Ion diffusion potentials across mycoplasma membranes determined by a novel method using a carbocyanine dye

Area of Science:

• Microbiology
• Biophysics
• Cellular Electrophysiology

Background:

• Transmembrane ion fluxes are critical for cellular functions.
• Mycoplasma species possess unique membrane properties that influence their electrophysiology.
• Accurate measurement of membrane potential is essential for understanding mycoplasma physiology.

Purpose of the Study:

• To investigate the impact of transmembrane ion fluxes on mycoplasma membrane potential.
• To establish a reliable method for calibrating membrane potential measurements in mycoplasmas.
• To determine the specific contribution of different ions (K+, Na+, Cl-) to the membrane potential.

Main Methods:

• Utilized a potential-sensitive carbocyanine dye for fluorescence-based membrane potential measurement.
• Calibrated membrane potential using potassium (K+) diffusion potentials in the presence of valinomycin.
• Employed the null-point method with calcium (Ca2+) ionophore A23187 to validate calibration assumptions.
• Applied nonlinear least-squares computer-assisted methods for data analysis.

Main Results:

• Developed and validated a calibration curve relating fluorescence intensity to membrane potential.
• Determined specific constants for Mycoplasma mycoides subsp. capri and enterococci.
• Demonstrated that K+ and Na+ diffusion potentials significantly contribute to the membrane potential.
• Found that Cl- diffusion does not influence the membrane potential.

Conclusions:

• Established a method for accurately measuring mycoplasma membrane potential.
• Quantified the significant roles of K+ and Na+ in establishing membrane potential.
• Estimated the resting membrane potential of mycoplasmas under growth conditions to be -68 mV.