Submitted on March 18, 2003
Accepted on June 12, 2003
Functional effects of novel anti-ClC-3 antibodies on native volume-sensitive osmolyte and anion channels (VSOACs) in cardiac and smooth muscle cells
Ge-Xin Wang1, William J. Hatton1, Grace L. Wang1, Juming Zhong1, Ilia Yamboliev1, Dayue Duan1, and Joseph R. Hume1*
1 Department of Pharmacology, University of Nevada, Reno, NV, USA; Center of Biomedical Research Excellence, University of Nevada, Reno, NV, USA
* To whom correspondence should be addressed. E-mail: joeh{at}med.unr.edu.
Functional effects of novel anti-ClC-3 antibodies on native volume-sensitive Cl- channels in cardiac and smooth muscle cells. Am J Physiol Heart Circ Physiol XXX:XXX-XXX, 2003. - Whether ClC-3 encodes volume-sensitive organic osmolyte and anion channels (VSOACs) remains controversial. We have shown previously that native VSOACs in some cardiac and vascular myocytes were blocked by a commercial anti-ClC-3 carboxyl terminus antibody (Alm C592-661 Ab), although recent studies have raised questions related to the specificity of Alm C592-661 Ab. Therefore, we have developed three new anti-ClC-3 Abs and investigated their functional effects on native VSOACs in freshly isolated canine pulmonary artery smooth muscle cells (PASMCs) and guinea-pig cardiac myocytes. These new Abs produced a common prominent immunoreactive band with an apparent molecular weight of 90-92 kDa in guinea-pig heart and PASMCs, and a similar molecular weight immunoreactive band was observed in brain from homozgygous Clcn3+/+ mice, but not from homozygous Clcn3-/- mice. VSOACs elicited by hypotonic cell swelling in PASMCs and guinea-pig atrial myocytes were nearly completely abolished by intracellular dialysis with two new anti-ClC-3 Abs specifically targeting the ClC-3 carboxy (C670-687 Ab) and amino terminus (A1-14 Ab). This inhibition of native VSOACs can be attributed to a specific interaction with endogenous ClC-3, since: 1) pre-absorption of the Abs with corresponding antigens prevented the inhibitory effects, 2) extracellular application of a new Ab raised against an extracellular epitope (Ex133-148) of ClC-3 failed to inhibit native VSOACs in PASMCs, 3) intracellular dialysis with an Ab targeting Kv1.1 potassium channels failed to inhibit native VSOACs in guinea-pig atrial myocytes, and 4) anti-ClC-3 C670-687 Ab had no effects on swelling-induced augmentation of the slow component of the delayed rectifying potassium current (IKs) in guinea-pig ventricular myocytes, although VSOACs in the same cells were inhibited by the Ab. These results confirm that endogenous ClC-3 is an essential molecular entity responsible for native VSOACs in PASMCs and guinea-pig cardiac myocytes.
