ClC-3-independent, PKC-dependent Activity of Volume-sensitive Cl^- Channel in Mouse Ventricular Cardiomyocytes

Abstract

Volume-sensitive outwardly rectifying (VSOR) Cl^- channels are activated during osmotic swelling and involved in the subsequent volume regulation in most animal cells. To test the hypothesis that the ClC-3 protein is the molecular entity corresponding to the VSOR Cl^- channel in cardiomyocytes, the properties of VSOR Cl^- currents in single ventricular myocytes isolated from ClC-3-deficient (Clcn3^-/-) mice were compared with those of the same currents in ClC-3-expressing wild-type (Clcn3^+/+) and heterozygous (Clcn3^+/-) mice. Basal whole-cell currents recorded under isotonic conditions in ClC-3-deficient and -expressing cells were indistinguishable. The biophysical and pharmacological properties of whole-cell VSOR Cl^- currents in ClC-3-deficient cells were identical in ClC-3-expressing cells. The VSOR Cl^- current density, which is an indicator of the plasmalemmal expression of functional channels, was essentially the same in cells isolated from these 3 types of mice and C57BL/6 mice. Activation of protein kinase C (PKC) by a phorbol ester was found to upregulate VSOR Cl^- currents in ClC-3-deficient and -expressing cardiomyocytes. This effect is opposite to the reported downregulatory effect of PKC activators on ClC-3-associated Cl^- currents. We thus conclude that functional expression of VSOR Cl^- channels in the plasma membrane of mouse cardiomyocytes is independent of the molecular expression of ClC-3.

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

“…This difference may be related to the relative amounts of ClC-3 and ICl swell protein that are inserted into the plasma membrane in these two cell types at any one time. Activation of ICl swell by ClC-3 overexpression alone is consistent with previous work, demonstrating that in these same murine VSM cells, Ad-ClC-3 expression alone enhances H 2 O 2 -dependent NF-B activation, even in the absence of other stimuli (21 (15)(16)(17), the size of the ICl swell elicited by hypotonic conditions was not altered in ClC-3 null VSM cells (Fig. 4).…”

“…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).…”

Acidic extracellular pH-activated outwardly rectifying chloride current in mammalian cardiac myocytes

“…I Cl,Ca (9,11,24) requires an elevation of [Ca 2ϩ ] i for its activation. I Cl,vol (8,9,11,29) exhibits a time-dependent inactivation at positive potentials and is sensitive to both DIDS and tamoxifen. I Cl,CFTR (9,11) shows little rectification under symmetrical [Cl Ϫ ] conditions and is insensitive to DIDS.…”

Role of the Vesicular Chloride Transporter ClC-3 in Neuroendocrine Tissue

“…It was suggested to mediate swelling-activated Cl Ϫ currents (Duan et al, 1997) or (different) Ca 2ϩ -activated Cl Ϫ currents Wang et al, 2006) in plasma membranes. Clcn3 Ϫ/Ϫ mice lacked neither current (Stobrawa et al, 2001;Arreola et al, 2002;Gong et al, 2004). ClC-3 was also proposed to have roles in neutrophil and smooth muscle reactive oxygen species generation (Moreland et al, 2006;Miller et al, 2007) and in ␤ cell insulin secretion (Barg et al, 2001).…”