CALCIUM-REGULATED CHANNELS AND THEIR BEARING ON PHYSIOLOGICAL ACTIVITIES IN CHARACEAN CELLS

Calcium is involved in the regulation of cytoplasmic streaming, membrane excitation, turgor regulation and salt tolerance in the giant internodal cells of the Characeae. To analyse the mechanism of Ca2+ action, model systems were used, namely, tonoplast-free and plasma membrane-permeabilized cells. In the former, the plasma membrane remained intact and its activity could be investigated by manipulating the cytoplasmic and external media, whereas in the latter, the tonoplast remained intact and its activity could be studied by altering the bathing solution. Studies using these model systems have established the presence of voltage-dependent Ca2+-channels in the plasma membrane and Ca2+-dependent ion channels in both the plasma membrane and the tonoplast. To further analyse Ca2+ action on the basis of single channel activities, patch-clamp techniques were applied to plasmolysed protoplasts and isolated cytoplasmic drops. Channel activities were measured using both cell-attached and excised membrane patch modes. A fresh-water member of the Characeae, Nitellopsis, becomes salt-tolerant if millimolar amounts of Ca2+ are present in the external medium. Under these conditions, excised patches of the plasma membrane exhibit K+-channel activity with unitary conductances of 25-50 pS and a permeability ratio (P(Na)/P(K)) of 0.28. These K+ channels were closed by external Ca2+ when ATP was present on the cytoplasmic side of the membrane. ATP could be replaced with AMP, which suggests that ATP acted neither as an energy source nor as a substrate for protein phosphorylation, but rather as an effector. In the tonoplast, K+ channels having a unitary conductance of 75 pS and a P(Na)/P(K) ratio of 0.2 were not activated by Ca2+ when it was present on the cytoplasmic side of the excised patches, but these channels were activated by Ca2+ injected into the cytoplasmic drop, which suggested the involvement of an unknown cytoplasmic factor(s) that mediates the Ca2+ signal. The brackish water Characeae, Lamprothamnium, can regulate elevated turgor induced by hypotonic treatment only when millimolar amounts of Ca2+ are present in the external medium. In this situation, elevated turgor may first activate Ca2+ channels and the increased level of Ca2+ in the cytoplasm may then activate K+ and Cl- channels in both the plasma membrane and the tonoplast. In the cytoplasmic-drop-attached mode, single K+ channel current-voltage measurements established that the K+ channel exhibited a unitary conductance of 50 pS for negative shifts of the voltage, while under positive shifts in the voltage 100 pS channel conductance was observed. The channel with a P(Na)/P(K) of 0.02 is highly selective for K+ against Na+ and this channel is directly activated by Ca2+ added to the cytoplasmic side of the excised patch. These results suggest that, in Nitellopsis and Lamprothamnium, Ca2+ regulation of channels in both the plasma membrane and the tonoplast may form the molecular basis for Ca2+-regulated physiological functions such as salt tolerance and turgor regulation in characean cells. The mode of Ca2+ regulation is discussed in light of current findings.