Functional Characteristics and Molecular Identification of Swelling-activated Chloride Conductance in Adult Rabbit Heart Ventricles

Outwardly rectifying swelling-activated chloride conductance （ICl, Swell） in rabbit heart plays a critical role in cardioprotection following ischemic preconditioning （IP）. But the functional characterization and molecular basis of this chloride conductance in rabbit heart ventricular myocytes is not clear. Candidate chloride channel clones （e.g. ClC-2, ClC-3, ClC-4 and ClC-5） were determined using RT-PCR and Western blot analysis.Whole cell ICl,Swell was recorded from isolated rabbit ventricular myocytes using patch clamp techniques during hypo-osmotic stress. The inhibitory effects of 4,4＇ isothiocyanato-2,2-disulfonic acid （DIDS）, 5-nitro-2（3-phenylroylamino） benzoic acid （NPPB） and indanyloxyacetic acid 94 （LAA-94） on ICl,Swell were examined. The expected size of PCR products for ClC-2, ClC-3 and ClC-4 but not for ClC-5 was obtained. ClC-2 and ClC-3 expression was confirmed by automated fluorescent DNA sequencing. RT-PCR and Western blot showed that ClC-4 was expressed in abundance and ClC-2 was expressed at somewhat lower levels. The biological and pharmacological properties of ICl,Swell, including outward rectification, activation due to cell volume change, sensitivity to DIDS, LAA-94 and NPPB were identical to those known properties of ICl,Swell in exogenously expressed systems and other mammals hearts. It was concluded that ClC-3 or ClC-4 might be responsible for the outwardly rectifying part of ICl,Wwell and may be the molecular targets of cardioprotection associated with ischemic preconditioning or hypo-osmotic shock.

Protein Kinase C Enhances the Swelling-Induced Chloride Current in Human Atrial Myocytes

Swelling-activated chloride currents（ICl.swell） are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C（PKC） in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate（PDBu） enhanced ICl.swellin a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.