D2O-INDUCED ION-CHANNEL ACTIVATION IN CHARACEAE AT LOW IONIC-STRENGTH

Effects of D2O were studied on internodal cells of the freshwater alga Nitellopsis obtusa under plasmalemma perfusion (tonoplast-free cells) with voltage clamp, and on Ca2+ channels isolated from the alga and r econstituted in bilayer lipid membranes (BLM). External application of artificial pond water (APW) with D2O as the solvent to the perfused p lasmalemma preparation led to an abrupt drop of membrane resistance (R (m) = 0.12 +/- 0.03 k Ohm . cm(2)), thus preventing further voltage cl amping. APW with 25% D2O caused a two-step reduction of R(m): first, d own to 2.0 +/- 0.8 k Ohm . cm(2), and then further to 200 Ohm . cm(2), in 2 min. It was shown that in the first stage, Ca2+ channels are act ivated, and then, Ca2+ ions entering through them activate the Cl- cha nnels. The Ca2+ channels are activated irreversibly. If 100 mM CsCl wa s substituted for 200 mM sucrose (introduced for isoosmoticity), no ef fect of D2O on R(m) was observed. Intracellular H2O/D2O substitution a lso did not change R(m). In experiments on single Ca2+ channels in BLM H2O/ D2O substitution in a solution containing 100 mM KCl (trans side ) produced no effect on channel activity, while in 10 mM KCl, at negat ive voltage, the open channel probability sharply increased. This effe ct was irreversible. The single channel conductance was not altered af ter the H2O/D2O substitution. The discussion of the possible mechanism of D2O action on Ca2+ and Cl- channels was based on an osmotic-like s tress effect and the phenomenon of higher D-bond energy compared to th e H-bond.