Biphasic voltage relaxation pattern observed in cells of Eremosphaera viridis after injection of charge-pulses of short duration: detection of tip clogging of intracellular microelectrodes by charge-pulse technique.
Charge pulse experiments performed on the peat-bog alga Eremosphaera viridis revealed an unusual voltage relaxation behaviour. Injection of charge pulses of 1 microseconds duration resulted in an immediate charging of the membranes (time constant of the order of 40 ns). Nevertheless, the potential-measuring microelectrode recorded an exponential increase in membrane voltage with a time constant of about 1.3 ms. The maximum voltage value was recorded after about 3 ms, followed by an exponential decay with a time constant of about 9.6 ms. This biphasic time course was independent of the amplitude of the injected charge and of the location of the impaled microelectrodes in the vacuole. Centrifuged cells in which the chloroplasts and the other organelles were pelleted in one part of the cells showed the same electrical response. Electrical breakdown of the cell membranes resulted in the disappearance of the biphasic voltage response. In this case only the decaying relaxation process could be recorded with a time constant of 3 ms. After resealing of the membranes the original biphasic relaxation response was restored. Increasing concentrations of KCl in the bathing medium reduced both time constants almost correspondingly. The experimental findings were evaluated with an electrical equivalent circuit. Theoretical analysis with reference to the experimental data suggested that the delayed voltage response of the potential-recording electrode resulted from a membrane seal across the tip of this electrode. The resistance of this seal was calculated to be about 400 M omega. The specific resistances and capacitances of tonoplast and plasmalemma membranes were calculated from the decaying part of the biphasic relaxation curves. The average values were found to be 2.58 omega.m2 and 5 mF.m-2. The investigations reported here suggest that charge pulse experiments can be generally used for the detection of membrane and cytoplasmic material clogging of the tip of intracellular microelectrodes, a problem with which most electrophysiologists are faced when interpreting data obtained from impaled microelectrodes.
Wehner
G
G
Lehrstuhl für Biotechnologie, Würzburg, F.R.G.
Friedmann
B
B
Zimmermann
U
U
D002462:Cell Membrane / Q000502:physiology*; D004594:Electrophysiology / Q000295:instrumentation / Q000379:methods; D056890:Eukaryota / Q000502:physiology*; D008433:Mathematics; D008564:Membrane Potentials; D008839:Microelectrodes; D008954:Models, Biological; D013997:Time Factors