Molecular Cell Physiological Study on Volume-Sensitive C1^- Channel

大多数哺乳动物细胞在短暂的渗透性肿胀或收缩后，即使在非等渗条件下也能调整其体积。当细胞内渗透压升高或细胞外渗透压降低迫使细胞肿胀时，细胞可表现出调节性体积减小(RVD)。在许多细胞类型中，包括大多数上皮细胞，RVD是通过激活K^+和C1^-通道而实现的。本研究应用膜片钳技术和分子生物学技术，研究了人肠上皮细胞407和人表皮样癌KB细胞中该容量敏感的c1~(2-)通道的电生理特性和分子特性。1994年进行的单通道研究表明，该通道在肠上皮细胞407细胞中具有中等的单位电导，并在大的正电位下表现出失活动力学。1996年进行的全细胞膜片钳研究表明，ATP，而不是环状AMP，通过与细胞外侧可到达的孔位结合，起到无镁形式的开放通道阻滞剂的作用。Varverde等人于1997年对细胞大小和全细胞C_1~-电流进行了同步测量，结果表明该通道是由细胞体积扩张激活的，而不是由膜伸展激活的。(1992)，我们研究了mdr1表达与该通道活性之间的可能关系。1995年进行的这项反义研究明确表明，取消肠道407细胞内源性MDR1的表达并不能取消C_1~-通道的活性。1997年在KB细胞中进行的外源性mdr1过表达研究表明，c1~-电流的最大幅度不受mdr1过表达的影响。因此，内源性表达和外源性过表达的mdr1本身不可能是通道蛋白。

Most mammalian cell types can readjust their volume after transient osmotic swelling or shrinkage even under anisotonic conditions. When cells are forced to swell by increased intracellular osmolality or decreased extracellular osmolality, they can exhibit a regulatory volume decrease (RVD). In many cell types, including most epithelial cells, RVD is attained by activation of K^+ and C1^- channels. In this study, the electrophysiological properties and molecular identity of this volume-sensitive C1^- channel were investigated in human intestinal epithelial Intestine 407 cells or human epidermoid KB cells by applying both patch-clamp and molecular biological techniques.Single-channel studies, performed in 1994, showed that this channel has an intermediate unitary conductance and exhibits inactivation kinetics at large positive potentials in Intestine 407 cells. Whole-cell patch-clamp studies, performed in 1996, demonstrated that ATP,but not cyclic AMP,acts as an open-channel blocker in the Mg-free form by binding to the pore site toward which ATP is accessible from the extracellular side. Simultaneous measurements of cell size and whole-cell C1^- currents, performed in 1997, clearly showed that this channel is activated by cell volume expansion but not by membrane stretch.Since P-glycoprotein (MDR1) was proposed to be the molecular identity of volume-sensitive C1^- channel by Varverde et al. (1992), we have examined a possible relation between the MDR1 expression and this channel activity. The antisense study, performed in 1995, definitely showed that abolition of endogenous MDR1 expression in Intestine 407 cells cannot abolish the C1^- channel activity. The study by exogenous MDR1 overexpression in KB cells, performed in 1997, indicated that the maximum amplitude of C1^- currents is not affected by overexpression of MDR1. Thus, it is concluded that both endogenously expressing and exogenously overexpressed MDR1 cannot be themselves the channel protein.