Functional inhibition of native volume‐sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti‐ClC‐3 antibody

Abstract

Intracellular dialysis of NIH/3T3 cells with a commercially available anti‐ClC‐3 polyclonal antibody (Ab) for ≈30 min completely inhibited expressed guinea‐pig ClC‐3 currents (IgpClC‐3), while intracellular dialysis with antigen‐preabsorbed anti‐ClC‐3 Ab failed to affect IgpClC‐3. Anti‐ClC‐3 Ab was used as a selective probe to examine the relationship between endogenous ClC‐3 expression and native volume‐sensitive outwardly rectifying anion channels (VSOACs) in guinea‐pig cardiac cells, canine pulmonary arterial smooth muscle cells (PASMCs) and Xenopus laevis oocytes. Intracellular dialysis or injection of anti‐ClC‐3 Ab abolished native VSOAC function in cardiac cells and PASMCs and significantly reduced VSOACs in oocytes. In contrast, native VSOAC function was unaltered by antigen‐preabsorbed anti‐ClC‐3 Ab. It is suggested that endogenous ClC‐3 represents a major molecular entity responsible for native VSOACs in cardiac and smooth muscle cells and Xenopus oocytes. Anti‐ClC‐3 Ab should be a useful experimental tool to directly test the relationship between endogenous ClC‐3 expression and native VSOAC function, and help resolve existing controversies related to the regulation and physiological role of native VSOACs in a wide variety of different cells.

ClC-3 Is a Fundamental Molecular Component of Volume-sensitive Outwardly Rectifying Cl− Channels and Volume Regulation in HeLa Cells and Xenopus laevis Oocytes

“…ClC-3, a ubiquitously expressed member of the ClC chloride channel superfamily, has been proposed to encode one type of native VSOAC in guinea pig ventricular myocytes, canine pulmonary arterial smooth muscle cells (pulmonary arterial smooth muscle cells), and X. laevis oocytes (16,27,28). Since ClC-3 was first cloned and expressed by Kawasaki et al in 1994, contradictory data has been reported for the functional expression of the ClC-3 chloride channel.…”

“…Investigation into the expression levels of both mRNA and protein in various tissues has led to conflicting results (24,33,36,60). Expression of cloned ClC-3 either from human (32,36,43), rat (24,33,34), or guinea pig (16,27,28) has led to differing biophysical and pharmacological properties or the inability to detect expressed ClC-3 chloride currents (36,37,61). There is also contradictory data as to whether the protein is an intracellular channel (35) or plasma membrane channel (33,43).…”

“…Oocytes were collected as previously described (28). Oocytes were removed and incubated in ND96 solution containing, in mM: 96 NaCl, 2 KCl, 1.8 CaCl 2 , 2.8 MgCl 2 , 5 Hepes, 2.5 sodium pyruvate, and 1 mg/ml gentamicin, pH 7.5.…”

“…Immunoblotting-Crude and membrane protein from HeLa cells and X. laevis oocytes were extracted as described previously (28). Protein content in HeLa cells and X. laevis oocytes was determined by the Bradford method (38) or the bicinchoninic acid method (39), respectively.…”

“…Furthermore, Wang et al (17) showed in bovine epithelial cells that ClC-3 antisense treatment delayed the rate of activation of native VSOAC and reduced its amplitude by up to 60% in a dose-dependent manner. Also, a polyclonal anti-ClC-3 antibody (Ab) was shown to functionally inhibit native VSOACs in guinea pig cardiac cells, canine pulmonary arterial smooth muscle cells, and Xenopus laevis oocytes (28). Although the ClC-3 hypothesis has received additional experimental support from other laboratories, (29 -32) its role as a volume sensitive chloride channel has recently become controversial (33)(34)(35)(36).…”