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Membrane permeabilization by thrombin-induced platelet microbicidal protein 1 is modulated by transmembrane voltage
S P Koo1, A S Bayer, B L Kagan
1Department of Medicine, Division of Infectious Diseases, St. John's Cardiovascular Research Center, LAC-Harbor UCLA Medical Center, Torrance, California 90509, USA. KOO@AFP76.HUMC.EDU
Abstract:
Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide generated from rabbit platelets when they are exposed to thrombin in vitro. It has potent microbicidal activity against a broad spectrum of bacterial and fungal pathogens, including Staphylococcus aureus. Previous in vitro studies involving whole staphylococcal cells and planar lipid bilayers (as artificial bacterial membrane models) suggested that membrane permeabilization by tPMP-1 is voltage dependent (S.-P. Koo, M. R. Yeaman, and A. S. Bayer, Infect. Immun. 64:3758-3764, 1996; M. R. Yeaman, A. S. Bayer, S. P. Koo, W. Foss, and P. M. Sullam, J. Clin. Investig. 101:178-187, 1998). Thus, the aims of the present study were to specifically characterize the electrophysiological events associated with membrane permeabilization by tPMP-1 by using artificial planar lipid bilayer membranes. We assessed the influence of transmembrane voltage polarity and magnitude on the initiation and modulation of tPMP-1 membrane permeabilization at various concentrations of tPMP-1 (range, 1 to 100 ng/ml) added to the cis side of the membranes. The incidence of membrane permeabilization induced by tPMP-1 at all of the concentrations tested was more frequent at -90 mV than at +90 mV. It is noteworthy that membrane permeabilization due to 1-ng/ml tPMP-1 was successfully initiated at -90 mV but not at +90 mV. Further, the mean onset times of induction of tPMP-1 activity were comparable under the various conditions. Modulation of ongoing membrane permeabilization was dependent on voltage and tPMP-1 concentration. Membrane permeabilization at a low tPMP-1 concentration (1 ng/ml) was directly correlated with trans-negative voltages, while a higher tPMP-1 concentration (100 ng/ml) induced conductance which was more dependent on trans-positive voltages. Collectively, these data indicate that the mechanism of tPMP-1 microbicidal activity at the bacterial cytoplasmic membrane may involve distinct induction and propagation stages of membrane permeabilization which, in turn, are modulated by transmembrane potential, as well as peptide concentration.
Insights
Thrombin-induced platelet microbicidal protein 1 (tPMP-1) permeabilizes bacterial membranes in a voltage-dependent manner. Its microbicidal activity involves distinct stages modulated by transmembrane potential and peptide concentration.
Area of Science:
- Biochemistry
- Microbiology
- Biophysics
Background:
- Thrombin-induced platelet microbicidal protein 1 (tPMP-1) exhibits broad-spectrum antimicrobial activity.
- Previous studies suggested tPMP-1-induced membrane permeabilization is voltage-dependent.
- Understanding the electrophysiological basis of tPMP-1's action is crucial for its therapeutic potential.
Purpose of the Study:
- To characterize the electrophysiological events of tPMP-1-induced membrane permeabilization.
- To investigate the influence of transmembrane voltage polarity and magnitude on tPMP-1 activity.
- To determine the role of peptide concentration in modulating membrane permeabilization.
Main Methods:
- Utilized artificial planar lipid bilayer membranes as models for bacterial membranes.
- Assessed tPMP-1 (1-100 ng/ml) effects under varying transmembrane voltages (-90 mV to +90 mV).
- Measured membrane permeabilization incidence, onset times, and modulation by voltage and peptide concentration.
Main Results:
- tPMP-1-induced membrane permeabilization was more frequent at -90 mV than +90 mV.
- Low tPMP-1 concentration (1 ng/ml) initiated permeabilization at -90 mV but not +90 mV.
- Membrane permeabilization was modulated by voltage polarity, magnitude, and tPMP-1 concentration, with distinct responses at low vs. high peptide concentrations.
Conclusions:
- tPMP-1's mechanism involves distinct induction and propagation stages of membrane permeabilization.
- Transmembrane potential and peptide concentration are key modulators of tPMP-1's antimicrobial action.
- These findings provide insights into the voltage-gated nature of tPMP-1's interaction with bacterial membranes.