Park K, Arreola J, Begenisich T, Melvin J E
Rochester Institute for Biomedical Sciences, Center for Oral Biology, University of Rochester Medical Center, Rochester, NY 14642, USA.
J Membr Biol. 1998 May 15;163(2):87-95. doi: 10.1007/s002329900373.
Rat parotid acinar cells express Cl- currents that are activated in a time-dependent manner by hyperpolarized potentials. ClC-2, a member of the ClC gene family, codes for a voltage-gated, inward rectifying anion channel when expressed in Xenopus oocytes. In the present study, we found that cDNA derived from individual parotid acinar cells contained sequence identical to that reported for ClC-2 in rat brain and heart. A polyclonal antibody generated against the N-terminal cytoplasmic domain of ClC-2 recognized an approximately 100 kD protein on western blots of both brain and parotid gland. ClC-2 expressed in oocytes has different kinetics from the currents found in parotid acinar cells. Since the ClC-2 channel was cloned from and its transcripts are expressed in mammalian tissue, we compared the channel properties of acinar cells to a mammalian expression system. We expressed ClC-2 channels in human embryonic kidney cells, HEK 293, using recombinant ClC-2 DNA and ClC-2 DNA fused with DNA coding for jellyfish green fluorescent protein (GFP). Confocal microscopy revealed that the expressed ClC-2-GFP chimera protein localized to the plasma membrane. Whole cell Cl- currents from HEK 293 cells expressing ClC-2-GFP were similar, if not identical, to the Cl- currents recorded from cells transfected with ClC-2 cDNA (no GFP). The voltage-dependence and kinetics of ClC-2 channels expressed in HEK 293 cells were quite similar to those in acinar cells. Channels in parotid acinar and HEK 293 cells activated at more positive membrane potentials and with a faster time course than the channels expressed in Xenopus oocytes. In summary, we found that ClC-2 message and protein are expressed in salivary cells and that the properties of voltage-activated, inward rectifying Cl- channels in acinar cells are similar to those generated by the ClC-2-GFP construct expressed in HEK 293 cells. The properties of the ClC-2 anion channel seem to be dependent on the type of cell background in which it is expressed.
大鼠腮腺腺泡细胞表达氯离子电流，该电流在超极化电位作用下以时间依赖性方式被激活。ClC-2是ClC基因家族的成员之一，当在非洲爪蟾卵母细胞中表达时，编码一种电压门控内向整流阴离子通道。在本研究中，我们发现来自单个腮腺腺泡细胞的cDNA包含与大鼠脑和心脏中报道的ClC-2序列相同的序列。针对ClC-2的N端胞质结构域产生的多克隆抗体在脑和腮腺的蛋白质免疫印迹中识别出一种约100 kD的蛋白质。在卵母细胞中表达的ClC-2具有与腮腺腺泡细胞中发现的电流不同的动力学特性。由于ClC-2通道是从哺乳动物组织中克隆出来的，其转录本也在哺乳动物组织中表达，因此我们将腺泡细胞的通道特性与哺乳动物表达系统进行了比较。我们使用重组ClC-2 DNA和与编码水母绿色荧光蛋白（GFP）的DNA融合的ClC-2 DNA，在人胚肾细胞HEK 293中表达ClC-2通道。共聚焦显微镜显示，表达的ClC-2-GFP嵌合蛋白定位于质膜。表达ClC-2-GFP的HEK 293细胞的全细胞氯离子电流与用ClC-2 cDNA（无GFP）转染的细胞记录的氯离子电流相似（如果不是完全相同的话）。在HEK 293细胞中表达的ClC-2通道的电压依赖性和动力学特性与腺泡细胞中的非常相似。腮腺腺泡细胞和HEK 293细胞中的通道比在非洲爪蟾卵母细胞中表达的通道在更正的膜电位下激活，且激活过程更快。总之，我们发现ClC-2信息和蛋白在唾液细胞中表达，并且腺泡细胞中电压激活的内向整流氯离子通道的特性与在HEK 293细胞中表达的ClC-2-GFP构建体产生的特性相似。ClC-2阴离子通道的特性似乎取决于其表达的细胞背景类型。
