title: Functional effects of novel anti-ClC-3 antibodies on native volume-sensitive osmolyte and anion channels in cardiac and smooth muscle cells.
abstract: 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 carboxy terminal antibody (Alm C592-661 antibody), although recent studies have raised questions related to the specificity of Alm C592-661 antibody. Therefore, we have developed three new anti-ClC-3 antibodies 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 antibodies produced a common prominent immunoreactive band with an apparent molecular mass of 90-92 kDa in the guinea pig heart and PASMCs, and a similar molecular mass immunoreactive band was observed in the brain from homozygous 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 antibodies specifically targeting the ClC-3 carboxy (C670-687 antibody) and amino terminus (A1-14 antibody). This inhibition of native VSOACs can be attributed to a specific interaction with endogenous ClC-3, because 1) preabsorption of the antibodies with corresponding antigens prevented the inhibitory effects, 2) extracellular application of a new antibody raised against an extracellular epitope (Ex133-148) of ClC-3 failed to inhibit native VSOACs in PASMCs, 3) intracellular dialysis with an antibody targeting Kv1.1 potassium channels failed to inhibit native VSOACs in guinea pig atrial myocytes, and 4) anti-ClC-3 C670-687 antibody had no effects on swelling-induced augmentation of the slow component of the delayed rectifying potassium current in guinea pig ventricular myocytes, although VSOACs in the same cells were inhibited by the antibody. These results confirm that endogenous ClC-3 is an essential molecular entity responsible for native VSOACs in PASMCs and guinea pig cardiac myocytes.
authors.affiliation: Center of Biomedical Research Excellence, Department of Pharmacology, University of Nevada School of Medicine, Reno, NV 89557-0046, USA.
authors.forename: Ge-Xin
authors.initials: GX
authors.lastname: Wang
authors.forename: William J
authors.initials: WJ
authors.lastname: Hatton
authors.forename: Grace L
authors.initials: GL
authors.lastname: Wang
authors.forename: Juming
authors.initials: J
authors.lastname: Zhong
authors.forename: Ilia
authors.initials: I
authors.lastname: Yamboliev
authors.forename: Dayue
authors.initials: D
authors.lastname: Duan
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Hume
mesh_terms: D000818:Animals; D000838:Anions / Q000378:metabolism*; D000906:Antibodies / Q000494:pharmacology; D015153:Blotting, Western; D048429:Cell Size / Q000502:physiology; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D051668:Delayed Rectifier Potassium Channels; D003956:Dialysis; D004285:Dogs; D006168:Guinea Pigs; D006325:Heart Atria; D007425:Intracellular Membranes / Q000378:metabolism; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D051662:Kv1.1 Potassium Channel; D051379:Mice; D018345:Mice, Knockout / Q000235:genetics; D009131:Muscle, Smooth, Vascular / Q000737:chemistry / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D015221:Potassium Channels / Q000276:immunology; D024642:Potassium Channels, Voltage-Gated; D011651:Pulmonary Artery
chemical_list: D000838:Anions; D000906:Antibodies; D018118:Chloride Channels; C115376:ClC-3 channel; D051668:Delayed Rectifier Potassium Channels; D007473:Ion Channels; C496947:Kcna1 protein, mouse; D010446:Peptide Fragments; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D051662:Kv1.1 Potassium Channel