DeCoursey T E, Cherny V V
Department of Molecular Biophysics and Physiology, Rush Presbyterian St. Luke's Medical Center, Chicago, illinois 60612, USA.
Biophys J. 1996 Jul;71(1):182-93. doi: 10.1016/S0006-3495(96)79215-9.
The single-channel proton conductance of the voltage-gated H(+)-selective channel, like that of the F0 component of the H(+)-ATPase, is nearly constant over a wide range of pH encompassing the physiological range. To examine the possible contributions of buffer diffusion and buffer-channel proton transfer reactions to this phenomenon, the effects of buffer concentration on voltage-activated H+ currents were explored in voltage-clamped rat alveolar epithelial cells. Changes in the external buffer concentration ([B]o), evaluated using the whole-cell configuration, had only small effects on H+ currents (IH). Lowering [B]o from 100 to 1 mM did not alter the voltage-activation curve or reversal potential (Vrev) but reduced IH, typically by 10-30%. Changes in internal buffer concentration ([B]i), examined in inside-out patches, usually altered IH more distinctly and subtly changed the kinetics. Overall, the effects of changing buffer concentration were small and subtle. The maximum attenuation of the single-channel H+ current at 1 mM buffer was estimated to be approximately 20% at either mouth of the H+ channel. Therefore, the rate-determining step in H+ permeation is neither deprotonation of buffer at the inner mouth of the channel nor protonation of buffer at the external surface. Evidently the rate of H+ permeation through the channel is itself small enough that diffusion of buffer in bulk solution does not directly limit the conductance significantly.
电压门控H⁺选择性通道的单通道质子电导，与H⁺-ATP酶F0组分的单通道质子电导一样，在涵盖生理范围的很宽pH范围内几乎是恒定的。为了研究缓冲液扩散和缓冲液-通道质子转移反应对这一现象的可能贡献，在电压钳制的大鼠肺泡上皮细胞中探讨了缓冲液浓度对电压激活的H⁺电流的影响。使用全细胞模式评估的外部缓冲液浓度（[B]o）变化对H⁺电流（IH）仅有微小影响。将[B]o从100 mM降至1 mM不会改变电压激活曲线或反转电位（Vrev），但会降低IH，通常降低10% - 30%。在膜片外翻模式下检测的内部缓冲液浓度（[B]i）变化通常对IH的改变更明显，并且会微妙地改变动力学。总体而言，改变缓冲液浓度的影响微小且微妙。在H⁺通道的任一端口，1 mM缓冲液时单通道H⁺电流的最大衰减估计约为20%。因此，H⁺通透的限速步骤既不是通道内口处缓冲液的去质子化，也不是外表面处缓冲液的质子化。显然，H⁺通过通道的通透速率本身足够小，以至于缓冲液在本体溶液中的扩散不会直接显著限制电导。
