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The presence of a Ca 2+ channel in the plasmalemma of tonoplast-free Nitellopsis obtusa cells was demonstrated and its characteristics were studied using current-and voltage-clamp techniques. A long-lasting inward membrane current (lm), recorded using a step voltage clamp, consisted of a single component without time-dependent inactivation. Increasing either [Ca2+]o or [C1-]o 1) enhanced the maximum amplitude of inward I,, ((Ira)p) and 2) shifted the peak voltage ((Vm)p) at (Im)p to more positive values under ramp-shaped voltage clamping and 3) depolarized the peak value of action potentials. This behavior is consistent with predictions based on the Nernst equation for Ca z+ but not for C1 . DIDS (4,4'-diisothiocyano-2,2'-disulfonic acid stilbene) did not suppress (Im)p in tonoplast-free cells, in contrast with its effect on normal cells. La 3+ and nifedipine blocked (lm)~, irreversibly. On the other hand, Ca 2~ channel agonist, BAY K 8644 irreversibly enhanced (l,,,)p. Both Sr -'+ influx and K ~ efflux increased upon excitation. The charge carried by Sr 2+ influx was compensated for by K ~ efflux. It is concluded that only the Ca 2+ channel is activated during plasmalemma excitation in tonoplast-free cells. In terms of the magnitude of (1,,,)~,, Sr 2+ could replace Ca 2+, but Mn 2+, Mg 2+ and Ba 2+ could not.External pH affected (Im)p and the membrane conductance (gm) at (l,~)p ((gin)p). Increasing the external ionic strength caused increases in both (Im)p and (grn)p, and shifted (Vm)p to positive values. At the same time, Sr 2+ influx increased. Thus Ca 2+ channel activation seems to be enhanced by increasing external ionic strength. The possible involvement of surface potential is discussed. Key WordsCa 2+ channel 9 I-V relation 9 membrane excitation 9 Nitellopsis obtusa 9 tonoplast-free cell

Summary. Electric characteristics of internodal Chara australis cells, from which the tonoplast had been removed by vacuolar perfusion with media containing EGTA, were studied in relation to intracellular concentrations of ATP and Mg 2 § using the ordinary microelectrode method and the open-vacuole method developed by Tazawa, Kikuyama and Nakagawa (1975. Plant Cell Physiol. 16:611). The concentration of ATP was decreased by introducing hexokinasc and glucose into the cell and that of Mg z+ by introducing EDTA or CyDTA. The membrane potential decrease and the membrane resistance increase were both significant when the ATP or Mg 2+ concentration was decreased. An ATPdependent membrane potential was also found in other species of Characeae, Nitella axillaris and N. pulchella. Excitability of the membrane was also completely lost by reducing the ATP or Mg 2+ concentration. Both membrane potential and excitability were recovered by introducing ATP or Mg 2+ into ATP-or Mg 2+-depleted cells.The time course of membrane potential recovery was followed by the open-vacuole method. Recovery began as soon as intracellular perfusion with medium containing ATP and Mg 2 + was started. Reversible transition of the membrane potential between polarized and depolarized levels by controlling the intracellular concentration of ATP or Mg 2. could be repeated many times by the open-vacuole method, when the excitability was suppressed by addition of Pb 2+ to the external medium.The ineffectiveness of an ATP analog, AMP-PNP, and the synergism of ATP and Mg 2+ in maintaining the membrane potential and excitability strongly suggest that ATP act via its hydrolysis by Mg2+-activated ATPase. The passive nature of the membrane, as judged from responses of the membrane potential to changes of the external K + concentration, was not altered by lowering the ATP concentration in the cell. The mechanism of membrane potential generation dependent on ATP is discussed on the basis of an electrogenic ion pump. Involvement of the membrane potential generated by the ion pump in the action potential is also discussed.Close correlation of the membrane potential with the metabolic activities of cells has been reported for many plant materials and the involvement of an ion pump in generating the membrane potential has been suggested (cf Slayman, 1970). The membrane potential was found to

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