Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0641
Submitted 9 October 2003 ; accepted in final form 2 December 2003
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ABSTRACT |
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bladder epithelium; epithelial transport; tight junctions; antibiotics; cationic proteins
Colistin is a decapeptide with a fatty acid tail (6-methyloctanoic acid or 6-heptanoic acid) and a molecular mass of 1,200 daltons. The decapeptide is composed of a heptapeptide ring with a tripeptide tail. The heptapeptide ring has a hydrophilic and a hydrophobic domain, and the tripeptide tail is hydrophilic. The fatty acid tail is attached to the tripeptide via an amide linkage. Colistin, at physiological pH, has a net charge of +5 due to the presence of the amino acid 2,4-diaminobutyric acid. The cationic nature and the fatty acid tail make colistin amphipathic in nature, i.e., readily water and lipid soluble. Of interest is that colistin is different from polymyxin B (PMB) by a single amino acid (leucine instead of phenylalanine in the cyclic heptapeptide). Although the amino acid difference is minor, it has been demonstrated (5) that there is a difference in potency between PMB and colistin to strains of P. aeruginosa, with PMB being more potent. Similarly, Nord and Hoeprich (10) compared the bactericidal activity and toxicity to mice of the two drugs and found PMB to be the more potent of the two drugs; however, for equivalent bactericidal activity, colistin was as toxic as PMB (10).
The bactericidal activity of polymyxin is caused by its ability to increase the permeability of the bacterial membrane. Three key features for bactericidal activity are the fatty acid tail, positively charged amino acids, and the peptide ring (see Ref. 17 for review). Loss of any one of these features results in loss of bactericidal activity. Bactericidal activity can be decreased by altering fatty acid chain length (see Ref. 12 for a review), decreasing the number of charged residues, altering the length of the ring, altering the length of the charged side chain, and altering the amino acid composition of the hydrophobic domain (14).
Previous studies (1, 2) have demonstrated that PMB is toxic to the urinary bladder epithelium. The proposed mechanism of PMB toxicity is by increasing membrane permeability, resulting in an increased influx of cations, anions, and water, leading to cell swelling and lysis. These authors performed a detailed analysis of the interaction of PMB with the urinary bladder membrane. The purpose of this study is to compare and contrast the effect of colistin to PMB on the mammalian urinary bladder epithelium, with the goal of determining whether colistin is less toxic to the bladder than PMB and the basis for any difference in toxicity.
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MATERIALS AND METHODS |
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The animal experiments were performed in accordance with the University of Texas Medical Branch animal care and use committee. Male New Zealand White rabbits (3 kg) were euthanized by lethal intravenous injection of sodium pentobarbital. Urinary bladders were excised and washed in a NaCl Ringer solution(see Solutions). The smooth muscle was dissected away, and the epithelium was mounted on a ring of 2-cm2 exposed area. The ring was transferred to a temperature-controlled, modified Ussing chamber (7) where the serosal side of the epithelium was held against a nylon mesh by a slight excess of solution in the mucosal chamber. Both the mucosal and serosal chambers initially held a bathing solution of NaCl Ringer and were aerated with 95% O2-5% CO2 while integral water jackets maintained the temperature of the bathing solution at 37°C. Both the mucosal and serosal chambers were stirred by magnetic spin bars at the bottom of each chamber.
Solutions
NaCl Ringer contained (in mM) 111.2 NaCl, 25 NaHCO3, 10 glucose, 5.8 KCl, 2.0 CaCl2, 1.2 KH2PO4, and 1.2 MgSO4. In KCl Ringer, all Na+ salts were substituted with the appropriate K+ salts. Unless otherwise noted, all experiments were performed by using KCl Ringer as the mucosal bathing solution. Colistin was prepared as a stock solution by dissolving colistin in distilled H2O. Colistin was added in microliter quantities to the mucosal solution. Colistin was purchased from Sigma Chemical (St. Louis, MO).
Electrical Measurements
All electrical measurements were made under voltage-clamp conditions unless otherwise noted. The transepithelial voltage (Vt) was measured with Ag-AgCl wires placed adjacent to either side of the epithelium (serosal solution ground) while current (I) was passed from Ag-AgCl electrodes placed in the rear of each chamber. Both sets of electrodes were connected to an automatic voltage clamp (Warner Instruments). The transepithelial resistance (Rt) and its inverse, the transepithelial conductance (Gt), were calculated, using Ohm's Law, from the current required to clamp the epithelium 10 mV from the holding voltage.
Data Acquisition
Current and voltage outputs of the voltage clamp were connected to an analog-to-digital converter (Scientific Solutions) interfaced with a computer, which calculated values for resistance and short-circuit current (Isc). Vt and I were continuously monitored on an oscilloscope. All data were printed out with the time of data acquisition and additionally stored on the hard disk.
Equivalent Circuit Analysis
The method of Yonath and Civan (19) was used to differentiate between an increase in the conductance of the cell membrane or the tight junctions. The Gt (µS/cm2) was plotted as a function of Isc (µA/cm2) when Vt = 0 mV in the presence of added protein. This plot was then fit by the equation
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Current-Voltage Relationship
The steady-state difference current-voltage (I/V) relationship of the protein-induced conductance was calculated by using the method of Tzan et al. (15). This method involves measuring the transepithelial I/V relationships in both the absence and presence of added protein; the difference between these two relationships is the voltage dependence of the current flowing through the protein-induced conductance. First, the tissue was voltage clamped to Vt = 0 mV, and the transepithelial current responses to computer-generated voltage pulses 30 ms in duration and of increasing magnitude and alternating polarity were measured. Next, the transepithelial potential was voltage clamped to 70 mV, protein was added to the mucosal solution and equilibrated for 5 min, and then the transepithelial potential was clamped to 0 mV. The conductance was allowed to reach a steady state before the I/V relationship was again measured. The difference between the I/V relationships in the presence and absence of added protein was then fit by the constant field equation to determine the relative ionic permeabilities of the protein-induced conductance.
Data Analysis and Statistics
Curve fitting was done on a personal computer by using NFIT (Island Products, Galveston, TX). Statistics were calculated by using INSTAT (GraphPad Software, San Diego, CA). Data are shown as means ± SE.
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RESULTS |
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It was previously demonstrated that PMB increases the Gt of the rabbit urinary bladder epithelium in a voltage-dependent manner (1). PMB was without effect on Gt when the apical membrane voltage was cell interior positive (by 15 mV); however, when the apical membrane voltage was made cell interior negative, there was a rapid increase in Gt. This voltage sensitivity had the advantage of allowing an accurate determination of the kinetics of the increase in membrane conductance. The standard experimental protocol used was as follows. Vt was clamped to 70 mV (this yielded an apical membrane voltage of 15 mV cell interior positive), and colistin was added to the apical membrane bathing solution from a concentrated stock solution. After a 5-min incubation (to allow for diffusion through the unstirred layer), Vt was clamped to 0 mV (an apical voltage of 55 mV cell interior negative) and the time course of Gt followed. Figure 1A shows that at a Vt of 70 mV, colistin did not alter Gt. When Vt was subsequently clamped to 0 mV, there was a time-dependent increase in Gt.
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Time Course of Gt Increase
The time course of Gt in response to 40 µM colistin was fit by an equation that was the sum of an exponential, a line, and the baseline Gt
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To measure the increase in conductance with increased time resolution, we recorded the time course of the colistin-induced increase in transepithelial current at intervals of 5 ms. In brief, the protocol was to first measure the transepithelial current when Vt was changed from 70 mV to 0 mV in the absence of bath colistin. Next, the transepithelial current (in the presence of colistin) was recorded when Vt was changed from 70 mV to 0 mV. The difference between the colistin-induced current and control current is then the time-dependent increase in the colistin-induced current. Figure 1B is an example of the colistin-induced current. In this example, there is a delay of 115 ms followed by an increase in current that is linear after
800 ms. This delay in the increase in current after the voltage was changed from 70 mV to 0 mV suggests that there are a number of kinetic steps before colistin increases the current flow across the epithelium.
Site of Action
The above data demonstrate that colistin increases Gt in a time- and voltage-dependent manner. The method of Yonath and Civan (19) was used to determine whether colistin was altering the conductance of the tight junctions or the cell membrane. If colistin is increasing the conductance of the tight junction, then Gt will increase in the absence of an increase in Isc. If colistin increases the cell membrane conductance, there will be a direct relationship between the increase in Gt and the increase in Isc. As shown in Fig. 2, the relationship between Gt and Isc is linear, suggesting that colistin is increasing the apical membrane conductance. The inverse slope of a plot of Gt vs. Isc is equal to the cell electromotive force (Ec) and was 45.3 mV. Because the basolateral electromotive force (EMF) is approximately 55 mV (9), the apical EMF is equal to 9.7 mV (cell interior ground). The ion selectivity of the colistin-induced conductance was determined from this apical membrane EMF, published intracellular ion activities (8), and the constant field equation. The chloride-to-potassium selectivity for the colistin-induced conductance is 1.26 ± 0.44 (n = 6). This suggests that colistin introduces a nonselective conductance at the apical of the urinary bladder epithelium.
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Voltage Dependence
An interesting observation is that when the apical membrane potential is clamped such that the cell interior is made electrically positive, colistin does not cause an increase in the membrane potential. In contrast, when the apical membrane voltage is made cell interior negative, there is a large increase in the membrane conductance. The relationship between membrane voltage and induced conductance is shown in Fig. 3. The smooth curve through the data points is the best fit of Eq. 3 to the data
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Colistin Concentration and Gt
Most of the previous experiments were performed by using 40 µM colistin. We next determined the dependence of the rate of conductance change to the bath concentration of colistin. The experimental protocol was to voltage clamp the epithelium to 70 mV, add colistin to the mucosal solution, and, after a 5-min incubation, voltage clamp the epithelium to 0 mV. After the initial rate of increase of Gt was measured, the voltage was clamped back to 70 mV. When Gt had decreased to baseline, a higher concentration of colistin was added to the mucosal solution. After a 5-min incubation, the voltage was clamped to 0 mV, the rate of conductance increase was measured, the voltage was clamped to 70 mV, and Gt was allowed to decrease to baseline. This procedure was repeated until a complete dose-response curve was recorded. The concentration-conductance curve for colistin was sigmoidal (Fig. 4), suggesting that colistin may associate with multiple binding sites before it can alter the apical membrane conductance. The data were fit by a kinetic scheme for multiple, highly cooperative sites (Hill equation)
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Effect of Divalent Cations
In previous studies it was demonstrated that millimolar concentrations of Ca2+ or Mg2+ reduce the magnitude of conductance change caused by cationic proteins (16). This effect of divalent cations can occur by three different mechanisms: rapid and reversible block of the conductance after it is induced (conductive block), rate of reversal (divalent cations alter the time course of the reversal of the colistin-induced conductance), and competition for a colistin membrane binding site. We investigated each of these possibilities in turn.
Conductive block. To determine whether Ca2+ or Mg2+ are conductive blockers, we used the standard protocol (see Effect of Colistin on Gt). After colistin had increased the conductance by 200 µS/cm2, it was removed from the mucosal bath, and then either Ca2+ or Mg2+ was added to the mucosal bath from a concentrated stock solution. Addition of either Ca2+ or Mg2+ resulted in a rapid decrease in the colistin-induced conductance. The relationship between the magnitude of the conductance decrease and concentration of either Ca2+ or Mg2+ is shown in Fig. 5. The data were fit by the inhibition form of the Michaelis-Menten equation. The best-fit value for the Ca2+ Ki was 0.81 ± 0.12 mM (n = 10), with Ca2+ blocking 85 ± 6% of the colistin-induced conductance. The fraction of the colistin-induced conductance that was not blocked by Ca2+ (15%) is not significantly different from zero. This suggests that Ca2+ completely blocks the colistin-induced conductance in a reversible manner. The best-fit value for the Mg2+ Ki was 8.6 ± 1.4 mM (n = 5), with Mg2+ blocking 72 ± 3% of the colistin-induced conductance. The fraction of colistin-induced conductance not blocked by Mg2+ (27%) is significantly different from zero. Thus Mg2+ cannot completely block the colistin-induced conductance. A possible explanation for this observation is that there are two populations of conductances, only one of which is blocked by Mg2+, whereas both are blocked by Ca2+.
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Rate of reversal. The effect of divalent cations on the rate of reversal was studied by using the following protocol. After colistin had increased the transepithelial conductance by 200300 µS/cm2, the mucosal solution was replaced with a colistinfree solution and the time course of the conductance decrease was followed. A kinetic model (originally developed for PMB) was used to describe the time course of colistin reversal (2). In brief, when colistin is removed from the bath, a membraneassociated (nonactive) pool of colistin can either dissociate from the membrane and enter the bath, or it can form a membrane conductance (). This active membrane conductance can leave the membrane with a rate constant kao or enter a stable conductive state (
) with a rate constant of kas. If the colistin-induced conductance is completely reversible, then kas will be zero. Figure 6 shows that at a holding voltage of 0 mV and in the absence of mucosal Ca2+ or Mg2+, the colistin-induced conductance is only partially reversed. The best-fit value for kao is 0.015 ± 0.004 s1, and that for kas is 0.004 ± 0.001 s1 (n = 7). Figure 7 shows the effect of bath Ca2+ and Mg2+ on the rate constant for the active conductance leaving the membrane (kao) or entering the stable state (kas). Of interest is that both rate constants are insensitive to Ca2+ and Mg2+.
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Binding site. The magnitude of the colistin-induced conductance saturates at high colistin concentrations (Fig. 4). This suggests that there is a membrane binding site for colistin. To determine the affinity of Ca2+ and Mg2+ for the binding site, the standard experimental protocol was employed, using a constant bath colistin concentration at different bath divalent cation concentrations. With the use of the values from Fig. 5, the rate of colistin-induced conductance increase as a function of bath divalent cation concentration was corrected for conductive block, and this corrected conductance is . The corrected data were then fit by a kinetic model that describes the binding of colistin or a divalent cation to a common binding site. The equation that describes the effect of divalent cations on the
is
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Reversal
We have demonstrated that removal of colistin from the mucosal bath at 0 mV resulted in a partial reversal of the colistin-induced conductance. In addition, Ca2+ or Mg2+ did not alter the time course of the conductance decrease or the fraction of the colistin-induced conductance that remained in the membrane (when Vt was held at 0 mV, 15%).
In the presence of bath colistin (but the absence of bath Ca2+ and Mg2+), the colistin-induced conductance can be reversed by changing Vt from 0 mV to 70 mV. The time course of the reversal was composed of a rapid decrease in Gt (in less than 1 s), followed by a slow decrease to the pre-colistin levels (Fig. 9). The slow decrease was described by an exponential function with a time constant of 0.082 ± 0.021 s1 (n = 6). Thus, at 70 mV and in the presence of bath colistin, the colistin-induced conductance is completely reversed. The ratio of the colistin-induced conductance immediately after Vt was changed from 70 mV to the colistin-induced conductance at 0 mV was 0.20 ± 0.04 (n = 12) in the presence of 200 µM colistin. Thus, immediately after Vt changed from 0 mV to 70 mV, there was an 80% decrease in the colistin-induced conductance. The ratio of colistin-induced conductance at 70 mV and 0 mV (G70/G0) is dependent on the mucosal bath composition (Table 2). Table 2 also shows the ratio of the fast conductance decrease to the slow conductance decrease (Gfast/Gslow). Thus, in the absence of bath colistin, G70/G0 is close to unity and Gfast/Gslow is close to zero; i.e., in the absence of bath colistin, there is no fast component. In the presence of colistin and 2 mM Ca2+ or 1.2 mM Mg2+, the ratio is 0.58.
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The effect of Ca2+ or Mg2+ concentration on the conductance ratio was determined by using the following protocol. First, with Vt held at 70 mV, either Ca2+ or Mg2+ was added to the mucosal bath, followed by 200 µM colistin. After a 5-min incubation, the voltage was changed to 0 mV, the conductance was allowed to increase by 100 µS/cm2, and the voltage was then changed to 70 mV and Gt recorded. Figure 10 shows the effect of Ca2+ and Mg2+ on Gfast/Gslow. Of interest is that Ca2+ has a greater effect on the conductance ratio than Mg2+.
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Long-Term Exposure
Although the colistin-induced conductance can be completely reversed by changing the voltage from 0 mV to 70 mV, this is only true for short exposure times. In this experiment the tissues were exposed to colistin in Ca2+- and Mg2+-containing KCl Ringer and held at 0 mV for a specified period of time, and then colistin was removed from the bath, Vt was changed to 70 mV, and the new steady-state conductance was recorded. The longer the tissue was exposed to colistin at 0 mV, the greater was the new steady-state conductance (Fig. 11). Thus colistin has produced an irreversible change in the barrier function of the bladder epithelium. After 120 min of exposure, three of the four tissues did not reach a new steadystate conductance; i.e., Gt continued to increase. The continued increase in conductance after the removal of colistin suggests that long-term exposure to colistin is toxic to the bladder epithelial cells.
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DISCUSSION |
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Colistin was found to increase the apical membrane conductance only when the apical membrane voltage was cell interior negative. The time course of the increase (induced by 40 µM colistin) had a short delay followed by one of three different responses: an exponential, the sum of an exponential and very slow linear increase, or a rapid linear increase. Ninety-one percent of the time courses contained an exponential. This finding suggests that, at steady state, there is an equal rate of conductance formation and dissociation. The very slow linear increase might be due to the direct effect of colistin or might be a secondary effect, e.g., disruption of tight junctions. The initial rate of the conductance increase for either of the exponentials was similar to the rate of increase of the rapid linear increase. The delay suggests that the voltage-dependent formation of the conductance is a multiple step process with a series of nonconductive steps. The relationship between the initial rate of conductance increase and colistin concentration was sigmoidal, with a Hill coefficient of 2. This suggests that two molecules of colistin are required to induce a conductance.
After the holding voltage was changed from 0 mV to 70 mV, there was a rapid decrease in the conductance, followed by a slower decrease. The rapid decrease required the presence of colistin in the bath (Table 2). In the presence of colistin, increasing bath Ca2+ or Mg2+ reduced the magnitude of the rapid decrease of the conductance (see Fig. 10). Speculation on the mechanism of the voltage sensitivity described above is not warranted until a molecular model of how colistin forms a membrane conductance is developed.
Comparison of PMB and Colistin
Table 3 compares some of the properties of PMB (1, 2) and colistin (present study). Similarities between PMB and colistin include voltage sensitivity, anion/cation selectivity, conductive block by Ca2+ and Mg2+, affinity of Ca2+ and Mg2+ for the membrane binding site, Hill coefficient, and reversal of the induced conductance by voltage and washing. This finding suggests that the basic molecular mechanisms by which these two molecules induce a conductance are the same.
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There are a number of differences between PMB and colistin. Some of the differences are minor, such as the dissociation constant of Mg2+ conductive block, the ability of Ca2+ to reverse the induced conductance at 0 mV, the rate of reversal at 0 mV (in the absence of Ca2+ or Mg2+), and the degree of rapid reversal when the voltage is changed from 0 mV to 70 mV. Some are major, such as the time course of the induced conductance, the dissociation constant, and the maximum rate of conductance increase. Which of the above differences can account for the difference in bactericidal activity? At low concentrations, PMB should be more toxic to bacteria than colistin because PMB has a higher binding affinity than colistin (66 vs. 272 µM) and the conductance increase by PMB is linear (does not saturate), whereas the colistin conductance saturates. At high concentrations, colistin should be more toxic to bacteria than PMB because the maximum rate of conductance change is greater for colistin than for PMB. Thus the bactericidal activity will be dependent on the concentration. At the Km for PMB (66 µM), the rate of conductance change for PMB is 78 µS·cm2·s1, whereas for colistin it is 13 µS·cm2·s1. At the Km for colistin (272 µM), the rate of conductance change for PMB will be 143 µS·cm2·s1, and for colistin it is 137.5 µS·cm2·s1. At very high concentrations of colistin or PMB, the rate of conductance change will be equal to the corresponding (see Table 3).
Structure of Polymyxins
Polymyxins are a class of cationic peptide antibiotics with at least 11 members. The two best-studied polymyxins are PMB and colistin. Structurally, PMB and colistin are similar; they are decapeptides that have five positively charged amino acids (diaminobutyric acid) and contain one of two fatty acid tails. The relative proportion of the fatty acid tails for PMB is 65% 6-methyloctanoic acid (PMB1) and 35% 6-methylheptanoic acid (PMB2) (6), and colistin is 70% 6-methyloctanoic acid (colistin A) and 30% 6-methylheptanoic acid (colistin B) (3). The only difference is that PMB contains a D-phenylalanine at position 6, whereas colistin contains a D-leucine. This difference results in PMB being more bactericidal than colistin at low concentrations, whereas at high concentrations they have equivalent bactericidal activity (5, 10).
Results from the present study suggest that the increased activity of PMB at low concentrations is due to a higher binding affinity of PMB compared with colistin. Viljanen and colleagues (18) compared the binding of the peptide portion of PMB and colistin to bacterial membranes and found that PMB had a higher binding affinity. Synthesis of PMB nonapeptide (PMBN, a PMB lacking the fatty acid tail and terminal amino acid) in which D-phenylalanine was replaced with L-phenylalanine increased the dissociation constant (decreased the binding affinity) to bacterial lipopolysaccharide (by a factor of 3) (14). These authors concluded that the configuration of the phenylalanine is important for membrane association. Recent studies in which PMBN was used have led to the proposal that, in addition to the electrostatic binding activity of PMBN, there is an important interaction of the hydrophobic amino acids (D-phenylalanine-leucine) of the PMBN with the hydrophobic lipid environment of the bacterial membrane (13). Structural studies and molecular modeling (11) of PMB interaction with lipid A suggest that there is a deep penetration of D-phenylalanine-leucine into the hydrophobic core of lipid A, resulting in an increased hydrophobic interaction. Similar studies for colistin suggested that there is less penetration of leucine-leucine side chain into the lipid A hydrophobic core, resulting in reduced hydrophobic interactions (11). The affinity of polymyxin to a cell membrane is due to a hydrophobic interaction of the tail with the lipid membrane, an electrostatic interaction of the five positive charges with the negatively charged lipid membrane, and, last, an interaction of the hydrophobic portion of the heptapeptide ring with the lipid membrane. The last of these three interactions produces a higher binding affinity for PMB compared with colistin.
Although PMB has a higher binding affinity than colistin, the maximal rate of conductance change is less. In the most simple model, the maximal rate of conductance change is equal to the product of the number of conductive units formed per unit of time (this is equal to rate of formation of conductive units minus rate of loss), the conductance of a single unit, and the open probability of a single unit. The data in this study do not allow one to differentiate among these possibilities.
In summary, colistin and PMB share many features in common, including voltage sensitivity, a membrane binding site for which they compete with divalent cations, and divalent cation block. They differ in their dissociation constant (PMB has a lower value than colistin) and the maximum rate at which they induce a membrane conductance change (at saturating concentrations, colistin induces a greater rate of membrane conductance change). These differences are due to a single amino acid. The lower dissociation constant for PMB compared with colistin can be explained by a stronger hydrophobic interaction between the heptapeptide ring and the lipid environment of the membrane. The greater rate of conductance change can be due to differences in the rate of formation of the conductance and to the size or open probability of an individual conductive unit. The lower dissociation constant for PMB is in agreement with the observation that PMB is more bactericidal at low concentrations than colistin, but at high concentrations they are similar.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-51382.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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