Pottosin I I, Andjus P R, Vucelić D, Berestovsky G N
Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow region.
J Membr Biol. 1993 Nov;136(2):113-24. doi: 10.1007/BF02505756.
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+.
我们使用膜片钳技术研究了H₂O/D₂O替代对裸藻属液滴膜中大电导Ca(2+)激活K⁺通道的渗透和门控的影响。该通道的选择性顺序为：K⁺ > Rb⁺ >> Li⁺、Na⁺、Cs⁺和Cl⁻。发现在对称的100 mM KCl中该通道的电导为130 pS。与H₂O相比，在D₂O中该单通道电导降低了15%。由于零电压下Ca²⁺结合亲和力降低了1.4倍，D₂O中胞质Ca²⁺对通道电导的阻断作用减弱。电压依赖性通道门控受D₂O影响主要是由于激活步骤中Ca²⁺与通道结合的变化。D₂O中Ca²⁺结合的希尔系数为3，H₂O中约为1。开放通道构象中Ca²⁺结合常数在H₂O和D₂O中分别为0.6和6 μM，而关闭构象中的结合受D₂O影响较小。H₂O/D₂O替代未使通道电压依赖性的斜率产生显著变化，但在1 mM内部Ca²⁺存在时导致高达60 mV的偏移。
