The Membrane Potential of Enzymatically IsolatedNitella expansaProtoplasts as Compared with their Intact Cells

With the enzymatically isolated Nitella protoplasts, sufficient insertions of micro-electrodes to make a stable measurement of the membrane potential by the conventional method could not be made because of an ‘elasticity’ of the outer membrane. We developed an effective method in which a micro-electrode could be inserted after the outer membrane was punctured by passing an electrical impulse through the micro-electrode. In this method, Ca ions play a crucial role in the ‘punching’ and ‘healing’ processes of the protoplast membrane. The effects of the cations K⁺, Na⁺, Ca²⁺ and the anions Cl⁻, $NO3-$, $SO42-$, on the membrane potentials of Nitella expansa protoplasts were compared with those of intact cells. The membrane potential of protoplasts was less negative than that of intact cells when concentrations of Na or K, in the presence of Ca, were below certain levels which increased with increasing Ca concentration; and it tended to become identical to that of intact cells when Na or K concentrations were beyond those levels. Beyond those levels for K the membrane potentials of both protoplasts and intact cells typically seemed to be the Nernst potentials in the presence of 0⋅1 to 30 mol m⁻³ Ca²⁺. However, for Na, the difference in potentials between intact cells and protoplasts decreased at much higher concentrations than for K. Increase of Ca always gave less negative protoplast potentials than those in intact cells. Replacement of Ca by Mg did not change the membrane potential of intact cells, although it was deleterious to protoplasts. The cell wall potential of intact cells was also measured by the micro-electrode technique and was revealed as a simple Donnan potential, assuming the fixed negative charge density of 0⋅8 equivalent per dm³. The membrane potential of intact cells seems to be a significant reflection of the plasmalemma potential which is thought to be measured directly in their protoplasts in terms of ionic selectivity and concentration dependency of the ion species examined. In addition, increased sensitivity to calcium in protoplast potentials compared to intact cells is suggested, though the membrane potential of intact cells seems to be largely preserved in their enzymatically isolated protoplasts.