q. Zhu G.Y. Yang J. Cui N.R. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Exposure of the oocytes to 15% CO2 for 4–5 min produced a fast and reversible inhibition of these inward rectifying currents (Fig.1 A). The inhibition of HIR currents by CO2 was mediated by decreases in pH, since selectively lowering intra- and extracellular pH (pHi, pHo) to the corresponding levels (pHi 6.6, pHo6.2) seen during 15% CO2 exposure inhibited the HIR currents to the same degree as hypercapnia (8Zhu G.Y. Chanchevalap S. Cui N.R. Jiang C. J. Physiol. (Lond. ). 1999; 516: 699-710Crossref Scopus (69) Google Scholar, 11Qu Z.Q. Zhu G.Y. Yang J. Cui N.R. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Macroscopic currents recorded in excised patches were similarly inhibited (Fig.1 B). The consistent pH sensitivity seen in whole-cell recordings and excised patches suggests that proton-mediated channel inhibition is independent of other cytosolic factors. The IRK1 channel, however, did not respond to 15% CO2 and pHi6.0–7.4 (Fig. 2).Figure 2Dependence of the CO2/pH sensitivity on certain parts of the HIR channel protein. A, because HIR responds to hypercapnia (15% CO2) but Kir2.1 (IRK1) does not, chimeras were constructed between these two channels to identify the intracellular pH-sensing domain. Whole-cell currents were studied in two-electrode clamp. Their sensitivity to 15% CO2 was examined as described for Fig. 1 A and are presented as percentage inhibition of the currents. The HIH carrying both the N (AA 1–50) and C termini (AA 171–445) from HIR and the rest of its structures from IRK1 (AA 87–178) showed CO2 sensitivity almost identical to the wild-type HIR. In IHH, the entire HIR N-terminal region (AA 1–50) was substituted with its counterpart in IRK1 (AA 1–86). The IHH lost sensitivity to CO2. When IRK1 N-terminal domain (AA 1–86) was replaced with the corresponding sequence in HIR (AA 1–50), the mutant channel (HII) became CO2-sensitive. Extension of the mutation to include the N terminus through the M2 region of HIR (HHI; AA 171–445 in HIR were replaced with AA 179–428 in IRK1) did not significantly change the CO2sensitivity. B, concentration-dependent inhibition of K+ currents by acidic pH in inside-out patches. Currents were studied in inside-out patches under conditions described in Fig. 1 B. The current amplitude can be expressed as a function of pHi using the Hill equation:y = 1/[1 + (pK a/x) h ], where pK a (apparent pK) is the midpoint pH value for channel inhibition, and h is the Hill coefficient. Although HIR currents showed strong pHi sensitivity with pK a 6.76 (h = 3.1), IRK1 and IHH had almost no response to a pHi change from 7.4 to 5.8 (pK a 4.95, h 0.9, and pK a5.25, h 0.9, respectively). Although its pHi sensitivity decreased (pK a 6.45,h 2.2), HII responded to pHi changes more like HIR than IRK1. With both N and C termini from HIR and the rest of the sequences from IRK1, HIH had almost identical pH sensitivity (pK a6.77, h 3.1) to HIR. Data are presented as means ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effect of CO2 and pH on HIR currents depended on certain portions of the channel protein. A substitution of the N terminus of HIR (HIRn) with that of IRK1 (IHH, see “Materials and Methods” for nomenclature) completely abolished the CO2/ pHi sensitivities (Fig. 2), whereas a replacement of the N terminus of IRK1 (IRKn) with HIRn rendered the mutant channel (HII) substantial CO2/ pHi sensitivities, indicating that the pH-dependent gating mechanism is related to the N terminus. Although the recombinant HIR-IRK channels with the N terminus from HIR remained pH-sensitive, the apparent pK (pH value for 50% channel inhibition, pK a) value of the HII was ∼0.3 pH units lower than that of the wild-type HIR (Fig. 2 B). Thus, it is possible that other parts of the HIR protein are also involved in the pH sensing. To test this possibility, we extended the recombinant channel to include the N terminus as well as the M1, H5, and M2 regions of HIR (HHI). When this construct was expressed, we did not see any significant increase in CO2 sensitivity over the HII (Fig.2 A). A chimera (HIH) was then constructed which contained both HIRn and the C terminus of HIR (HIRc) and other sequences (M1 through M2) from IRK1. This mutant expressed inward rectifying currents with the amplitude and rectification comparable with those of HIR. When the HIH was exposed to 15% CO2 or acidic pH, we found that this channel was inhibited to exactly the same degree as the wild-type HIR (Fig. 2). The function of the HIRc in pH sensing was not simply contributive, because introducing the HIRc alone to the IRK1 (IIH, IHH) failed to produce any CO2/ pHi sensitivity (Fig.2). Thus, the HIRc is also required for pH sensing, although its role strictly depends on the presence of HIRn. The requirement of both the N and C termini for pHi sensing suggests that the channel gating may result from interactions of both the C and N termini. If the N and C termini can interact in pH sensing, there should be amino acid sequences critical for the interaction. To locate the sequences, several N-terminal mutants were constructed based on HIH and IRK1. We chose to use HIH rather than HIR, because the extracellular pH sensor was removed in this mutant (4Coulter K.L. Perier F. Radeke C.M. Vandenberg C.A. Neuron. 1995; 15: 1157-1168Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 11Qu Z.Q. Zhu G.Y. Yang J. Cui N.R. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) without affecting baseline currents or the pHi sensitivity, as mentioned above. HIRn and IRKn were divided into three segments at two conserved areas. Each chimera carried one or two segments from HIR and the rest from IRK1. The pHi sensitivity was then studied (Fig.3). If an area is not involved in interactions with the HIRc, mutation of this sequence should not affect the distinct pH sensitivity of channels carrying HIRc or IRKc as shown in Fig. 2 B (HIH versus HII); if it is involved, on the other hand, substitution of this sequence with that in IRK1 should produce a channel with identical pH sensitivity regardless of the presence of HIRc or IRKc. When a short sequence near the M1 domain was mutated to that in IRK1, the pH sensitivity of this mutant channel could no longer be enhanced by HIRc with a marked reduction in pH sensitivity (N51–60HIH versus N1–76IRK, Fig.3 A). This short motif contained about 10 amino acids with only three residues (TYM) different from those in IRK1. In contrast, mutations of other N-terminal domains did not eliminate the effect of HIRc in augmenting pH sensitivity although decreases in pH sensitivity were also observed (Fig. 3, B–D). These results suggest that the TYM motif that we described previously (11Qu Z.Q. Zhu G.Y. Yang J. Cui N.R. Li Y. Chanchevalap S. Sulaiman S. Haynie H. Jiang C. J. Biol. Chem. 1999; 274: 13783-13789Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) is indeed the N-terminal interaction site. The C terminus of HIR has 275 amino acids with a number of feature structures. At its C-terminal end, there is a potential PDZ-binding sequence. Immediately before this sequence, there are a cluster of negatively charged residues and a proline-rich motif. To determine whether these structures are involved in CO2 and pH sensing, several deletions of the C-terminal sequences were created by introducing a stop codon at positions 361, 381, or 393 (Fig.4 A). However, the CO2 sensitivity of these C-truncated HIR channels did not show any significant difference from the wild-type HIR (Fig.4 B), indicating that these structures are not necessary for the CO2 sensing. Several chimeras were constructed using C-terminal segments of HIR and IRK1, shown in Fig. 4 A. CO2 and pH sensitivities of these chimeras were then compared with those of HIH and HII. The substitution of a sequence from position 231 or 261 to the end of the C terminus (HIH231–445C, HIH261–445C) had no effect on the CO2 and pHi sensitivities of the mutant channels, which remained identical to HIH (Fig. 4, B andC). A chimera with sequence 196–445 replaced by that in IRK1 showed CO2 and pHi sensitivities similar to the HII, suggesting that the region between 196 and 230 is critical in CO2/ pHi sensing (Fig. 4, B andC). In region 196–230, there are only six residues that differ between HIR and IRK1, with four of them (PYMQ) clearly in contrast with their counterparts. Mutations of all four of these residues to those in IRK1 (HIH-SRIS) completely eliminated the C-terminal effect on enhancing pH sensitivity (Fig. 4, B andC). Introducing these four residues to HII significantly increased the CO2/ pHi sensitivity of the mutant channel over the HII (HII-PYMQ, Fig. 4, B and C). These results suggest, therefore, that the C-terminal domain for the N/C-terminal interaction is located at the PYMQ motif. Simultaneous mutations of both the N-terminal TYM and C-terminal PYMQ sequences (TYM-PYMQ) rendered the mutant channel the pHisensitivity identical to that of N51–60HIH (Fig. 4 C), suggesting the mutual dependence of these two motifs. Although the TYM and PYMQ motifs are critical in channel gating, there is no titratable residue within them. Apparently, the proton-binding sites are located in areas other than these two motifs. If protons mediate the N/C-terminal interaction, there should be proton-binding sites in the channel protein. Histidine has a pK value 6.04 in its side chain, which is close to the physiological pH level. This property makes the histidine residue the highly promising proton-binding site (10Chanchevalap, S., Yang, Z. J., Cui, N. R., Qu, Z., Zhu, G. Y., Liu, C. X., Giwa, L. R., Abdulkadir, L., and Jiang, C. J. Biol. 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