Single-Channel Properties of Volume-Sensitive Cl− Channel in ClC-3–Deficient Cardiomyocytes

Abstract

It is controversial whether the ClC-3 protein, which is one of the voltage-dependent chloride channel ClC family members, is a candidate for the volume-sensitive outwardly rectifying (VSOR) Cl -channel per se or its regulator. Here, for the first time, we examined the single-channel properties of the VSOR Cl -channel in ventricular myocytes isolated from ClC-3-deficient mice. The single-channel current induced by cell swelling exhibited Cl -selectivity, mild outward rectification, and an intermediate unitary con-The volume-sensitive outwardly rectifying (VSOR) Cl-channel is ubiquitously expressed and plays an essential role in cell volume regulation in most animal cells [1][2][3]. It has recently been reported that this channel is involved in important cell functions such as proliferation [4] and apoptosis [5]. In spite of the importance of its functions, the molecular identification of this channel has not yet been achieved, although the ClC-3 protein, which is one of the members of the voltage-dependent chloride channel ClC family, has been considered as the candidate for the VSOR Cl Okazaki, ductance (around 38 pS). A Cl -channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), reversibly inhibited the outward current. These single-channel properties were identical with those in ClC-3 expressing wild-type ventricular myocytes. These results indicate that the single-channel activity of the VSOR Cl -channel is independent of the expression of ClC-3 proteins in mouse ventricular myocytes. [The Japanese Journal of Physiology 55: [379][380][381][382][383] 2005]

Overexpression of CLC-3 in HEK293T cells yields novel currents that are pH dependent

“…Other investigators have also found no difference in ICl swell between ClC-3 wild-type and null cells (17,48,50). This, however, contradicts studies showing that ClC-3 antibodies (9,11,55) and antisense oligonucleotides (22,51) inhibit ICl swell n a variety of cell types.…”

Activation of Swelling-activated Chloride Current by Tumor Necrosis Factor-α Requires ClC-3-dependent Endosomal Reactive Oxygen Production

“…Unfortunately, other investigators have either been unable to express ClC-3 currents (8 -10) or have observed currents that were insensitive to changes in cell volume (10 -14). Importantly, ClC-3 null cells have consistently exhibited normal appearing whole-cell (15)(16)(17) and single channel (18) ICl swell currents. The regulation of ICl swell was, however, clearly altered in ClC-3 null cells (6).…”

ClC-3 and IClswell are Required for Normal Neutrophil Chemotaxis and Shape Change

“…It was proposed that ClC-3 is a swelling-induced chloride conductance in NIH/3T3 cells stably transfected with ClC-3 cDNA (37). In addition, anti-ClC-3 antibodies and ClC-3 RNA interference strategies elicit impaired ICl swell activity in several cell types (38,47). However, the overexpression or absence of ClC-3 has no impact on the RVD in multiple cell types, including neurons (48), cardiac myocytes (38), and epithelial cells (49), indicating that ClC-3 is not ICl swell (50,51).…”

“…In addition, anti-ClC-3 antibodies and ClC-3 RNA interference strategies elicit impaired ICl swell activity in several cell types (38,47). However, the overexpression or absence of ClC-3 has no impact on the RVD in multiple cell types, including neurons (48), cardiac myocytes (38), and epithelial cells (49), indicating that ClC-3 is not ICl swell (50,51). Most recently, Picollo and Pusch (52) reported that ClC-4 and -5, proteins in a subbranch of the ClC (chloride channel) gene superfamily that have 90% sequence homology to ClC-3, are in fact chloride/proton antiporters (52,53).…”

“…Hypotonic Challenge-Given the finding that ClC-3 was required for normal cell shape change during chemotaxis and given the prior controversy over the relationship of ClC-3 to ICl swell (37,38), we investigated the RVD in response to hypotonic challenge in ClC-3-deficient PMNs. Complementary assays were performed using Coulter counter and flow cytometry techniques to assess a change in mean cell volume in response to hypotonic challenge.…”