Deoxycholic Acid Blocks Vibrio cholerae OmpT but Not OmpU Porin
2006; Elsevier BV; Volume: 281; Issue: 29 Linguagem: Inglês
10.1074/jbc.m602426200
ISSN1083-351X
AutoresGuillaume Duret, Anne H. Delcour,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoOmpT and OmpU are general diffusion porins of the human intestinal pathogen Vibrio cholerae. The sole presence of OmpT in the outer membrane sensitizes cells to the bile component deoxycholic acid, and the repression of OmpT in the intestine may play an important role in the adaptation of cells to the host environment. Here we report a novel important functional difference between the two porins, namely the sensitivity to deoxycholic acid. Single channel recordings show that submicellar concentrations of sodium deoxycholate induce time-resolved blocking events of OmpT but are devoid of any effect on OmpU. The effects are dose-, voltage-, and pH-dependent. They are elicited by deoxycholate applied to either side of the membrane, with some asymmetry in the sensitivity. The voltage dependence remains even when deoxycholate is applied symmetrically, indicating that it is intrinsic to the binding site. The pH dependence suggests that the active form is the neutral deoxycholic acid and not the negatively charged species. The results are interpreted as deoxycholic acid acting as an open-channel blocker, which may relate to deoxycholic acid permeation. OmpT and OmpU are general diffusion porins of the human intestinal pathogen Vibrio cholerae. The sole presence of OmpT in the outer membrane sensitizes cells to the bile component deoxycholic acid, and the repression of OmpT in the intestine may play an important role in the adaptation of cells to the host environment. Here we report a novel important functional difference between the two porins, namely the sensitivity to deoxycholic acid. Single channel recordings show that submicellar concentrations of sodium deoxycholate induce time-resolved blocking events of OmpT but are devoid of any effect on OmpU. The effects are dose-, voltage-, and pH-dependent. They are elicited by deoxycholate applied to either side of the membrane, with some asymmetry in the sensitivity. The voltage dependence remains even when deoxycholate is applied symmetrically, indicating that it is intrinsic to the binding site. The pH dependence suggests that the active form is the neutral deoxycholic acid and not the negatively charged species. The results are interpreted as deoxycholic acid acting as an open-channel blocker, which may relate to deoxycholic acid permeation. Vibrio cholerae is an enteric pathogen responsible for cholera. After ingestion of contaminated water by the human host, the bacteria will encounter various micro-environments before colonizing the gut and producing an infection (1.Krukonis E.S. DiRita V.J. Curr. Opin. Microbiol. 2003; 6: 186-190Crossref PubMed Scopus (126) Google Scholar, 2.Lee S.H. Hava D.L. Waldor M.K. Camilli A. Cell. 1999; 99: 625-634Abstract Full Text Full Text PDF PubMed Google Scholar). Important aspects of the physiology and pathogenesis of the bacterial cell rely on detection of and response to the extracellular environment (3.Skorupski K. Taylor R.K. Mol. Microbiol. 1997; 25: 1003-1009Crossref PubMed Scopus (202) Google Scholar). The flow of hydrophilic solutes through the outer membrane of this Gram-negative bacterium is largely controlled by the general diffusion porins OmpU and OmpT. The pore-forming ability of these proteins has been demonstrated by liposome swelling assays (4.Chakrabarti S.R. Chaudhuri K. Sen K. Das J. J. Bacteriol. 1996; 178: 524-530Crossref PubMed Scopus (92) Google Scholar), antibiotic flux assays in live cells (5.Wibbenmeyer J.A. Provenzano D. Landry C.F. Klose K.E. Delcour A.H. Infect Immun. 2002; 70: 121-126Crossref PubMed Scopus (86) Google Scholar), and electrophysiology (6.Simonet V.C. Baslé A. Klose K.E. Delcour A.H. J. Biol. Chem. 2003; 278: 17539-17545Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Recently, we have demonstrated that the two porins are functionally distinct on the basis of their electrophysiological properties (6.Simonet V.C. Baslé A. Klose K.E. Delcour A.H. J. Biol. Chem. 2003; 278: 17539-17545Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). As other general diffusion porins, OmpU and OmpT exist as trimers of monomeric high conductance channels that remain in the open state for >90% of the time. They do exhibit a spontaneous gating activity, evidenced by the random transitions of the monomeric channels between ion conducting (open) and non-conducting (closed) states. The frequency of these random transitions is fairly low for OmpU (every ∼4 s, comparable with that of the Escherichia coli porin OmpF) but much higher for OmpT (every ∼0.3 s), indicating that OmpT may be a much more dynamic pore. General diffusion porins are characterized by their tendency to inactivate at high transmembrane voltage, a phenomenon whose functional relevance and molecular mechanism remain elusive. OmpT was found to be more voltage-sensitive than OmpU and inactivates at a threshold voltage of ∼90 mV, a voltage that is about half the voltage required to inactivate OmpU and that might be in the range attainable by Donnan potentials in physiological conditions. Finally, OmpT was shown to be relatively non-selective with a ratio of permeability for potassium to chloride (PK/PCl) of ∼4 (similar to E. coli OmpF), while OmpU is a much more cation-selective channel (PK/PCl ∼14). The conductance of the monomers is similar for the two porins (∼350 picosiemens for OmpT and ∼300 picosiemens for OmpU in 150 mm KCl), but OmpU has a tendency to display transitions to various states of lower conductance (6.Simonet V.C. Baslé A. Klose K.E. Delcour A.H. J. Biol. Chem. 2003; 278: 17539-17545Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). OmpU allows the passage of larger sugars than OmpT does (4.Chakrabarti S.R. Chaudhuri K. Sen K. Das J. J. Bacteriol. 1996; 178: 524-530Crossref PubMed Scopus (92) Google Scholar) but, however, displays a smaller rate of β-lactam antibiotic flux (5.Wibbenmeyer J.A. Provenzano D. Landry C.F. Klose K.E. Delcour A.H. Infect Immun. 2002; 70: 121-126Crossref PubMed Scopus (86) Google Scholar). Since the three-dimensional structure of OmpU and OmpT is unknown, and as the transport rate of a pore depends on both size and the thermodynamics of the interactions between permeating solutes and the channel wall, it remains difficult at this point to assess whether the pore of OmpU has larger dimensions than that of OmpT or vice versa. The two porins also appear to confer different physiological properties to bacterial cells. In particular, the sensitivity of cells to bile and bile components is an important factor for this intestinal pathogen. One of the major components of bile is deoxycholic acid, a cholesterol derivative with detergent-like properties. Previous work has shown that mutant V. cholerae cells that express exclusively OmpT (and no OmpU) grow more poorly in presence of deoxycholate than cells that express exclusively OmpU (7.Provenzano D. Lauriano C.M. Klose K.E. J. Bacteriol. 2001; 183: 3652-3662Crossref PubMed Scopus (86) Google Scholar). This phenotype is similar to the behavior of E. coli bile acid-sensitive ompC mutants versus E. coli cells that express only OmpC (8.Thanassi D.G. Cheng L.W. Nikaido H. J. Bacteriol. 1997; 179: 2512-2518Crossref PubMed Google Scholar) and has been attributed to the fact that some porins (V. cholerae OmpT and E. coli OmpF) allow a better permeation of deoxycholate than others (V. cholerae OmpU and E. coli OmpC). The outer membranes of Gram-negative bacteria are intrinsically more resilient than phospholipid bilayers to the action of detergents, such as bile acids, due to the presence of lipopolysaccharides in the outer leaflet (9.Nikaido H. Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2900) Google Scholar). Permeation of bile acids does occur though, as several promoters are regulated by these bile components (10.Gupta S. Chowdhury R. Infect. Immun. 1997; 65: 1131-1134Crossref PubMed Google Scholar, 11.Provenzano D. Schuhmacher D.A. Barker J.L. Klose K.E. Infect. Immun. 2000; 68: 1491-1497Crossref PubMed Scopus (120) Google Scholar, 12.Rosenberg E.Y. Bertenthal D. Nilles M.L. Bertrand K.P. Nikaido H. Mol. Microbiol. 2003; 48: 1609-1619Crossref PubMed Scopus (226) Google Scholar, 13.Hung D.T. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3028-3033Crossref PubMed Scopus (116) Google Scholar) and multidrug resistance pumps play important roles in detergent extrusion (8.Thanassi D.G. Cheng L.W. Nikaido H. J. Bacteriol. 1997; 179: 2512-2518Crossref PubMed Google Scholar, 14.Bina J.E. Mekalanos J.J. Infect. Immun. 2001; 69: 4681-4685Crossref PubMed Scopus (143) Google Scholar). Although general diffusion porins are poorly permeable to hydrophobic compounds (9.Nikaido H. Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2900) Google Scholar), it is anticipated that amphipaths such as deoxycholate have a finite flux rate through these pores. Once in the periplasm, detergents molecules can partition in the cytoplasmic membrane, thus compromising cellular integrity and growth. It is important to note, however, that many studies have used submicellar concentrations of sodium deoxycholate or other bile salts and have shown a variety of effects, from pump-mediated efflux to biofilm formation (8.Thanassi D.G. Cheng L.W. Nikaido H. J. Bacteriol. 1997; 179: 2512-2518Crossref PubMed Google Scholar, 12.Rosenberg E.Y. Bertenthal D. Nilles M.L. Bertrand K.P. Nikaido H. Mol. Microbiol. 2003; 48: 1609-1619Crossref PubMed Scopus (226) Google Scholar, 13.Hung D.T. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3028-3033Crossref PubMed Scopus (116) Google Scholar, 14.Bina J.E. Mekalanos J.J. Infect. Immun. 2001; 69: 4681-4685Crossref PubMed Scopus (143) Google Scholar, 15.Hung D.T. Zhu J. Sturtevant D. Mekalanos J.J. Mol. Microbiol. 2006; 59: 193-201Crossref PubMed Google Scholar). The expression of the ompU and ompT genes is under the control of the transcriptional regulator ToxR, a membrane bound environmental sensor that plays a major role in regulating expression of virulence factors, such as cholera toxin and toxin-coregulated pilus (16.Klose K.E. Int. J. Med. Microbiol. 2001; 291: 81-88Crossref PubMed Scopus (36) Google Scholar). ToxR was shown to be an activator of ompU expression and a repressor of ompT expression (17.Crawford J.A. Kaper J.B. DiRita V.J. Mol. Microbiol. 1998; 29: 235-246Crossref PubMed Scopus (104) Google Scholar, 18.Li C.C. Crawford J.A. DiRita V.J. Kaper J.B. Mol. Microbiol. 2000; 35: 189-203Crossref PubMed Scopus (86) Google Scholar). The activity of ToxR is itself modulated by a variety of external factors, such as pH, bile salts, osmolarity, and temperature (3.Skorupski K. Taylor R.K. Mol. Microbiol. 1997; 25: 1003-1009Crossref PubMed Scopus (202) Google Scholar, 10.Gupta S. Chowdhury R. Infect. Immun. 1997; 65: 1131-1134Crossref PubMed Google Scholar, 13.Hung D.T. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3028-3033Crossref PubMed Scopus (116) Google Scholar). Once inside the host, activation of ToxR would turn on ompU expression and turn off ompT expression. Presumably this switch in porin expression has an important role in the adaptive success of the cells in the host environment. Interestingly, Klose and collaborators (19.Provenzano D. Klose K.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10220-10224Crossref PubMed Scopus (177) Google Scholar) have also shown that engineered cells that express solely ompT from a ToxR-activated ompU promoter have attenuated virulence properties, as they become defective in colonization and virulence factor production. Although these porins are not required for virulence (7.Provenzano D. Lauriano C.M. Klose K.E. J. Bacteriol. 2001; 183: 3652-3662Crossref PubMed Scopus (86) Google Scholar), it appears that the type of porin present in the outer membrane may have an impact on the ability of V. cholerae cells to cause disease. To continue our characterization of functional differences between OmpU and OmpT that may have important physiological consequences, we have investigated the effect of the bile salt sodium deoxycholate (DC) 2The abbreviations used are: DC, sodium deoxycholate; octyl-POE, N-octyloligo-oxyethylene; MES, 2-(N-morpholino)ethanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid., 3In solution, deoxycholate exists as a mixture of ionized and protonated deoxycholate. on the channel properties of OmpU and OmpT. Our results demonstrate that OmpT is reversibly blocked by protonated deoxycholate, presumably as it transits through the pore, while OmpU is impervious to the presence of DC. To our knowledge, this is the first report of block of a pore-forming protein by deoxycholic acid. Chemicals, Media, and Buffer Composition—The OmpU and OmpT porin samples were prepared from the porin-deficient V. cholerae strain KKV884 (7.Provenzano D. Lauriano C.M. Klose K.E. J. Bacteriol. 2001; 183: 3652-3662Crossref PubMed Scopus (86) Google Scholar) expressing the ompU or ompT gene cloned into a pBAD30 plasmid. Cells were grown in Luria-Bertani broth (1% tryptone, 1% NaCl, and 0.5% yeast extract) with appropriate antibiotics (0.1 mg/ml ampicillin, 0.1 mg/ml streptomycin) and l-arabinose (0.01% for ompT expression, 0.05% for ompU expression). Tryptone and yeast extract were from Difco Laboratories. N-Octyl-oligo-oxyethylene (octyl-POE) was purchased from Axxora. Other chemicals were from Sigma. For electrophysiology, the following buffers were used: buffer A (150 mm KCl, 10 μm CaCl2, 0.1 mm K-EDTA, 5 mm HEPES (pH 7.2)), buffer B (= buffer A + 20 mm MgCl2), buffer K(= buffer A where HEPES is substituted by 5 mm MES and the pH is 5.2), buffer L (600 mm KCl, 10 μm CaCl2, 0.1 mm K-EDTA, 5 mm HEPES (pH 7.2)), and buffer M (= buffer A where HEPES is substituted by 5 mm CHES, and the pH is 9.2). A 25 mm stock solution of sodium deoxycholate was made in the appropriate buffer in a plastic tube. The solution was diluted to the desired concentration and immediately perfused into the patch clamp chamber. The solutions were stable for at least 1 h, while each individual experiment with fresh DC sample lasted for 10–15 min. Protein Purification—Purification of OmpU and OmpT protein was essentially done as described (6.Simonet V.C. Baslé A. Klose K.E. Delcour A.H. J. Biol. Chem. 2003; 278: 17539-17545Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Protein extraction with the detergent octyl-POE was done at 4 °C. A first column chromatography was done on an anion exchange column (Mono Q HR10/10, Amersham Biosciences), and the protein eluted between 130 and 210 mm NaCl (in 1% octyl-POE, 10 mm sodium phosphate buffer (pH 7.6)). Subsequently, OmpT- or OmpU-containing fractions were further purified by size exclusion chromatography on a HiLoad 26/20 Superdex 200 prep grade column (Amersham Biosciences) in 1% octyl-POE, 50 mm NaCl, 10 mm sodium phosphate buffer (pH 7.6). Proteins were identified by Western blot. Protein visualization and purity were assessed by silver staining after SDS-PAGE. Pure protein was kept at –80 °C in 1% octyl-POE, 10 mm sodium phosphate buffer (pH 7.6), and 50 mm NaCl, prior to use in electrophysiology. Protein concentration was determined with the bicinchoninic assays (Pierce). Reconstitution into Liposomes and Patch Clamp Electrophysiology—Pure protein was reconstituted into soybean phospholipids (Azolectin, from Sigma) at protein-to-lipid ratio of 1:3,000 to 1:5,000 (w/w), and patch clamp experiments were performed on unilamellar blisters emerging from liposomes when placed in buffer B, as described (6.Simonet V.C. Baslé A. Klose K.E. Delcour A.H. J. Biol. Chem. 2003; 278: 17539-17545Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 20.Delcour A.H. Martinac B. Adler J. Kung C. Biophys. J. 1989; 56: 631-636Abstract Full Text PDF PubMed Scopus (178) Google Scholar). Patch pipettes of ∼10 megaohm resistance were filled with buffer A or buffer A + DC and brought into contact with the blister membrane to generate seals of 0.5–1.0 gigaohm. All experiments were conducted on excised patches produced by brief air exposure. After excision, the bath solution was exchanged for buffer A or other buffers, as dictated by the experiment. An Axopatch 1D amplifier (Axon Instrument) was used to monitor currents under voltage clamp conditions. The current was filtered at 1 KHz, digitized at 1.25-ms sampling intervals (ITC-18, Instrutech), and stored on a PC computer using the Acquire software (Bruxton). Data Analysis—Analysis of patch clamp traces was done with a program specifically developed in the laboratory and written by Arnaud Baslé using Microsoft Visual Studio C. Amplitude histograms were constructed by scanning current records and counting the number of sample points at each current value. The open probability (Po) was calculated as the ratio of the observed integrated current obtained over a 1-min-long recording to the total current expected for the same duration if the current value remained at the fully open level. The dose-response curve was fitted to the Hill equation below using SigmaPlot (Marquardt-Levenberg algorithm), Po(DC)/Po(CON)=1/{1+([DC]/IC50) n} (Eq. 1) where Po(DC) and Po(CON) are the open probabilities in presence and absence of DC, respectively, IC50 is the inhibitory concentration at which Po is reduced by 50% relative to control, and n is the Hill coefficient. Based on the fact that cells expressing solely OmpU or OmpT have different sensitivities to DC in growth and that antibiotic flux through OmpU or OmpT are differently affected by crude bile, we hypothesized that DC might show differential effects on the pore properties of OmpU and OmpT. To test this hypothesis, we recorded the electrophysiological behavior of purified OmpU or OmpT pores in the absence or the presence of DC with the use of the patch clamp technique. Fig. 1 shows traces of the activity of a single trimer of OmpT in symmetric buffer A (see "Experimental Procedures" for composition) before and after applying 0.008% DC to the bath of the same patch. In all experiments, the voltages indicated on the figures represent pipette voltage. Traces A and B of Fig. 1 demonstrate that DC has a profound effect on the channel behavior when the pipette voltage is positive. In the absence of DC, the current trace dwells at a baseline level that corresponds to the total current flowing through three monomers (level marked "3o"). Frequent transitions to the level marked "2o" correspond to spontaneous random closures of a single monomer. The amplitude histograms shown to the right of the traces illustrate the distribution of current values between these two levels. In the absence of DC, most current values distribute as a peak of the largest conductance. The short trace right below trace A shows the details of a small segment of trace A marked by a horizontal bar. In the presence of 0.008% DC (trace B), the current steps to all possible current levels for this trimer, including a fully closed level. The corresponding amplitude histogram shows the almost even distribution of current values among these four levels. The expanded trace below trace B shows the clean square pulse pattern of closures and openings that suggests that each monomer is fully and reversibly blocked by DC. Indeed, the conductance of OmpT was measured by voltage-current plots and was found to be unaffected by DC (data not shown). This kinetic pattern is similar to the transient block of OmpF observed in the presence of ampicillin (21.Nestorovich E.M. Danelon C. Winterhalter M. Bezrukov S.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9789-9794Crossref PubMed Scopus (276) Google Scholar) or of LamB observed in the presence of maltodextrins (22.Schwarz G. Danelon C. Winterhalter M. Biophys. J. 2003; 84: 2990-2998Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 23.Kullman L. Winterhalter M. Bezrukov S.M. Biophys. J. 2002; 82: 803-812Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), as these solutes translocate through the channels. As for these solutes and their respective channels, the effect of DC on OmpT was found to be voltage-dependent and much less pronounced at negative pipette potentials. The effect was reversible upon application of buffer without DC. In contrast, OmpU was completely insensitive to DC in this concentration range (Fig. 2). As was done for Fig. 1, a patch containing a single trimer of OmpU was sequentially studied in the absence and the presence of 0.008% DC, and the traces obtained at + and –50 mV pipette voltages are represented. The kinetic pattern of the channel was unchanged in the presence of DC, and no other effect was detected. This is not due to the artifactual lack of access of DC to the patch, as the subsequent perfusion of 0.1 mm spermine in the same patch was fully effective at inhibiting OmpU, in a manner similar to the inhibition of OmpF by this polyamine (24.Iyer R. Delcour A.H. J. Biol. Chem. 1997; 272: 18595-18601Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). This insensitivity of OmpU-containing patches to DC also serves as a control for unspecific effects that this amphipathic molecule might have on pores and membranes. We have found that patches were stable for at least 1 h at concentrations less than 0.01% (∼250 μm). Higher concentrations lead to membrane breakdown. These low concentrations are well below the critical micellar concentration for DC at room temperature (2–6 mm (25.Matsuoka K. Moroi Y. Biochim. Biophys. Acta. 2002; 1580: 189-199Crossref PubMed Scopus (141) Google Scholar)) and do not introduce noise or other artifacts into the electrophysiological traces. To quantify the effect of DC on OmpT, we have measured the Po of a single full trimer at various voltages and various concentrations. Fig. 3 shows the concentration dependence of the ratio of Po with and without DC obtained from traces of either an OmpU (closed symbols) or an OmpT (open symbols) trimer at +50 mV. The open probability of OmpU is so reproducibly maintained close to 1.0 that the error bars are within the thickness of the symbols. The solid line is a linear regression fit yielding a horizontal line. For OmpT, a concentration dependence is evident in the range of 0.001% to 0.01% DC. The solid line is a fit to the Hill equation (Equation 1) given under "Experimental Procedures," where the IC50 is 0.008% DC, and the Hill coefficient is 2.5. These data are supportive of a model where the minimum number of binding sites is 3, and each monomer is blocked by a single molecule of DC, with, surprisingly, some amount of cooperativity. Cooperativity has been postulated for the simultaneous activation of porin trimers reconstituted in bilayers as clusters (26.Schindler H. Rosenbusch J.P. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3751-3755Crossref PubMed Scopus (253) Google Scholar) but not for substrate binding or modulation nor between monomers of the same trimer. The effect of bath-applied DC on OmpT in symmetric buffer A is also voltage-dependent, as illustrated in Figs. 1 and 4. In the absence of DC, the open probability of OmpT remains close to 1.0 in the voltage range of –50 mV to +70 mV (Fig. 4, filled circles). At higher positive or negative voltages, the channels inactivate, leading to a decreased Po, even in the absence of DC (6.Simonet V.C. Baslé A. Klose K.E. Delcour A.H. J. Biol. Chem. 2003; 278: 17539-17545Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). For this reason, the investigation was limited to a voltage range where Po remains constant, so the analysis of DC effect would not be complicated by channel inactivation. Bath-applied DC is quasi-ineffective in the negative voltage range, but the block becomes increasingly stronger as the transmembrane voltage increases in the positive range (Fig. 4, open circles). We initially thought that the voltage dependence of bath-applied DC was due to negatively charged deoxycholate molecules being driven from the bath into the pore at pipette positive potentials. If this scenario is correct, we would expect to see the opposite voltage dependence when DC is present in the pipette and absent in the bath. We did observe an inhibitory effect of DC when applied from the pipette side, supporting the model that the site of DC action is within the pore. Surprisingly, we found that the trend of the voltage dependence was the same regardless of where DC is applied, i.e. the effect is always more pronounced at positive pipette voltages than at negative ones. There is some sidedness to the efficacy of DC, though, as the reduction of Po is more pronounced when DC is in the pipette (Fig. 4, closed squares) than when it is in the bath (Fig. 4, open circles). These results suggest that the asymmetric voltage dependence of the DC effect is intrinsic to the channel, and indeed this asymmetric voltage dependence is still observed when the same concentration of DC is applied to both sides of the membrane (Fig. 4, open squares). Since there is no voltage dependence of Po in this voltage range, the voltage dependence of the DC effect is not due to the increased availability of open pores at positive pipette voltages. Thus it seems that the configuration of the DC binding site within the pore displays voltage dependence. These results also suggest that the active molecular species of DC is not the negatively charged molecules. The pKa of deoxycholic acid being 6.2, the ratio of unprotonated to protonated molecules would be 10:1 at the pH of buffer A (pH 7.2). Therefore, there is a non-negligible amount of protonated deoxycholate molecules present at pH 7.2. If indeed the neutral protonated deoxycholate is the active species, its effect should be decreased at higher pH and increased at lower pH. The results of Fig. 5 and Fig. 6 show that it is indeed the case. Fig. 5 shows traces obtained from the same patch at +50 mV pipette voltage in the presence of 0.006% DC at three different pH values, where the ratios R of negatively charged to protonated deoxycholate are widely different: pH 9.2 (R = 1000), pH 7.2 (R = 10), and pH 5.2 (R = 0.1). At pH 9.2, there is only 0.1% of protonated deoxycholate, and no effect on the channel is observed (top trace). At pH 5.2 (bottom trace), there is 90% of protonated deoxycholate, and the effect is much more pronounced than at pH 7.2 (∼9% protonated deoxycholate). It is important to note that the perfusion sequence for this experiment was buffer A + DC (pH 7.2), then buffer M + DC (pH 9.2), and finally buffer K + DC (pH 5.2). Therefore the observed effect is not due to a time dependence. These results also re-emphasize the reversibility of the phenomenon, since the channel completely recovers from block once buffer A + DC (pH 7.2) is exchanged for buffer M + DC (pH 9.2). The enhancement of the effect of DC with increasingly acidic pH is illustrated in Fig. 6, which compares the values of Po at these three different pH values. The top part shows that by themselves, the pH 5.2 and pH 9.2 buffers have no effect on the open probability.FIGURE 6Effect of pH on the OmpT open probability in presence of DC. The Po of a single trimer of OmpT in the absence (A) or the presence (B) of 0.006% bath-applied DC is plotted against the pipette potential. Symbols represent the averages of Po (±S.E.) from four separate patches, except for the cases of buffer M in the bath (n = 3) and of buffer A + DC in the bath (n = 5). When not seen on the graph, the error bars lie within the thickness of the symbols. In all cases, the pipette buffer was buffer A. The pH values of bath solutions are as follows: pH 9.2 (•, ○), pH 7.2 (▴, ▵), and pH 5.2 (▪, □).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also attempted similar experiments on OmpU to investigate whether the 10-fold increase in concentration of protonated deoxycholate might induce some inhibition. Unfortunately, OmpU is sensitive to acidic pH, and its open probability is decreased to ∼10% at pH 5.2 in the absence of DC. Perfusion of 0.006% DC at pH 5.2 had no additional effect, but of course these experiments are more difficult to interpret since the channel is already mostly in the closed state. Here we report that the general diffusion porins OmpU and OmpT of the outer membrane of V. cholerae are differentially sensitive to channel block by deoxycholic acid in a physiological range. As bile and the bile component deoxycholic acid are known to affect a variety of physiological responses of this intestinal pathogen (10.Gupta S. Chowdhury R. Infect. Immun. 1997; 65: 1131-1134Crossref PubMed Google Scholar, 11.Provenzano D. Schuhmacher D.A. Barker J.L. Klose K.E. Infect. Immun. 2000; 68: 1491-1497Crossref PubMed Scopus (120) Google Scholar, 13.Hung D.T. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3028-3033Crossref PubMed Scopus (116) Google Scholar, 15.Hung D.T. Zhu J. Sturtevant D. Mekalanos J.J. Mol. Microbiol. 2006; 59: 193-201Crossref PubMed Google Scholar, 27.Schuhmacher D.A. Klose K.E. J. Bacteriol. 1999; 181: 1508-1514Crossref PubMed Google Scholar, 28.Chatterjee A. Chaudhuri S. Saha G. Gupta S. Chowdhury R. J. Bacteriol. 2004; 186: 6809-6814Crossref PubMed Scopus (49) Google Scholar), this distinct sensitivity of the two channels is likely to have an important cellular consequence. Deoxycholic acid constitutes about 20% of the bile acids present in bile. Bile acids are secreted as glycine or taurine conjugates but deconjugation by enzymes of the bacterial flora occurs to produce the unconjugated form used here. Bile acid concentrations are on the order of 40 mm in bile (29.Begley M. Gahan C.G. Hill C. FEMS Microbiol. Rev. 2005; 29:
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