| IntroductionVolume-sensitive outwardly rectifying (VSOR) CF channel is ubiquitously expressed in all kinds of animal cells including mammalian cardiomyocytes. This channel serves as the pathway for volume-regulatory anion efflux, which plays an importment role in the regulatory volume decrease (RVD) caused by osmotic swelling. In addition, it has become recently evident that this channel is essentially involved in cell proliferation or cell cycle progression and in apoptosis. However, its molecular entity has not firmly been identified yet.Among a number of candidates proposed so far, the C1C-3 protein is the last one which has not been decided. C1C-3 is one of nine members of the mammalian CF channel gene, CLC, family. Duan et al proposed the C1C-3 hypothesis on the basis of observations that C1C-3 was cloned from a guinea pig ventricle when transfected into NIH/3T3 cells, which exhibited basally active CF currents that showed the same properties as those of the native VSOR CF current in guinea pigcardiomyocytes and that site-directed mutagenesis altered rectification and anion selectivity. The same laboratory has provided additional evidence that several different types of anti-ClC-3 antibody inhibited native VSOR Cl~ currents in cardiac and smooth muscle cells as well as Xenopus oocytes and that endogenous VSOR Cl~ currents in HeLa cells and Xenopus oocytes were suppressed by ClC-3 antisense oligonucleotides and antisense cRNA, respectively. Similar suppressing effects of ClC-3 antibodies and antisense oligonucleotides were also observed in other laboratories.Nevertheless, the hypothesis that ClC-3 was used as a volume-sensitive CV channel has been controversial from the beginning. There have been many observations at variance with this hypothesis: First, a number of groups failed to functionally express mouse, rat and human ClC-3 (mClC-3, rClC-3 and hClC-3) as well as the guinea pig isoform of ClC-3 (gpClC-3). Second, the activity of VSOR Cl- channel was not affected by transfection with the gene for hClC-3 (hClcn3) into mammalian cells. Third, heterologous expression with hClC-3 or rClC-3 exhibited activation of Cl- currents distinct from the VSOR Cl" current.Furthermore, it is now obvious that ClC-3 is not the identity of VSOR Cl- channel in some types of cells such as rat lacrimal cells as well as mouse hepatocytes, pancreas and parotid acinar cells for the following reasons: 1) Majid et al. observed functional expression of VSOR Cl- channels in rat lacrimal cells that lack molecular expression of ClC-3. 2) Stobrawa et al. demonstrated that targeted disruption of the ClC-3 gene never impairs the activity of VSOR Cl- channel in mouse hepatocytes and pancreatic acinar cells. 3) The magnitude and the kinetics of swelling-activated Cl- currents in parotid acinar cells isolated from ClC-3-deficient mice were equivalent to those from wild-type mice. These results indicated that ClC-3 was not the molecular entity of VSOR Cl- channel in rat lacrimal cells, mouse hepatocytes, pancreatic acinar cells and parotid acinar cells.However, there still remains a possibility that ClC-3 is responsible for the molecular entity of VSOR Cl- channel in cardiomyocytes. To test this possibility, thus, the present study was made on single ventricular myocytes isolated from ClC-3-deficient mice and the wild-type of littermates. MethodsMice with homozygous disruption of Clcn3 gene (Clcn3-/- mice) were produced. ClC-3-deficient mice are chimera between C57BL/6J and 129/SV mice. Wild-type (Clcn3+/+), heterozygous (Clcn3+/-), and homozygous (Clcn3-/-) mice were obtained by intercrossing heterozygous mice at approximately Mendelian ratio. Single mouse ventricular myocytes were isolated from male mice by perfusing collagenase. RT-PCR was used to test expression of ClC-3 mRNA and its deficiency in cardiomyocytes from three groups. The amplified PCR product is 328 bp. Cl- currents were recorded by the conventional whole-cell patch-clamp technique to investigate the effects of ClC-3 or PKC on VSOR Cl- currents. Results1. Card...
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