Perfusion of charophyte cells: A critical analysis of the method

The data obtained by different types of intracellular perfusion were compared. As the ligated cells cannot be space-clamped, the efficiency of compartment-clamping was evaluated, showing that the difference I/V (current-voltage) profile between space-clamped and compartment-clamped data could be approximated by a straight line. The time-dependence of the clamp currents was not affected by the clamp technique. The comparison of different sets of data was quantified by fitting the I/V curves with a mathematical model (Beilby and Walker, 1996). The I/V curves of ligated cells perfused with 1 mM ATP showed the closest similarity to intact cells with resting potentials of -220 ± 10 mV (7 cells) and similar model parameter values. The cells under open-end perfusion with ATP showed less hyperpolarized resting p.d.s (potential differences): -175 ± 12 mV (4 cells). For both preparations the -ATP data were similar with resting p.d.s at -80±12 mV (5 ligated cells, 7 open-end cells). The excited state was more pronounced in open-end cells (resting level: -59±12 mV, 5 cells) than in ligated cells (resting level: -65 ± 12 mV, 7 cells). In open-end cells the pump responded faster to changes of ATP concentration than the excitation channels. The cells stabilized with Pb(NO₃)₂ were strongly depolarized both with ATP: -80 ± 10 mV (6 cells) and without: 0 ± 10 mV (6 cells). Most model parameters differed from those in the intact cells. The excited state was abolished.