| ClC-3 encodes a volume regulated, outwardly rectifying chloride channel in heart. Using molecular probes, the abundance and distribution of ClC-3 was investigated relative to ClC-2, another volume-regulated chloride channel and member of the ClC family, and normalized to beta-actin. Total RNA from several species (dog, guinea pig and rat) was isolated from atria and ventricle tissue. Quantitative-RT-PCR indicated that transcriptional levels of ClC-3 were between 1.8% and 10.2% of beta-actin in atria, and 3.4% and 8.6% in ventricle. ClC-2 was expressed significantly less (P < .05) in each tissue (0.04--0.08% of beta-actin in atria, and 0.03--0.18% of beta-actin in ventricle). In an effort to link functional ClC-3 channels to ICl.Vol in a transient expression system as well as native currents, intracellular dialysis of a polyclonal antibody specific to the C-terminus was used. Dialysis of the anti-ClC-3 Ab functionally inhibited transiently expressed ClC-3 currents in NIH/3T3 cells as well as endogenous swelling activated currents in guinea pig cardiac myocytes, canine pulmonary artery smooth muscle cells, and Xenopus laevis oocytes, suggesting ClC-3 is a major component of ICl.Vol in these cells. Finally, the functional role of the ClC-3 N-terminus in channel regulation was investigated. Mutation of a consensus protein kinase C (PKC) phosphorylation site (74) within the amino (N-) terminus of ClC-3 yields a constitutively active channel that is no longer sensitive to changes in cell volume, suggesting a fundamental role of the N-terminus in regulation of ClC-3 channel activity by cell volume. The present study was designed to further investigate the molecular mechanism for this "N-type" regulation. Wild-type ClC-3 tagged with a green fluorescence protein (GFP) at the C-terminus was used as a template to construct deletion mutants. The entire N-terminal 100 amino acid residues (ClC-3DeltaNT) were removed and the truncated channel was functionally expressed in NIH/3T3 cells. ClC-3DeltaNT yielded a constitutively active functional channel, which showed no response to PKC and changes in cell volume. Deletion of a cluster of negatively charged amino acids 16--211 (ClC-3Delta16-21) within the N-terminus also yielded a constitutively active open channel phenotype, indicating that these amino acids are involved in the N-type regulation. Delivery of a phosphorylated peptide, corresponding to the N-terminal residues 12--61 to the intracellular side of the channel, inhibited the whole-cell and single-channel currents of the ClC-3DeltaNT channel, further confirming the essential role of the N-terminus in volume regulation of channel activity. These data strongly suggest that the N-terminus of the ClC-3 channel acts as a blocking particle inhibiting the flow of anions through the channel pore. This "N-type" regulation of the ClC-3 channels may be an important transducing mechanism linking changes in cell volume and channel protein phosphorylation to channel gating.;Collectively, these data demonstrate that ClC-3 is a sarcolemmal chloride channel present at significant amounts in cardiac myocytes. These channels, regulated by a change in phosphorylation state of serine 51 within the ClC-3 N-terminus in an N-type mechanism similar to that previously described for Na+ and K+ channels, may represent a molecular entity underlying the ICl.Vol in many tissues. |
