Separation of tonoplast and plasma membrane potential and resistance in cells of oat coleoptiles

Abstract

Membrane potential and resistance were recorded from parenchymal cells of oat (Avena) coleoptiles, using one and two intracellular electrodes. Membrane potential is largest (−100 mV) in impalements with low input resistance (2–4 MΩ), and is less negative (−50 mV) in penetrations with high input resistance (> 20 mΩ). The interpretation is that the electrode lodges in the vacuole which is positive to the cytoplasm (but still negative to the external solution), and that measurements of net membrane potential are compromised to varying degrees by leakage shunts introduced across the high resistance vacuolar membrane by the electrode. This conclusion is supported by several additional lines of evidence. (1) It is possible to convert large-R/small-V impalements into small-R/large-V penetrations by passing excess current through the electrode or by briefly ‘ringing’ the capacitance neutralization circuit in the amplifier. The cells usually recover their resistance in a few minutes, with a concomitant decrease in the negativity of the membrane potential. (2) Changes in external [K] affect the measuree potential by an amount that is independent of the input resistance of the impalement. This is consistent with an effect of [K] o on the potential of the plasma membrane and the occurrence of leakage shunts primarily at the tonoplast. (3) Quantitatively, the effects of a change in [K] o on resistance indicate that nearly 90 percent of the input resistance of unshunted cells resides in the tonoplast. (4) The effects of metabolic inhibitors (DNP, CN−) on potential are smaller in large-R than in small-R impalements. This observation suggests there are electrogenic pumps contributing to the membrane potential at both the plasmalemma and tonoplast. Finally, we conclude that with an electrode in the vacuole it is possible to record potentials that are dominated by the contribution of the plasma membrane, provided care is taken to select impalements combining both large, negative potential and low input resistance.