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• W2012455985 abstract "Protons are involved in gating Kir2.3. To identify the molecular motif in the Kir2.3 channel protein that is responsible for this process, experiments were performed using wild-type and mutated Kir2.3 and Kir2.1. CO2 and low pHi strongly inhibited wild-type Kir2.3 but not Kir2.1 in whole cell voltage clamp and excised inside-out patches. This CO2/pH sensitivity was completely eliminated in a mutant Kir2.3 in which the N terminus was substituted with that in Kir2.1, whereas a similar replacement of its C terminus had no effect. Site-specific mutations of all titratable residues in the N terminus, however, did not change the CO2/pH sensitivity. Using several chimeras generated systematically in the N terminus, a 10-residue motif near the M1 region was identified in which only three amino acids are different between Kir2.3 and Kir2.1. Mutations of these residues, especially Thr53, dramatically reduced the pH sensitivity of Kir2.3. Introducing these residues or even a single threonine to the corresponding positions of Kir2.1 made the mutant channel pH-sensitive. Thus, a critical motif responsible for gating Kir2.3 by protons was identified in the N terminus, which contained about 10 residues centered by Thr53. Protons are involved in gating Kir2.3. To identify the molecular motif in the Kir2.3 channel protein that is responsible for this process, experiments were performed using wild-type and mutated Kir2.3 and Kir2.1. CO2 and low pHi strongly inhibited wild-type Kir2.3 but not Kir2.1 in whole cell voltage clamp and excised inside-out patches. This CO2/pH sensitivity was completely eliminated in a mutant Kir2.3 in which the N terminus was substituted with that in Kir2.1, whereas a similar replacement of its C terminus had no effect. Site-specific mutations of all titratable residues in the N terminus, however, did not change the CO2/pH sensitivity. Using several chimeras generated systematically in the N terminus, a 10-residue motif near the M1 region was identified in which only three amino acids are different between Kir2.3 and Kir2.1. Mutations of these residues, especially Thr53, dramatically reduced the pH sensitivity of Kir2.3. Introducing these residues or even a single threonine to the corresponding positions of Kir2.1 made the mutant channel pH-sensitive. Thus, a critical motif responsible for gating Kir2.3 by protons was identified in the N terminus, which contained about 10 residues centered by Thr53. The modulation of membrane excitability is a significant cellular property, which not only provides cells with an important strategy for responding to their external stimuli but also enables them to monitor their internal environment and intermediary metabolism (1Jiang C. Haddad G.G. Lister G. Tissue Oxygen Deprivation: Developmental Molecular and Integrated Function. Marcel Dekker, New York1996: 595-616Google Scholar). Inward rectifier K+ channels play a part in this process in which several intracellular molecules such as nucleotides and protons are involved (2Jan L.Y. Jan Y.N. J. Physiol. (London). 1997; 505: 267-282Crossref Scopus (173) Google Scholar, 3Nichols C.G. Lopatin A.N. Annu. Rev. Physiol. 1997; 59: 171-191Crossref PubMed Scopus (668) Google Scholar). Whereas the concentration of protons is normally controlled by several buffering and feedback-controlling systems, pH can fall beyond its normal levels under certain pathophysiological conditions. For instance, CO2 retention, or hypercapnia, can cause a reduction in intra- and extracellular pH leading to respiratory acidosis (4Roos A. Boron W.F. Physiol. Rev. 1981; 61: 296-434Crossref PubMed Scopus (2297) Google Scholar). It is known that CO2 sensing, which plays an important role in the feedback regulation of CO2 and pH homeostasis in mammalian systems, is carried out by chemoreceptors, especially the central chemoreceptors (5Mitchell R.A. Berger R.A. Am. Rev. Respir. Dis. 1975; 111: 206-224PubMed Google Scholar, 6Loeschcke H.H. J. Physiol. (Lond.). 1982; 332: 1-24Crossref Scopus (276) Google Scholar, 7Schlaefke M.E. Rev. Physiol. Biochem. Pharmacol. 1981; 90: 171-244Crossref PubMed Google Scholar, 8Fitzgerrald R.S. Lahiri S. Fishman A.P. Handbook of Physiology. American Physiological Society, Baltimore1986: 313-362Google Scholar). Through these chemoreceptors, a high PCO2 level stimulates respiratory neuronal networks in the brain stem and enhances respiratory motor output. During this process, the information of PCO2 levels received by these chemoreceptor cells may first be conveyed to the change in their membrane excitability and then passed to the respiratory neuronal networks through chemical or electrical synaptic transmissions (9Euler C.Von. Fishman A.P. Handbook of Physiology. American Physiological Society, Baltimore1986: 1-67Google Scholar, 10Feldman J.L. Bloom F.E. Handbook of Physiology. American Physiological Society, Baltimore1986: 463-524Google Scholar). Hence, the change in membrane excitability constitutes an important step in the CO2 sensing. The alteration in membrane excitability with hypercapnia is known to be mediated by specific ion channels. CO2 has been shown to enhance the firing activity of locus ceruleus neurons by suppressing a proton- and polyamine-sensitive inward rectifying K+current (11Pineda J. Aghajanian G.K. Neuroscience. 1997; 77: 723-743Crossref PubMed Scopus (190) Google Scholar). Our previous studies have shown that CO2induces a depolarization in Lymnaea snail neurons by inhibiting a K+ channel with modest inward rectification (12Jiang C. Zhu G.Y. Hayashi F. Haddad G.G. Abstr. Soc. Neurosci. 1997; 23: 171.5Google Scholar). Recently, we have further demonstrated that CO2inhibits specific inward rectifier K+ channels including Kir2.3 (13Zhu G. Chanchevalap S. Cui N.R. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar). Because the Kir2.3 is expressed in the central nervous system and contributes to the maintenance of membrane excitability (14Perier F. Radeke C.M. Vandenberg C.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6240-6244Crossref PubMed Scopus (123) Google Scholar), detailed studies of the modulation of Kir2.3 during hypercapnia may bring about an understanding of the molecular mechanisms for CO2 sensing. Hypercapnia can modulate channel activity directly by acting on channel protein and indirectly by recruiting second messengers and other intermediate molecules. Our previous studies showed that the Kir2.3 inhibition during hypercapnia is largely mediated by decreases in the intra- and extracellular pH (pHi and pHo) and demonstrated that this channel has two pH sensors on either side of the plasma membranes (13Zhu G. Chanchevalap S. Cui N.R. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar). Whereas the pHo sensor has been well studied (15Coulter K.L. Perier F. Radeke C.M. Vandenberg C.A. Neuron. 1995; 15: 1157-1168Abstract Full Text PDF PubMed Scopus (120) Google Scholar), the pHi sensor, which contributes to more than 80% of channel inhibition, works at near physiological pH levels, and seemingly plays a more important role during hypercapnia, is still not understood. Therefore, we designed these experiments using a molecular genetic approach combined with patch clamp experiments to have an intervention of the structural-functional relationship of the Kir2.3 in CO2/pHi sensing. Frogs (Xenopus laevis) were anesthetized by bathing in 0.3% 3-aminobenzoic acid ethyl ester. A few lobes of the ovaries were removed after a small abdominal incision (∼5 mm).Xenopus oocytes were treated with 2 mg/ml collagenase (Type I, Sigma) in OR2 solution (82 mm NaCl, 2 mmKCl, 1 mm MgCl2, and 5 mm HEPES) for 90 min at room temperature. After washing, the oocytes were then incubated at 18 °C in ND-96 solution containing (in mm) 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES, and 2.5 sodium pyruvate with 100 mg/liter Geneticin added. A vector for eukaryotic expression (pcDNA3.1, Invitrogen, Carlsbad, CA) was used to express Kir2.1 and Kir2.3 channels. Chimerical constructs between Kir2.1 and Kir2.3 were prepared by an overlap extension at the junction of the interested domains using polymerase chain reaction (Pfu or Taq DNA polymerases, Stratagene, La Jolla, CA). The resulting polymerase chain reaction products were subcloned into pcDNA 3.1. Site-specific mutations were made using a site-directed mutagenesis kit (Quickchange, Stratagene). Correct mutations in both the overlapping polymerase chain reaction and site-directed mutagenesis were confirmed with DNA sequencing. Whole cell currents were studied on the oocytes 2–5 days after injection. Two-electrode voltage clamp was performed using an amplifier (Geneclamp 500, Axon Instruments, Foster City, CA) at room temperature (22–24 °C). The extracellular solution contained 90 mmKCl, 3 mm MgCl2, and 5 mm HEPES (pH 7.4). Cells were impaled using electrodes filled with 3 mKCl. The current electrodes had a resistance of 0.3–0.6 megaohms. Current records were low-pass filtered (Bessel, 4-pole filter, 3 dB at 5 kHz), digitized at 5 kHz (12-bit resolution), and acquired using pClamp 6.0.3 (Axon Instruments) (16Jiang C. Sigworth F.J. Haddad G.G. J. Neurosci. 1994; 14: 5590-5602Crossref PubMed Google Scholar, 17Zhu G. Zhang Y. Xu H. Jiang C. J. Neurosci. Methods. 1998; 81: 73-83Crossref PubMed Scopus (87) Google Scholar). Oocytes were accepted for further experiments only if they expressed large inward rectifying currents (>3 μA), and their leak currents measured as the difference before and after a leak subtraction were less than 10% of the peak currents. Xenopus oocytes were placed in a semi-closed recording chamber (BSC-HT, Medical System, Greenvale, NY) in which oocytes were placed on a supporting nylon mesh, so that the perfusion solution bathed both the top and bottom surface of the oocytes. CO2(15% balanced with air) exposures were the same as described previously (13Zhu G. Chanchevalap S. Cui N.R. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar). A high concentration of KHCO3 was used for intracellular acidification (18Fakler 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, 19Doi 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). In these experiments, bicarbonate at concentrations of 20, 50, and 90 mm was applied to the bath solutions without changing the K+ concentration. Intracellular pH levels were measured when these perfusates were administrated to oocytes. Patch clamp experiments were performed at room temperature (about 25 °C) as described previously (16Jiang C. Sigworth F.J. Haddad G.G. J. Neurosci. 1994; 14: 5590-5602Crossref PubMed Google Scholar, 17Zhu G. Zhang Y. Xu H. Jiang C. J. Neurosci. Methods. 1998; 81: 73-83Crossref PubMed Scopus (87) Google Scholar). In brief, fire-polished patch pipettes (0.5–2 megaohms) were made from 1.2-mm borosilicate capillary glass (Sutter P-94/PC puller). Macroscopic currents were recorded from Giant inside-out patches. Current records were low-pass filtered (2,000 Hz, Bessel, 4-pole filter, −3 dB), digitized (10 kHz, 12-bit resolution), and stored on computer disk for later analysis (pClamp 6, Axon Instruments). Junction potentials between the bath and pipette solutions were appropriately nulled before seal formation. For macroscopic current recordings, the oocyte vitelline membranes were mechanically removed after exposing hypertonic solution (400 mosmol) 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 and recording pipette solution contained (in mm): 40 KCl, 75 potassium gluconate, 5 potassium fluoride, 0.1 sodium vanadate, 10 potassium pyrophosphate, 1 EGTA, 0.2 ADP, 10 PIPES, 1The abbreviations used are: PIPES, piperazine-N,N′-bis-(2-ethanesulfonic acid); H7, 1-[5-isoquinolinesulfonyl]-2-methylpiperazine dihydrochloride 10 glucose, and 0.1 spermine (FVPP solution, pH 7.4). This bath solution was chosen after several others had been tested regarding channel run-down in excised patches. In a control experiment, we found that macroscopic currents recorded from giant inside-out patches were very well maintained, showing less than 10% reduction over a 20-min period of recordings made in such a bath solution. A parallel perfusion system was used to administer agents to patches or cells at a rate of ∼1 ml/min with no dead space (16Jiang C. Sigworth F.J. Haddad G.G. J. Neurosci. 1994; 14: 5590-5602Crossref PubMed Google Scholar, 17Zhu G. Zhang Y. Xu H. Jiang C. J. Neurosci. Methods. 1998; 81: 73-83Crossref PubMed Scopus (87) Google Scholar). Low pH exposures were carried out using the same bath solutions that had been titrated to various pH levels as required by experimental protocols. PIPES buffer was used because of its buffering range from pH 5.8 to 7.4. Data are presented as means ± S.E. (n ≥ 4). Differences in means were tested with the Student's t test and were accepted as significant if p ≤ 0.05. Kir2.3 currents were studied by expressing these K+ channels in Xenopus oocytes. In the voltage clamp mode, whole cell Kir2.3 currents showed a strong inward rectification and were highly sensitive to extracellular Ba2+ (IC50 = 11 ± 2 μm,n = 4). Exposure of the oocytes to 15% CO2produced a marked inhibition of the whole cell Kir2.3 currents by 80 ± 2% (n = 7) (Fig.1 A). This effect was reversible and dependent on concentrations of CO2 (5, 10, and 15%, data not shown). Selective intracellular acidification without changing the extracellular pH also inhibited Kir2.3 to an extent comparable with that of CO2 when changes in pH levels were considered. The measured intracellular pH level during 15% CO2 exposure was 6.58 ± 0.13 (n = 6) using ion-selective microelectrodes (20Jiang C. Agulian S. Haddad G.G. J. Physiol. (Lond.). 1992; 448: 697-708Crossref Scopus (82) Google Scholar). Selective intracellular acidification to this pH level inhibited Kir2.3 by 74 ± 4% (n = 5). The inhibition of Kir2.3 by CO2 or intracellular acidification can be a direct effect of protons on the channel protein or an indirect effect through changes in concentrations of intracellular second messengers, protein kinases, phosphatases, and other cytosol-soluble factors. To delineate the mechanisms underlying the channel modulation, we performed experiments using cell-free excised patches. In the inside-out patch configuration, Kir2.3 currents were strongly inhibited when the cytosolic surface of the plasma membrane was exposed to low pH solutions. This inhibition was fast, reversible, and concentration-dependent (Fig.2). Another member of the Kir2 family, Kir2.1, however did not respond to either high CO2 (Fig.1 B) or pHi 6.6 (Fig.3), suggesting that this effect was rather specific. Because cytosol-soluble factors were vastly diluted or washed out under our experimental condition, the inhibition of Kir2.3 channels is unlikely to be mediated by second messengers and other cytosol-soluble factors. Moreover, because our intracellular solutions contained chemicals that were unfavorable to protein dephosphorylation (see “Materials and Methods”), the modulation of Kir2.3 channel activity by pH levels may not be related to the fast turn-over of protein phosphorylation and dephosphorylation.Figure 3The crucial role of the N-terminal region in pH sensing. A, because Kir2.3 (HIR) responds to CO2 and low pHi but Kir2.1 (IRK) does not, chimeras were constructed between these two channels to identify the intracellular pH-sensing domain. In IN-HIR, the Kir2.3 N-terminal region (from the N terminus to the beginning of M1) was substituted with the counterpart of Kir2.1. This IN-HIR recombinant lost its sensitivity to intracellular acidification. When the Kir2.1 N-terminal domain was replaced with the corresponding sequence in Kir2.3, the mutant channel became pHi-sensitive. Note that pHi sensitivity of these chimeras was studied by two-electrode clamp experiments in different oocytes. B, concentration-dependent inhibition of K+currents in inside-out patches. Whereas Kir2.3 currents showed strong pHi sensitivity with pK 6.77, Kir2.1 and IN-HIR had almost no response to a pHi change from 7.4 to 5.8. Although its pHi sensitivity decreased (pK6.45), HN-IRK responded to pHi changes in a manner more like Kir2.3 than Kir2.1. Data are presented as means ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our results in excised patches support an idea that the proton-sensitive mechanism for channel gating is located on the Kir2.3 channel protein. To further test this idea, we chose to use molecular genetic approaches to determine the molecular basis of Kir2.3 channel modulation by intracellular protons. According to the generally accepted membrane-spanning topology of inward rectifier K+ channels, the Kir2.3 channel should have its N and C termini in the cytosol. In addition, part of the pore (P or H5) sequence should be accessible from the intracellular side. Based on these characteristics of Kir channels, we constructed chimeras in which each of these three sequences was replaced by its counterpart in Kir2.1. CO2 and pH sensitivities of these recombinant HIR-IRK channels were then studied in whole cell voltage clamp and excised patches. Substitution of the N terminus in Kir2.3 with that in Kir2.1 (IN-HIR) completely eliminated the channel response to low pHi (Figs. 3 A and 5). A chimerical Kir2.1 channel carrying an N-terminal sequence of Kir2.3 (HN-IRK) became pH-sensitive, although the degree of the pH sensitivity was smaller than that of wild-type Kir2.3 (Figs. 3 A and 5). Switches of the C terminus (IRK-HC, Fig. 5) or the H5 region from Kir2.3 to Kir2.1 (IN-HIR, Figs. 3 A and 5), however, failed to produce any CO2/pH sensitivity in these mutant channels. Thus, these results indicate that the CO2/pHi-sensitive mechanism is located on the N terminus rather than the H5 and C terminus of Kir2.3. It is known that a large number of protein molecules can be modulated by pH levels. Binding of protons to certain titratable amino acids can cause changes in the electrical charges of these residues and thus alter the protein conformation and channel activity. If this is the case in Kir2.3, there should be amino acid residues on the channel protein acting as receptive sites for protons. To test this hypothesis, we examined titratable amino acids in the N terminus of Kir2.3, especially histidines in which the side chain has a pK value of 6.04. Using site-directed mutagenesis, these histidines were changed to aspartates in which positive charges at these positions were converted to negative charges during hypercapnia. A simultaneous replacement of all three histidines (H2D, H4D, H11D) had no effect on channel expression and sensitivity to CO2 and pHi (Fig. 4, A andB). Neither did replacements of individual ones, suggesting that these histidines do not constitute the pH sensor in Kir2.3. To further strengthen this idea, we deleted all of these histidines plus the other nine residues at the beginning of the N terminus and found that the CO2/pHi sensitivity was unaffected (see below). In addition to these histidines, several residues that exist only in Kir2.3 and are potentially titratable at nonphysiological (extremely high or low) pH levels were studied. After these residues were switched to those in Kir2.1, including R6G, R14T, K16Q, and R17Q, we found that the same pH sensitivity was retained in all of these mutant Kir2.3 channels. Thus, none of these residues appears to be the reception or initiation site for proton sensing. To understand the pH-dependent mechanisms for Kir2.3 gating, we divided the N-terminal region into three segments and reconstructed several N-terminal chimeras using Kir2.3 and Kir2.1 sequences. Most of these chimeras were expressed in oocytes as functional channels, although the current amplitude in some of them was reduced. To eliminate the extracellular pH sensor, the sequence from M1 through M2 in Kir2.3 was replaced with a corresponding one in Kir2.1. This mutant channel carrying N and C termini from Kir2.3 (HIH) showed CO2 and pHi sensitivities almost identical to the wild-type Kir2.3 (Fig. 6 B). Thus, several chimeras were constructed based on the HIH. The first 12 amino acids at the N terminus of Kir2.3 (N1–12IH) were not involved in the CO2 sensitivity. A truncation of these residues did not have any significant effect on the channel expression, base-line currents, and sensitivity to CO2 (70 ± 3%,n = 4, p > 0.05) (Fig.5). Although a deletion of the next 12 residues did not produce functional channels, chimera with the first 19 amino acids replaced by those in Kir2.1 (N1–19IH) showed a CO2 sensitivity very close to that of the wild-type Kir2.3 (61 ± 4%, n = 6). The middle part of the N-terminal region includes 20 residues with 9 of them different between Kir2.3 and Kir2.1. Substitution of this sequence in Kir2.3 (N25–44IH) moderately reduced the level of CO2 sensitivity (54 ± 6%, n = 4). Unlike the two N-terminal regions mentioned above, an amino acid sequence near the M1 membrane-spanning domain containing about 10 residues was critical for CO2sensitivity. Replacement of this short sequence alone (N51–60IH) was sufficient to eliminate 80% of the CO2 sensitivity of the mutant Kir2.3 (13 ± 2%, n = 4). When this short sequence was constructed to Kir2.1 (N77–86IRK), we found that the mutant channel gained a substantial sensitivity to CO2(18 ± 5%, n = 4) and pHi (Figs. 5and 7). Extension of this sequence to 41 residues prior to the M1 domain of Kir2.3 (N46–86IRK) gave the mutant Kir2.1 a CO2sensitivity (36 ± 3%, n = 4) nearly the same as the HN-IRK (40 ± 4%, n = 6, p > 0.05) (Fig. 5). Therefore, this 10-residue motif is critical for the sensing of CO2/pH in Kir2.3, although another 30 amino acids (total 41 residues) around this motif are also needed to achieve the full effect of the entire N terminus. There is a high sequence homology in these 10 residues between Kir2.3 and Kir2.1, in which Kir2.3 differs from Kir2.1 in only three residues (Thr53, Tyr57, and Met60). Mutation of all of them into residues in Kir2.1 (T53I, Y57W, and M60V) greatly reduced the pH sensitivity by 0.6 pH units and generally disabled this mutant channel in terms of CO2 sensing (Fig. 6). Replacements of two of these residues (T53I, Y57W) had the same effect (Fig. 6 B). Even with a single mutation (T53I), the CO2 and pH sensitivities were severely diminished. Mutation of either tyrosine or methionine alone (Y57W or M60V) also caused a reduction in the CO2 and pH sensitivities, though their effects were much less than that of the T53I mutation. When these three residues were constructed to Kir2.1 (N77–86IRK), the effect was dramatic. The mutant Kir2.1 started to be inhibited at pHi6.6 with a pK value of 6.0 and h (Hill coefficient) of 2.3 (n = 5). Its pH sensitivity became more like that of HN-IRK, a chimera carrying the full N terminus from Kir2.3, than of the wild-type Kir2.1 (Fig.7). Mutation of isoleucine 79 alone (I79T-IRK) had an effect on converting the pH sensitivity of the Kir2.1 as effectively as the N77–86IRK mutation (pK 5.93,h 2.3, n = 5) (Fig. 7), demonstrating the critical role of the threonine 53 of Kir2.3 in CO2 and pHi sensing. Because threonine 53 in Kir2.3 is a protein kinase C phosphorylation site (21Zhu G. Qu Z. Cui N. Jiang C. J. Biol. Chem. 1999; 274: 11643-11646Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), channel sensitivity to CO2 was studied in the presence of high concentration H7 (1-[5-isoquinolinesulfonyl]-2-methylpiperazine dihydrochloride, a blocker of protein kinases C and A; Sigma). After bathing the oocytes with 30 μm H7 for 20 min, exposure of the oocytes to 15% CO2 produced the same degree of inhibition of Kir2.3 currents (74 ± 3% control versus 72 ± 1% with H7, p > 0.05, paired Student's t test,n = 4), a result consistent with our studies of wild-type Kir2.3 using excised patches (see above). In our current studies, we have demonstrated that a molecular motif that is located on the N terminus consisting of about 10 amino acids is responsible for gating the Kir2.3 channel by high CO2 and low intracellular pH. Like Ca2+, nucleotides, and other second messengers, proton is an important regulator of cellular functions. A number of ion channels have been known to be modulated by protons; these include large conductance Ca2+-activated K+ channels, hcurrents, gap junctions, and γ-aminobutyric acid andN-monomethyl arginine receptor channels (22Traynelis S.F. Cull-Candy S.G. Nature. 1990; 345: 347-350Crossref PubMed Scopus (474) Google Scholar, 23Krishek B.J. Amato A. Connolly C.N. Moss S.J. Smart T.G. J. Physiol. (Lond.). 1996; 492: 431-443Crossref Scopus (85) Google Scholar, 24Morley G.E. Taffet S.M. Delmar M. Biophys. J. 1996; 70: 1294-1302Abstract Full Text PDF PubMed Scopus (235) Google Scholar, 25Bukauskas F.F. Peracchia C. Biophys. J. 1997; 72: 2137-2142Abstract Full Text PDF PubMed Scopus (105) Google Scholar, 26Wellner-Kienitz M.C. Shams H. Neuroscience. 1998; 87: 109-121Crossref PubMed Scopus (15) Google Scholar, 27Wellner-Kienitz M.C. Shams H. Scheid P. J. Neurophysiol. 1998; 79: 2885-2894Crossref PubMed Scopus (43) Google Scholar). Changes in the activity of these ion channels can take place under several physiological and pathophysiological conditions when intra- and extracellular pH levels are low. One of these conditions is high CO2 or hypercapnia. CO2 sensing by the central chemoreceptors is critical in feedback mechanisms controlling CO2 levels in mammals (5Mitchell R.A. Berger R.A. Am. Rev. Respir. Dis. 1975; 111: 206-224PubMed Google Scholar, 7Schlaefke M.E. Rev. Physiol. Biochem. Pharmacol. 1981; 90: 171-244Crossref PubMed Google Scholar, 8Fitzgerrald R.S. Lahiri S. Fishman A.P. Handbook of Physiology. American Physiological Society, Baltimore1986: 313-362Google Scholar, 28Scheid P. Oyamada Y. Ballantyne D. FASEB J. 1998; 12: 288Google Scholar), which may involve the modulation of K+ channel activity. Inhibition of inward rectifier K+ channels can produce depolarization and an increase in membrane excitability (2Jan L.Y. Jan Y.N. J. Physiol. (London). 1997; 505: 267-282Crossref Scopus (173) Google Scholar, 3Nichols C.G. Lopatin A.N. Annu. Rev. Physiol. 1997; 59: 171-191Crossref PubMed Scopus (668) Google Scholar). Although there are a number of ways to inhibit these K+ channels during hypercapnia, our current studies indicate that the mechanisms for the inhibition of Kir2.3 are related to the specific structure of the channel protein. We have found that fast and reversible inhibition of Kir2.3 activity by high CO2 or low pH can be seen in cell-free excised patches, indicating that cytosolic soluble factors such as second messengers, Mg2+, and polyamines are not players. Also, CO2/pH sensing is seen in Kir2.3 but not in Kir2.1, which has a nearly 80% homology to Kir2.3 in amino acid sequence, suggesting that a specific sequence in Kir2.3 protein underlies this sensitivity. Moreover, our data do not support the idea that the inhibition of Kir2.3 during hypercapnia or intracellular acidification is mediated by phosphorylation of channel proteins. There are chemical compounds in our intracellular solution to prevent channel rundown that are inhibitors of phosphatases and phosphodiesterases. This observation plus the lack of ATP and Mg2+ in our intracellular solution strongly suggests that the turnover of protein phosphorylation and dephosphorylation in gating Kir2.3 may not occur under such an experimental condition. Also our results have shown that the inhibition of Kir2.3 activity by CO2 is not affected by the blockade of protein kinases C and A. Therefore, specific structures on Kir2.3 channel protein seem to be responsible for CO2 and pH sensing. If protons have a direct effect on channel functions, they may bind to the channel protein through certain titratable amino acid residues and alter conformation and activity of the channel. Also, protons can affect channel activity if they are involved in the binding of the channel protein with another protein. In any case, the channel protein should have specific sequences for these interactions. To locate these sites, we have studied the pH sensitivity of Kir2.3 channel by selectively replacing amino acid residues and peptide sequences in this channel with those in Kir2.1, which is not sensitive to a pH change in physiological ranges. These molecular genetic interventions have led us to look closely at the structure-functional relationship in the Kir2.3 channel in CO2/pH sensing. Consistent with the idea that the sensing mechanism is related to the channel protein, we have found that CO2/pH sensitivity requires the presence of the N-terminal region of Kir2.3. To find proton-binding sites in the N terminus, we have examined all of the histidine residues by substituting them with aspartate, a negatively charged amino acid, or by deleting all of them with another nine residues at the very end of the N terminus. However, none of these mutants has shown a change in its sensitivity to CO2 and pH, indicating that these histidines are not the pH sensor. Interestingly, the extracellular pH sensor does not involve titratable histidine residues either. Although an extracellular domain including a histidine is crucial in pHo sensing, it does not seem to have a proton-binding site. Indeed, selective substitution of the histidine residue in this cluster has no effect on the pHosensitivity of Kir2.3 (15Coulter K.L. Perier F. Radeke C.M. Vandenberg C.A. Neuron. 1995; 15: 1157-1168Abstract Full Text PDF PubMed Scopus (120) Google Scholar). Thus, Coulter et al. (15Coulter K.L. Perier F. Radeke C.M. Vandenberg C.A. Neuron. 1995; 15: 1157-1168Abstract Full Text PDF PubMed Scopus (120) Google Scholar) believe that an unidentified cysteine residue may play a role. Using these chimerical and site-directed mutant Kir channels, we have identified a consensus sequence in the N-terminal region of Kir2.3 that is crucial for Kir2.3 gating by CO2 and pHi. This motif is located in a conserved region near the M1 membrane-spanning domain. Although there are only three residues that are different between Kir2.3 and Kir2.1, this motif may involve some neighboring residues. Mutation of any of these residues causes a marked reduction in CO2 sensitivity. Of these three residues, the Thr53 is the key. Mutation of this residue leads to an almost complete loss of channel sensitivity to pHi and CO2, whereas creation of this residue in Kir2.1 makes the mutant channel pH-sensitive. Interestingly, threonine is not a titratable amino acid, and thereby pH change should not affect either its charges or its polarity. Thus, protons may not act directly on this residue to change channel conformation and activity. Then how can this residue be involved in CO2/pH sensing and the modulation of channel activity? We believe that there are at least three potential ways by which this threonine residue can participate in the modulation of channel activity during hypercapnia. 1) This residue may be engaged in the maintenance of the pK value of another amino acid residue(s) titratable at high or low pH, as suggested previously in Kir1.1 (20Jiang C. Agulian S. Haddad G.G. J. Physiol. (Lond.). 1992; 448: 697-708Crossref Scopus (82) Google Scholar, 29Choe H. Zhou H. Palmer L.G. Sackin H. Am. J. Physiol. 1997; 273: F516-F529PubMed Google Scholar). 2) This residue may be involved in a motif that is required for protein-protein interactions in CO2/pH sensing. 3) Phosphorylation at this residue may occur during CO2/pHi exposure. Is it possible that protons bind to ionizable amino acids other than histidine residues in the N terminus? To answer this question, we have changed nonconserved lysine and arginine residues in the Kir2.3 N terminus into corresponding ones in the Kir2.1 (R6G, R14T, K16Q, and R17Q). Our data reveal that mutations of these residues do not affect CO2/pHi sensitivity. Also, we have systematically divided the N termini of Kir2.3 and Kir2.1 into several segments and rejoined them alternatively to construct chimerical N termini. These extensive interventions would allow us to interrupt the pH sensitivity of Kir2.3 if there were such a proton-binding site, whether on positively or negatively charged residues. Although some of these chimeras, such as the N51–60IH, are shown to be more critical than others in CO2/pH sensing, residues potentially titratable in physiological pH levels do not exist in the sequence of Kir2.3. Interestingly, there are three residues (Asp52, Arg54, and Arg56) that are titratable at extremely high or low pH in the short motif. We have tried to mutate some of them into neutral and polar residues (asparagine) in the Kir1.1 channel. Because they are highly conserved in all Kir channels, these mutations did not produce any functional channels (30Xu H.X. Chanchevalap S. Zhu G. Qu Z. Zhang Y. Haynie H. Jiang C. Abstr. Soc. Neurosci. 1998; 24: 148.8Google Scholar). Therefore, whether some of them are proton sensors with pK levels that can be affected by the Thr53 is still unknown. Because mutations of Thr53 and Tyr57 into nonpolar isoleucine and tryptophan dramatically reduced the CO2 sensitivity of Kir2.3, the polarity of positions 53 and 57 seems important in pH sensing. The periodicity of the three crucial residues (Thr53, Tyr57, and Met60) may suggest a helical structure in the region, but our search using the Chou-Fasman and Robson-Granier analyses did not show any helical structure in this short motif. Our results indicate amino acid sequences other than this motif are also needed to fulfill a complete CO2 and pHisensitivity. In addition to these 10 residues, we have observed that a sequence composed of a total 41 amino acids can produce an effect of CO2/pH as effectively as the entire N terminus. Also, the presence of the C terminus enhances the CO2/pH sensitivity of the mutant Kir channels, although it has no effect on CO2/pH sensing by itself. A simple explanation of these data would be that the C terminus interacts with the N terminus in gating the channel, as suggested recently in Kir1.1 by Schulte et al. (31Schulte U. Hahn H. Wiesinger H. Ruppersberg J.P. Fakler B. J. Biol. Chem. 1998; 273: 34575-34579Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Our data, however, do not support the idea that CO2/pH sensing in Kir2.3 is a result of protein phosphorylation, as has been described above. Therefore, it is possible that CO2/pH sensing in Kir2.3 is mediated by an interaction of amino acid residues on the Kir2.3, which is affected by protons and controls the channel activity. Clearly, further understanding of the molecular mechanisms for Kir2.3 modulation by protons may provide information of channel gating under a variety of pathophysiological conditions, and the demonstration of these critical residues in CO2/pH sensing in our current studies constitutes an important step toward this goal. We believe that the molecular basis of pH sensing in Kir2.3 may underlie the CO2 chemoreception in the central nervous system. Studies on central chemoreceptors have shown that a large number of these cells depolarize and their membrane excitability increases during hypercapnia. This change in membrane excitability is not mediated by a change in synaptic transmission, because the depolarization persists with the removal of extracellular Ca2+ or exposure to tetrodotoxin (32Dean J.B. Bayliss D.A. Erickson J.T. Lawing W.L. Millhorn D.E. Neuroscience. 1990; 36: 207-216Crossref PubMed Scopus (166) Google Scholar). Although several ionic mechanisms may be involved in the regulation of membrane excitability during hypercapnia, K+ channels, especially the inward rectifier K+ channels, seem to be the major player (11Pineda J. Aghajanian G.K. Neuroscience. 1997; 77: 723-743Crossref PubMed Scopus (190) Google Scholar, 12Jiang C. Zhu G.Y. Hayashi F. Haddad G.G. Abstr. Soc. Neurosci. 1997; 23: 171.5Google Scholar, 33Hayashi F. Jiang C. Takeda R. Haddad G.G. Abstr. Soc. Neurosci. 1992; 18: 348.10Google Scholar). The inhibition of these inward rectifier K+ channels during hypercapnia produces depolarization and increases membrane excitability (13Zhu G. Chanchevalap S. Cui N.R. Jiang C. J. Physiol. (Lond.). 1999; 516: 699-710Crossref Scopus (70) Google Scholar). With two pH sensors on Kir2.3, cells can sense pH changes on either side of the plasma membranes, in their internal and external environment. Because the primary sensor is a K+ channel, the changes in intra- and extracellular pH can be readily coupled to membrane excitability in the nerve cells that are endowed with this pH sensing mechanism and in the neuronal networks to which these cells project. Because Kir2.3 is expressed in the central nervous system (14Perier F. Radeke C.M. Vandenberg C.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6240-6244Crossref PubMed Scopus (123) Google Scholar, 34Karschin C. Karschin A. Mol. Cell. Neurosci. 1997; 10: 131-148Crossref PubMed Scopus (70) Google Scholar), the determination of molecular mechanisms in proton sensing may be applicable to the search for unidentified homologues of the Kir2.3 channel in brain stem neurons, which may eventually result in the identification of the CO2 central chemoreceptors. We thank Drs. Lily L. Jan and Carol A. Vandenberg for the gifts of Kir2.1 and Kir2.3 cDNAs." @default.
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• W2012455985 title "Identification of a Critical Motif Responsible for Gating of Kir2.3 Channel by Intracellular Protons" @default.
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