Regulation of Connexin Channels by pH
1999; Elsevier BV; Volume: 274; Issue: 6 Linguagem: Inglês
10.1074/jbc.274.6.3711
ISSN1083-351X
AutoresCarville G. Bevans, Andrew L. Harris,
Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoProtonated aminosulfonate compounds directly inhibit connexin channel activity. This was demonstrated by pH-dependent connexin channel activity in Good's pH buffers (MES (4-morpholineethanesulfonic acid), HEPES, and TAPS (3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino]-1-propanesulfonic acid)) that have an aminosulfonate moiety in common and by the absence of pH-dependent channel activity in pH buffers without an aminosulfonate moiety (maleate, Tris, and bicarbonate). The pH-activity relation was shifted according to the pK a of each aminosulfonate pH buffer. At constant pH, increased aminosulfonate concentration inhibited channel activity. Taurine, a ubiquitous cytoplasmic aminosulfonic acid, had the same effect at physiological concentrations. These data raise the possibility that effects on connexin channel activity previously attributed to protonation of connexin may be mediated instead by protonation of cytoplasmic regulators, such as taurine. Modulation by aminosulfonates is specific for heteromeric connexin channels containing connexin-26; it does not occur significantly for homomeric connexin-32 channels. The identification of taurine as a cytoplasmic compound that directly interacts with and modulates connexin channel activity is likely to facilitate understanding of cellular modulation of connexin channels and lead to the development of reagents for use in structure-function studies of connexin protein. Protonated aminosulfonate compounds directly inhibit connexin channel activity. This was demonstrated by pH-dependent connexin channel activity in Good's pH buffers (MES (4-morpholineethanesulfonic acid), HEPES, and TAPS (3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino]-1-propanesulfonic acid)) that have an aminosulfonate moiety in common and by the absence of pH-dependent channel activity in pH buffers without an aminosulfonate moiety (maleate, Tris, and bicarbonate). The pH-activity relation was shifted according to the pK a of each aminosulfonate pH buffer. At constant pH, increased aminosulfonate concentration inhibited channel activity. Taurine, a ubiquitous cytoplasmic aminosulfonic acid, had the same effect at physiological concentrations. These data raise the possibility that effects on connexin channel activity previously attributed to protonation of connexin may be mediated instead by protonation of cytoplasmic regulators, such as taurine. Modulation by aminosulfonates is specific for heteromeric connexin channels containing connexin-26; it does not occur significantly for homomeric connexin-32 channels. The identification of taurine as a cytoplasmic compound that directly interacts with and modulates connexin channel activity is likely to facilitate understanding of cellular modulation of connexin channels and lead to the development of reagents for use in structure-function studies of connexin protein. Changes in intracellular pH (pHi) 1The abbreviations used are: pHi, intracellular pH; Cx, connexin; CT, C-terminal; NT, N-terminal; P o, channel open probability; TSF, transport-specific fractionation (of liposomes); CL, cytoplasmic loop; MES, 4-morpholineethanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydrox-ymethyl)ethyl]amino]-1-propanesulfonic acid.affect gap junction conductance between cells (1Spray D.C. Burt J.M. Am. J. Physiol. 1990; 258: C195-C205Crossref PubMed Google Scholar). The sensitivity of junctional conductance to changes in pHi varies with cell type and connexin isoform (2Campos de Carvalho A.C. Braz. J. Med. Biol. Res. 1988; 21: 177-188PubMed Google Scholar, 3Liu S. Taffet S. Stoner L. Delmar M. Vallano M.L. Jalife J. Biophys. J. 1993; 64: 1422-1433Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 4Delmar M. Liu S. Morley G.E. Ek J.F. Pertsova R.N. Anumonwo J.M. Taffet S.M. Zipes D.P. Jalife J. Cardiac Electrophysiology: From Cell to Bedside. W. B. Saunders, New York1995: 135-143Google Scholar, 5Spray D.C. Citi S. Molecular Mechanisms of Epithelial Cell Junctions: From Development to Disease. R. G. Landes, New York1994: 195-215Google Scholar). Decrease of pHi from physiological levels typically produces a decrease in junctional conductance (6Iwatsuki N. Petersen O.H. J. Physiol. (Lond.). 1979; 291: 317-326Crossref Scopus (34) Google Scholar, 7Turin L. Warner A.E. J. Physiol. (Lond.). 1980; 300: 489-504Crossref Scopus (136) Google Scholar, 8Spray D.C. Harris A.L. Bennett M.V.L. Science. 1981; 211: 712-715Crossref PubMed Scopus (343) Google Scholar) and in permeability to large tracers (9Schuetze S.M. Goodenough D.A. J. Cell Biol. 1982; 92: 694-705Crossref PubMed Scopus (47) Google Scholar, 10Connors B.W. Benardo L.S. Prince D.A. J. Neurosci. 1984; 4: 1324-1330Crossref PubMed Google Scholar). The decrease in junctional conductance is usually reversible with return of pHi to normal physiological values. The molecular mechanisms that underlie this modulation of connexin channel activity are unclear and may differ among connexin isoforms and cell types. It has been proposed that the modulation is due to direct protonation of connexin (8Spray D.C. Harris A.L. Bennett M.V.L. Science. 1981; 211: 712-715Crossref PubMed Scopus (343) Google Scholar), changes in ionized calcium concentration (11Rink T.J. Tsien R.Y. Warner A.E. Nature. 1980; 283: 658-660Crossref PubMed Scopus (79) Google Scholar), and activation of calmodulin (12Peracchia C. Wang X.G. Braz. J. Med. Biol. Res. 1997; 30: 577-590Crossref PubMed Scopus (27) Google Scholar, 13Torok K. Stauffer K. Evans W.H. Biochem. J. 1997; 326: 479-483Crossref PubMed Scopus (95) Google Scholar, 14Peracchia C. Wang X.G. Li L.Q. Peracchia L.L. Pflügers Arch. 1996; 431: 379-387Crossref PubMed Scopus (80) Google Scholar). For connexin-43 and for connexin-32/connexin-38 chimerae, recent work strongly indicates a pH-dependent interaction between segments of the C-terminal domain and the single cytoplasmic loop that inhibits channel activity (3Liu S. Taffet S. Stoner L. Delmar M. Vallano M.L. Jalife J. Biophys. J. 1993; 64: 1422-1433Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 15Morley G.E. Ekvitorin J.F. Taffet S.M. Delmar M. J. Cardiovasc. Electrophysiol. 1997; 8: 939-951Crossref PubMed Scopus (69) Google Scholar, 16Ek J.F. Delmar M. Perzova R. Taffet S.M. Circ. Res. 1994; 74: 1058-1064Crossref PubMed Scopus (79) Google Scholar, 17Morley G.E. Taffet S.M. Delmar M. Biophys. J. 1996; 70: 1294-1302Abstract Full Text PDF PubMed Scopus (235) Google Scholar, 18Ekvitorin J.F. Calero G. Morley G.E. Coombs W. Taffet S.M. Delmar M. Biophys. J. 1996; 71: 1273-1284Abstract Full Text PDF PubMed Scopus (144) Google Scholar, 19Wang X.G. Peracchia C. Am. J. Physiol. 1996; 40: C1743-C1749Crossref Google Scholar, 20Wang X.G. Li L.Q. Peracchia L.L. Peracchia C. Pflügers Arch. 1996; 431: 844-852Crossref PubMed Scopus (55) Google Scholar, 21Wang X.G. Peracchia C. Biophys. J. 1997; 73: 798-806Abstract Full Text PDF PubMed Scopus (41) Google Scholar). In this study, we set out to investigate modulation of connexin channel activity as a function of pH, using in a reconstituted system connexin channels immunoaffinity-purified from native tissues. Channel activity was monitored using transport-specific fractionation (TSF) of liposomes into which connexin channels were reconstituted. Channel activity in this system was affected by changes in pH. To our surprise, the changes in channel activity were accounted for by the direct action of the protonated form of the aminosulfonate compounds used as pH buffers, rather than of proton concentration itself. There is no evidence for the direct action of pH alone on the activity of these connexin channels in this system in the absence of aminosulfonate compounds. The sensitivity to protonated aminosulfonates was connexin isoform-specific. Heteromeric channels containing connexin-26 (Cx26) in addition to connexin-32 (Cx32) were highly sensitive, whereas homomeric Cx32 channels were not. Taken together with recent work by others on the structural basis of pH sensitivity of connexin channels in cells, these data suggest testable hypotheses for cellular and molecular regulation of connexin channel activity. They also provide an opportunity for development of pharmacological and affinity reagents for structure-function studies of connexin channels. This is the first report of a noncovalent modulatory activity of a biological molecule on connexin channels (see Ref. 22Bevans C.G. Harris A.L. J. Biol. Chem. 1999; 274: 3720-3725Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Preliminary reports of this work have appeared in abstract form (23Bevans C.G. Harris A.L. Mol. Biol. Cell. 1997; 8: 96aGoogle Scholar, 24Bevans C. Harris A.L. Biophys. J. 1996; 70: A32Google Scholar, 25Green T.R. Fellman J.H. Eicher A.L. Pratt K.L. Biochim. Biophys. Acta. 1991; 1073: 91-97Crossref PubMed Scopus (131) Google Scholar). Egg phosphatidylcholine, bovine phosphatidylserine, azolectin (soybean l-phosphatidylcholine), and lissamine rhodamine B-labeled phosphatidylethanolamine were purchased from Avanti Polar Lipids. N-Octyl-d-glucopyranoside (octylglucoside) was from Calbiochem. Bio-Gel (A-0.5 m; exclusion limit, 500,000 Da) was purchased from Bio-Rad. CNBr-activated Sepharose beads were obtained from Amersham Pharmacia Biotech. Use and care of animals was according to institutional guidelines. Connexin was affinity-purified from an octylglucoside-solubilized crude membrane fraction of rat or mouse liver using a monoclonal antibody against Cx32 as described in Refs. 26Rhee S.K. Bevans C.G. Harris A.L. Biochemistry. 1996; 35: 9212-9223Crossref PubMed Scopus (40) Google Scholarand 27Bevans C.G. Kordel M. Rhee S.K. Harris A.L. J. Biol. Chem. 1998; 273: 2808-2816Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, with the modification that 5 mm EGTA was included in the homogenization and phosphate buffers. Rat liver yields homomeric Cx32, and mouse liver yields heteromeric Cx32/Cx26. Homomeric Cx26 channels were not available from a native tissue source; because Cx26 forms heteromeric channels with Cx32, such a source would have to lack Cx32. Homomeric Cx32 can be obtained from rat liver because the Cx26 content of rat liver is very low. There is no wild-type animal for which the converse is true. It was occasionally possible to obtain fractions of connexin from mouse liver that varied in Cx32/Cx26 ratio by taking advantage of the finding that more Cx26 elutes from the immunobeads (along with Cx32) in the initial fractions than in later fractions. Except where noted, the heteromeric Cx32/Cx26 channels were from pooled elution fractions. Gel electrophoresis, blotting, and staining of blots were carried out as described in Ref. 27Bevans C.G. Kordel M. Rhee S.K. Harris A.L. J. Biol. Chem. 1998; 273: 2808-2816Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar. The monoclonal antibody (M12.13) used in the immunoaffinity purification and for specific staining of Cx32 on Western blots is directed against a cytoplasmic domain of Cx32 (28Goodenough D.A. Paul D.L. Jesaitis L. J. Cell Biol. 1988; 107: 1817-1824Crossref PubMed Scopus (144) Google Scholar). TAPS, HEPES, MES, taurine, and maleate were obtained from Sigma. Because all TSF experiments were carried out at 37 °C, the pK a values for each compound used in calculations of protonated aminosulfonate concentrations were those either measured at 37 °C (TAPS, 6.0; HEPES, 7.1; MES, 8.1 (29Research Organics, Inc 1996/7 Catalog of Biochemicals. Research Organics, Inc., Cleveland, OH1996Google Scholar)) or calculated from the value at 25 °C and the measured enthalpy of ionization (taurine, 8.78 (30Christensen J.J. Hansen L.D. Izatt R.M. Handbook of Proton Ionization Heats and Related Thermodynamic Quantities. Wiley-Interscience, New York1976Google Scholar)). Unless otherwise noted, the pK a values given in the text are those at 37 °C. Liposome formation and protein incorporation followed the protocol of Mimms et al. (31Mimms L.T. Zampighi G.A. Nozaki Y. Tanford C. Reynolds J.A. Biochemistry. 1981; 20: 833-840Crossref PubMed Scopus (557) Google Scholar) as modified by Harris et al. (32Harris A.L. Walter A. Paul D.L. Goodenough D.A. Zimmerberg J. Mol. Brain Res. 1992; 15: 269-280Crossref PubMed Scopus (33) Google Scholar) and Rhee et al. (26Rhee S.K. Bevans C.G. Harris A.L. Biochemistry. 1996; 35: 9212-9223Crossref PubMed Scopus (40) Google Scholar). Liposomes were formed by gel filtration of a 1 mg/ml mixture of PC, PS, and rhodamine-labeled PE at a molar ratio of 2:1:0.03 in urea buffer (see below) containing 80 mm octylglucoside and immunoaffinity-purified connexin. The size of the liposomes was monodisperse with an approximate mean diameter of 900 Å, shown by HPLC gel filtration (33Ollivon M. Walter A. Blumenthal R. Anal. Biochem. 1986; 152: 262-274Crossref PubMed Scopus (69) Google Scholar). The protein/lipid ratio of the liposomes was typically 1:60 (w/w), corresponding to an amount of connexin equivalent to <∼1 hemichannel per liposome (see under "Data Analysis," below). Minor variation in the amount of protein used, the amount damaged in purification, the reconstitution efficiency, and the amount of lipid retained on the column produced variations in the percentage of liposomes containing active channels under standard conditions. The protein/lipid ratios used yielded functional channels in 30–50% of the liposomes. The procedure used to fractionate liposomes into two populations based on sucrose-permeability is described and fully characterized in Harris et al. (32Harris A.L. Walter A. Paul D.L. Goodenough D.A. Zimmerberg J. Mol. Brain Res. 1992; 15: 269-280Crossref PubMed Scopus (33) Google Scholar, 34Harris A.L. Walter A. Zimmerberg J. J. Membr. Biol. 1989; 109: 243-250Crossref PubMed Scopus (19) Google Scholar) and Rhee et al. (26Rhee S.K. Bevans C.G. Harris A.L. Biochemistry. 1996; 35: 9212-9223Crossref PubMed Scopus (40) Google Scholar). The principle of using a density shift to fractionate liposomes was adapted from Goldin and Rhoden (35Goldin S.M. Rhoden V. J. Biol. Chem. 1978; 253: 2575-2583Abstract Full Text PDF PubMed Google Scholar). Liposomes containing functional channels are separated from liposomes without functional channels by TSF achieved by centrifugation through an isoosmotic density gradient formed by urea and sucrose solutions. Urea buffer contained 10 mm KCl, 10 mm HEPES, 0.1 mm EDTA, 0.1 mm EGTA, 3 mm sodium azide, and 459 mm urea at pH 7.6. Sucrose buffer was identical except that an osmotically equivalent concentration of sucrose (400 mm) replaced the urea. Osmolality of urea and sucrose buffers was 500 mosmol/kg, and their specific gravities (d 420) 1.0056 and 1.0511. An aliquot of liposomes was layered on each 4.4 ml gradient. Gradients were centrifuged at 300,000 × g for 2–3 h in a swinging bucket rotor (Sorvall TST 60.4) at 37 °C. Liposome bands were recovered by aspiration. The distribution of the liposomes in the gradient was calculated from the specific intensity of rhodamine fluorescence (Perkin-Elmer 650–10S or L550B spectrofluorometer; 560 nm excitation; 590 nm emission) and the volume of each collected band. During the centrifugation, liposomes without functional channels move into the gradient a short distance, being buoyed by the (lighter) entrapped urea buffer and form a band in the upper part of the gradient. Liposomes with functional channels continuously equilibrate their internal solution with the external solution and move to a position in the lower part of the gradient corresponding to the density of the liposome phospholipid membrane. Equilibration of extraliposomal and intraliposomal osmolytes is rapid (milliseconds for these 900-Å-diameter liposomes). Therefore, even a channel that opens only infrequently for brief times will mediate full exchange of osmolytes and cause liposome movement to the characteristic lower position. Calculations show that the assay is insensitive to large changes in channel P o down to 0.001. It is formally possible that a modulatory compound could affect the proportion of liposomes that shift to the lower position by restricting the diameter of the pores, rather than moving P oclose to 0. However, for this to occur, the channels would have to become impermeable to urea and to sucrose (liposomes permeable to urea only move to an intermediate position (36Harris A.L. Bevans C.G. Werner R. Gap Junctions. IOS Press, Amsterdam1997: 60-64Google Scholar, 37Bevans C. Brutyan R.A. DeMaria C. Harris A.L. Biophys. J. 1995; 68: A204Google Scholar)). Such a change in diameter would effectively eliminate the ability of connexin channels to mediate molecular signaling between cells and therefore can be regarded as a decrease in channel activity. Previous work with the TSF system suggested that the channels distribute among the liposomes in a manner described by the Poisson distribution (26Rhee S.K. Bevans C.G. Harris A.L. Biochemistry. 1996; 35: 9212-9223Crossref PubMed Scopus (40) Google Scholar). This means that for a given protein-lipid ratio (λ) in the liposomes, a Poisson distribution accounts for the fraction of the liposomes that have functional channels. When λ is small, essentially all of the liposomes in which there are channels contain exactly one channel. However, as λ increases, the fraction of liposomes with two or more channels increases. When this is the case, a change in the fraction of liposomes in the lower TSF band does not reflect exactly the change in channel activity (e.g. liposomes with two channels will move to the lower band unless both channels are closed by a given concentration of ligand). This leads to an underestimate of the inhibitory effect of a test condition, which was corrected in the following manner: λ was estimated from the maximum activity (percentage of liposomes with active channels) for a given preparation of liposomes. Using the Poisson distribution, this λ was used to calculate the distribution of channels in the liposome population, which was used to compensate for the error introduced by some of the liposomes containing more than one channel. This calculation transforms the fraction of permeable liposomes in a population into an index of discrete single channel activity. For each preparation of connexin, the percentage of liposomes in the lower band of TSF data was normalized to the maximum value obtained for that preparation. The maximum value was almost always at the highest pH value for the series. This enabled comparison of modulatory effects across reconstitutions that produced different amounts of channel activity (fractions of liposomes with functional channels). Where several preparations were used, normalized data sets were combined for each buffer for calculation of means and standard errors. The activity data was fit with a four-parameter logistic function of the form f(x) = a/(1 + exp(b*(x −c))) + d using the Marquardt-Levenberg algorithm. A Hill equation was not used because the TSF-pH data does not arise from a titration curve, but rather from the superimposition of responses of an unknown number of channel forms with different properties. For this reason, a smooth function was fit to the data to determine a characteristic half-maximal channel activity parameter. Channel activity data was modeled assuming that protonated taurine binds to one or more sites on a connexin hemichannel to inhibit channel activity. The calculations assume: 1) the pK a37 °C of taurine is 8.78 (30Christensen J.J. Hansen L.D. Izatt R.M. Handbook of Proton Ionization Heats and Related Thermodynamic Quantities. Wiley-Interscience, New York1976Google Scholar), 2) the Cx32/Cx26 channel population consists of five equal subpopulations each having a different taurine dissociation constant (K d) or cooperativity (n Hill), and 3) channels withP o < 0.001 are binned as closed by the TSF. At each protonated taurine concentration the fraction of active channels is summed over all the subpopulations. Because the number of subpopulations, the way that the K d orn Hill values are distributed, and theP o cutoff value are not known, these calculations only demonstrate the general adequacy of the model rather than provide estimates of binding parameters. For each subpopulation of channels, a P o versus [protonated taurine] relation was calculated from a standard binding isothermK dn/(K dn + [protonated taurine]n) where K d is the dissociation constant and n is n Hillas defined above. The binning of the liposomes into active and inactive populations by the TSF was simulated according to (3Liu S. Taffet S. Stoner L. Delmar M. Vallano M.L. Jalife J. Biophys. J. 1993; 64: 1422-1433Abstract Full Text PDF PubMed Scopus (140) Google Scholar) above, giving a step function in the activityTSF versus[protonated taurine] relation for each subpopulation. For different binding parameters the step occurs at different [protonated taurine]. Summing the activityTSF versus [protonated taurine] step functions for each subpopulation produces an aggregate activityTSF versus [protonated taurine] relation that is a series of steps. The binding parametersK d or n Hill were adjusted so that the relation approximated the TSF data. Connexin was immunopurified from octylglucoside-solubilized crude plasma membranes from rat and mouse liver using a monoclonal antibody specific for Cx32, as previously described and characterized (26Rhee S.K. Bevans C.G. Harris A.L. Biochemistry. 1996; 35: 9212-9223Crossref PubMed Scopus (40) Google Scholar, 27Bevans C.G. Kordel M. Rhee S.K. Harris A.L. J. Biol. Chem. 1998; 273: 2808-2816Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar,38Harris A.L. Blank M. Vodyanoy I. Biomembrane Electrochemistry. American Chemical Society, Washington, D. C.1994: 197-224Google Scholar). Earlier biochemical and functional studies have characterized connexin purified in this way from rat liver as homomeric Cx32 hemichannels, and that from mouse liver as heteromeric Cx32/Cx26 hemichannels. The heteromeric channels are functionally heterogeneous with regard to permeability to large molecules, presumably due to heterogeneities of isoform stoichiometry and/or arrangement (27Bevans C.G. Kordel M. Rhee S.K. Harris A.L. J. Biol. Chem. 1998; 273: 2808-2816Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). The activities of channels formed by Cx32 and Cx32/Cx26 were explored and compared by TSF of liposomes. TSF has been well characterized (32Harris A.L. Walter A. Paul D.L. Goodenough D.A. Zimmerberg J. Mol. Brain Res. 1992; 15: 269-280Crossref PubMed Scopus (33) Google Scholar,34Harris A.L. Walter A. Zimmerberg J. J. Membr. Biol. 1989; 109: 243-250Crossref PubMed Scopus (19) Google Scholar, 36Harris A.L. Bevans C.G. Werner R. Gap Junctions. IOS Press, Amsterdam1997: 60-64Google Scholar) and effectively used in channel permeability studies (26Rhee S.K. Bevans C.G. Harris A.L. Biochemistry. 1996; 35: 9212-9223Crossref PubMed Scopus (40) Google Scholar, 27Bevans C.G. Kordel M. Rhee S.K. Harris A.L. J. Biol. Chem. 1998; 273: 2808-2816Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar,39Sugawara E. Nikaido H. J. Biol. Chem. 1994; 269: 17981-17987Abstract Full Text PDF PubMed Google Scholar, 40Walter G. Hutchinson M.A. Hunter T. Eckhart W. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4025-4029Crossref PubMed Scopus (34) Google Scholar). In brief, connexin channels are incorporated into the membranes of unilamellar liposomes. When centrifuged in an appropriate isoosmolar density gradient, solute exchange through active channels causes liposomes to become more dense and move to a position deep in the gradient. Liposomes without active channels remain in the upper part of the gradient. Any significant channel open probability (P o) results in sufficient osmolyte exchange to cause the change in density. Because the change in liposome density can result from brief channel openings, only when P ochanges above or below a very low value are changes in channel activity detected by TSF. TSF is therefore an essentially all-or-none assay of per-liposome channel activity. The technique is more fully described under "Experimental Procedures." The effects of test compounds on channel activity were assessed by exposing connexin-containing liposomes to the compounds during a TSF spin. The change in distribution of liposomes between the upper and lower positions, relative to a control gradient without the compound, is a quantitative measure of the fractional change in activity of the population of the channels. To assess the effects of pH on immunopurified, reconstituted connexin channels, activity was assessed in TSF gradients in which the pH was adjusted by addition of strong acid or base. In these initial studies, the reference condition was pH 7.6 in 10 mm HEPES. Activity was assessed at pH 5 and pH 9 and normalized to that at the reference condition. Experiments were carried out using homomeric Cx32, and two fractions of a population of heteromeric Cx32/Cx26 channels with different ratios of Cx32 to Cx26. The Cx32/Cx26 channels were sensitive to pH changes over this range, whereas the homomeric Cx32 channels were essentially insensitive (Fig.1). Furthermore, the Cx32/Cx26 channel population with the greater proportion of Cx26 was more sensitive than that with less Cx26. Thus, pH sensitivity correlated with Cx26 content. These results suggest that Cx26 is directly responsible for the sensitivity to pH or indirectly confers pH sensitivity on the heteromeric connexin channels. The pH-induced loss of activity with decreased pH in this system is fully reversible, as it is in cells. In the experiment shown in Fig. 1, the one-half maximal effect was near the pK a of HEPES, the pH buffer in the solutions. To test whether the pH sensitivity was buffer-specific, other pH buffers were used in place of HEPES in the TSF solutions, all at 10 mm. In MES and TAPS, the Cx32/Cx26 channels showed pH effects similar to those seen in HEPES. However, the pH range over which channel activity declined was different for each pH buffer (Fig.2, filled symbols). A smooth function was fit to each data set to define half-maximum values for activity in each buffer. It was found that the half-maximum activity value in each buffer was displaced toward the pK a of that buffer (see Fig. 3). Half-maximum values for MES, HEPES, and TAPS were at pH 6.1, 7.3, and 8.5, respectively, close to the corresponding buffer pK avalues of 6.0, 7.3, and 8.1 (29Research Organics, Inc 1996/7 Catalog of Biochemicals. Research Organics, Inc., Cleveland, OH1996Google Scholar, 30Christensen J.J. Hansen L.D. Izatt R.M. Handbook of Proton Ionization Heats and Related Thermodynamic Quantities. Wiley-Interscience, New York1976Google Scholar). (All TSF experiments were carried out at 37 °C, and all pK a values given are at that temperature.)Figure 3Chemical structures of the pH buffers.MES, HEPES, and TAPS are commonly used Good buffers (41Good N.E. Winget G.D. Winter W. Connolly T.N. Izawa S. Singh R.M.M. Biochemistry. 1966; 5: 467-477Crossref PubMed Scopus (2160) Google Scholar). They share the common structural motif of a protonatable amine moiety that is separated from an ionized sulfonate moiety by two or three methylene groups. This functional motif derives from the naturally occurring precursor compound, the β amino acid taurine.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Furthermore, pH buffers that were not in this chemical family, such as maleate and Tris (Fig. 2, open symbols) and bicarbonate (not shown), elicited no change in channel activity over the same pH range. The pH buffers in which activity was pH-dependent were all Good buffers (41Good N.E. Winget G.D. Winter W. Connolly T.N. Izawa S. Singh R.M.M. Biochemistry. 1966; 5: 467-477Crossref PubMed Scopus (2160) Google Scholar) and are all aminosulfonate compounds (Fig. 3). Chemical precursors of the Good buffers include the non-aminosulfonates 2-bromoethanesulfonate, isethionate, and 2-propanesulfonate. These compounds, and the amino acid glycine, were tested and were without effect. The data in Figs. 1 and 2 strongly suggest that the observed changes in connexin channel activity are not due to protonation of connexin, but to the action of protonated aminosulfonate compounds on connexin channels that contain Cx26. The data in Fig. 2 could be accounted for if either the deprotonated aminosulfonate acts as a channel agonist or protonated aminosulfonate acts as a channel antagonist. To help distinguish these possibilities, channel activity was assessed at pH 7.6 in 50 mm rather than 10 mm HEPES. At the higher concentration, channel activity was reduced to the level of activity at 10 mm HEPES at low pH (i.e. when it is fully protonated) (Fig. 2, asterisk). This indicates that the protonated (zwitterionic) form of aminosulfonate buffers acts as a channel antagonist, since in this experiment the concentrations of both the protonated and deprotonated species were increased equally. This point is made more rigorously in the following experiment. The amino acid taurine (2-aminoethanesulfonic acid), a chemical precursor of the Good buffers, is an aminosulfonate and also can function as a pH buffer. Activity of Cx32/Cx26 channels was assessed in 10 mm taurine solutions adjusted to pH values from 7 to 10.5 (Fig. 4 A, open circles). A smooth function fit to the data (curved line) shows that the half-maximum value for activity modulation by taurine approximates the pK a value for taurine, consistent with the findings for the other aminosulfonate buffers (Fig. 2). When taurine concentration wa
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