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J. Biol. Chem., Vol. 280, Issue 27, 25871-25880, July 8, 2005

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Functional Characterization of Novel Alternatively Spliced ClC-2 Chloride Channel Variants in the Heart^*

Fiona C. Britton¶, Guan-Lei Wang¶||, Z. Maggie Huang||, Linda Ye||, Burton Horowitz, Joseph R. Hume||, and Dayue Duan||**

From the Center of Biomedical Research Excellence and Departments of,^||Pharmacology and Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557

A novel volume-regulated hyperpolarization-activated chloride inward rectifier channel (Cl.ir) was identified in mammalian heart. To investigate whether ClC-2 is the gene encoding Cl.ir channels in heart, ClC-2 cDNAs cloned from rat (rClC-2) and guinea pig (gpClC-2) hearts were functionally characterized. When expressed in NIH/3T3 cells, full-length rClC-2 yielded inwardly rectifying whole-cell currents with very slow activation kinetics (time constants > 1.7 s) upon hyperpolarization under hypotonic condition. The single-channel rClC-2 currents had a unitary slope conductance of 3.9 ± 0.2 picosiemens. A novel variant with an in-frame deletion at the beginning of exon 15 that leads to a deletion of 45 bp (corresponding to 15 amino acids in -helices O and P, rClC-2_509–523) was identified in rat heart. The relative transcriptional expression levels of full-length rClC-2 and rClC-2_509–523 in rat heart were 0.018 ± 0.003 and 0.028 ± 0.006 arbitrary units, respectively, relative to glyceraldehyde-3-phosphate dehydrogenase (n = 5, p = nonsignificant). A similar partial exon 15 skipping with a deletion of 105 bp (35 amino acids in -helices O-Q, gpClC-2_509–543) was also identified in guinea pig heart. Expression of both rClC-2_509–523 and gpClC-2_509–543 resulted in functional channels with phenotypic activation kinetics and many properties identical to those of endogenous Cl.ir channels in native rat and guinea pig cardiac myocytes, respectively. Intracellular dialysis of anti-ClC-2 antibody inhibited expressed ClC-2 channels and endogenous Cl.ir currents in native rat and guinea pig cardiac myocytes. These results demonstrate that novel deletion variants of ClC-2 due to partial exon 15 skipping may be expressed normally in heart and contribute to the formation of endogenous Cl.ir channels in native cardiac cells.

Received for publication, March 15, 2005 , and in revised form, May 5, 2005.

^* The work was supported in part by National Center of American Heart Association Grant GIA 9950153N; National Heart, Lung and Blood Institute Grant HL63914; and National Center for Research Resources Grant P-20 RR-15581. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by American Heart Association Western Affiliate Fellowships.

Deceased.

^** To whom correspondence should be addressed: Dept. of Pharmacology, University of Nevada School of Medicine, Manville Medical Bldg., Rm. 9, 1664 N. Virginia St., MS 318, Reno, NV 89557-0270. Tel.: 775-784-4738; Fax: 775-784-1620; E-mail: dduan{at}med.unr.edu.

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