Optical response of the indicator chlortetracycline to membrane potential

Chlortetracycline is a fluorescent, Ca2+ indicator commonly used to monitor the internal Ca2+ concentration of membrane vesicles and organelles. We have found that the intensity of chlortetracycline fluorescence in the presence of Ca2(+)-loaded liposomes is dependent on the membrane potential of the vesicles as well as the intravesicular Ca2+ concentration. The fluorescence of chlortetracycline was lower when an inside-negative membrane potential was placed across the liposome membrane. Since chlortetracycline diffuses across the membrane in the zwitterionic form, the distribution of chlortetracycline across the membrane should not be strongly dependent on the membrane potential. However, because the proton permeability of phospholipid vesicles is relatively high, the intravesicular proton concentration is dependent on the membrane potential. The binding of Ca2+ to chlortetracycline is dependent on pH in the range of pH 6 to pH 8. Therefore, changes in the intravesicular pH as a result of a change in the membrane potential causes relatively large changes in the chlortetracycline fluorescence signal even when there isn't a change in the Ca2+ concentration.