The Membrane Potential of Enzymatically Isolated Nitella expansa Protoplasts as Compared with their Intact Cells

With the enzymatically isolated Nitella protoplasts, sufficientinsertions of micro-electrodes to make a stable measurementof the membrane potential by the conventional method could notbe made because of an ‘elasticity’ of the outermembrane. We developed an effective method in which a micro-electrodecould be inserted after the outer membrane was punctured bypassing an electrical impulse through the micro-electrode. Inthis 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^–,, , on the membrane potentials of Nitella expansa protoplasts were compared with those of intactcells. The membrane potential of protoplasts was less negativethan that of intact cells when concentrations of Na or K, inthe presence of Ca, were below certain levels which increasedwith increasing Ca concentration; and it tended to become identicalto that of intact cells when Na or K concentrations were beyondthose levels. Beyond those levels for K the membrane potentialsof both protoplasts and intact cells typically seemed to bethe Nernst potentials in the presence of 0•1 to 30 molm^–3 Ca²⁺. However, for Na, the difference in potentialsbetween intact cells and protoplasts decreased at much higherconcentrations than for K. Increase of Ca always gave less negativeprotoplast potentials than those in intact cells. Replacementof Ca by Mg did not change the membrane potential of intactcells, although it was deleterious to protoplasts. The cellwall potential of intact cells was also measured by the micro-electrodetechnique and was revealed as a simple Donnan potential, assumingthe fixed negative charge density of 0•8 equivalent perdm³. The membrane potential of intact cells seems to be a significantreflection of the plasmalemma potential which is thought tobe measured directly in their protoplasts in terms of ionicselectivity and concentration dependency of the ion speciesexamined. In addition, increased sensitivity to calcium in protoplastpotentials compared to intact cells is suggested, though themembrane potential of intact cells seems to be largely preservedin their enzymatically isolated protoplasts. Key words: Membrane potential, protoplasts, Nitella expansa, cell wall potential