Effects of D2O on permeation and gating in the Ca(2+)-activated potassium channel from Chara.

We studied the effects of H2O/D2O substitution on the permeation and gating of the large conductance Ca(2+)-activated K+ channels in Chara gymnophylla droplet membrane using the patch-clamp technique. The selectivity sequence of the channel was: K+ > Rb+ >> Li+, Na+, Cs+ and Cl-. The conductance of this channel in symmetric 100 mM KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D2O as compared to H2O. The blockade of channel conductance by cytosolic Ca2+ weakened in D2O as a result of a decrease in zero voltage Ca2+ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D2O primarily due to the change in Ca2+ binding to the channel during the activation step. The Hill coefficient for Ca2+ binding was 3 in D2O and around 1 in H2O. The values of the Ca2+ binding constant in the open channel conformation were 0.6 and 6 microM in H2O and D2O, respectively, while the binding in the closed conformation was much less affected by D2O. The H2O/D2O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large as 60 mV with 1 mM internal Ca2+.