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J. Biol. Chem., Vol. 279, Issue 8, 6893-6904, February 20, 2004

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SK3-1C, a Dominant-negative Suppressor of SK_Ca and IK_Ca Channels^*

Aaron Kolski-Andreaco, Hiroaki Tomita¶, Vikram G. Shakkottai||, George A. Gutman**, Michael D. Cahalan, J. Jay Gargus , and K. George Chandy

From the Physiology and Biophysics,^¶Psychiatry and Human Behavior,^**Microbiology and Molecular Genetics, and Pediatrics, University of California Irvine, Irvine, California 92697

Small conductance Ca²⁺-activated K⁺ channels, products of the SK1-SK3 genes, regulate membrane excitability both within and outside the nervous system. We report the characterization of a SK3 variant (SK3-1C) that differs from SK3 by utilizing an alternative first exon (exon 1C) in place of exon 1A used by SK3, but is otherwise identical to SK3. Quantitative RT-PCR detected abundant expression of SK3-1C transcripts in human lymphoid tissues, skeletal muscle, trachea, and salivary gland but not the nervous system. SK3-1C did not produce functional channels when expressed alone in mammalian cells, but suppressed SK1, SK2, SK3, and IKCa1 channels, but not BK_Ca or K_V channels. Confocal microscopy revealed that SK3-1C sequestered SK3 protein intracellularly. Dominant-inhibitory activity of SK3-1C was not due to a nonspecific calmodulin sponge effect since overexpression of calmodulin did not reverse SK3-1C-mediated intracellular trapping of SK3 protein, and calmodulin-Ca²⁺-dependent inactivation of Ca_V channels was not affected by SK3-1C overexpression. Deletion analysis identified a dominant-inhibitory segment in the SK3-1C C terminus that resembles tetramerization-coiled-coiled domains reported to enhance tetramer stability and selectivity of multimerization of many K⁺ channels. SK3-1C may therefore suppress calmodulin-gated SK_Ca/IK_Ca channels by trapping these channel proteins intracellularly via subunit interactions mediated by the dominant-inhibitory segment and thereby reduce functional channel expression on the cell surface. Such family-wide dominant-negative suppression by SK3-1C provides a powerful mechanism to titrate membrane excitability and is a useful approach to define the functional in vivo role of these channels in diverse tissues by their targeted silencing.

Received for publication, October 27, 2003 , and in revised form, November 20, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF438203.

^* This work was supported by grants from the National Institutes of Health (MH59222), Rockefeller Brothers Fund (to K. G. C.), and National Ataxia Foundation (to K. G. C.). 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 a National Institutes of Health predoctoral training grant in cellular and molecular neuroscience (1T32 N507444).

^|| Supported by a predoctoral fellowship from the American Heart Association Western States Affiliate.

To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Room 291, Joan Irvine Smith Hall, College of Medicine, University of California, Irvine, CA 92697. Tel.: 949-824-2133; Fax: 949-824-3143; E-mail: gchandy{at}uci.edu.

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