SK3-1C, a dominant-negative suppressor of SKCa and IKCa channels.
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 BKCa or KV 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 CaV 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 SKCa/IKCa 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.
Kolski-Andreaco
Aaron
A
Department of Physiology and Biophysics, University of California Irvine, Irvine, California 92697, USA.
Tomita
Hiroaki
H
Shakkottai
Vikram G
VG
Gutman
George A
GA
Cahalan
Michael D
MD
Gargus
J Jay
JJ
Chandy
K George
KG
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D005091:Exons; D017353:Gene Deletion; D020868:Gene Silencing; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D007438:Introns; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D016716:PC12 Cells; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics* / Q000378:metabolism*; D024681:Potassium Channels, Calcium-Activated / Q000235:genetics / Q000502:physiology; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D051640:Small-Conductance Calcium-Activated Potassium Channels; D014018:Tissue Distribution; D014162:Transfection
D002147:Calmodulin; D018076:DNA, Complementary; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C406328:KCNN3 protein, human; C496923:KCNN4 protein, human; C491575:Kcnn4 protein, rat; D008164:Luminescent Proteins; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D020033:Protein Isoforms; D012333:RNA, Messenger; D051640:Small-Conductance Calcium-Activated Potassium Channels; D049452:Green Fluorescent Proteins