Kolski-Andreaco Aaron, Tomita Hiroaki, Shakkottai Vikram G, Gutman George A, Cahalan Michael D, Gargus J Jay, Chandy K George
Department of Physiology and Biophysics, University of California Irvine, Irvine, California 92697, USA.
J Biol Chem. 2004 Feb 20;279(8):6893-904. doi: 10.1074/jbc.M311725200. Epub 2003 Nov 24.
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.
小电导钙激活钾通道是由SK1 - SK3基因编码的产物，可调节神经系统内外的膜兴奋性。我们报道了一种SK3变体（SK3 - 1C）的特性，它与SK3的不同之处在于利用了一个替代的第一外显子（外显子1C）来取代SK3所使用的外显子1A，但在其他方面与SK3相同。定量逆转录聚合酶链反应（RT - PCR）检测到SK3 - 1C转录本在人类淋巴组织、骨骼肌、气管和唾液腺中大量表达，但在神经系统中未检测到。当单独在哺乳动物细胞中表达时，SK3 - 1C不产生功能性通道，但能抑制SK1、SK2、SK3和IKCa1通道，而不抑制BKCa或KV通道。共聚焦显微镜显示SK3 - 1C在细胞内隔离SK3蛋白。SK3 - 1C的显性抑制活性并非由于非特异性钙调蛋白海绵效应，因为钙调蛋白的过表达并不能逆转SK3 - 1C介导的SK3蛋白在细胞内的捕获，并且钙调蛋白 - Ca2 +依赖性的CaV通道失活不受SK3 - 1C过表达的影响。缺失分析在SK3 - 1C的C末端鉴定出一个显性抑制片段，该片段类似于据报道可增强许多钾通道四聚体稳定性和多聚化选择性的四聚化卷曲螺旋结构域。因此，SK3 - 1C可能通过由显性抑制片段介导的亚基相互作用在细胞内捕获这些通道蛋白，从而抑制钙调蛋白门控的SKCa/IKCa通道，进而减少细胞表面功能性通道的表达。SK3 - 1C这种全家族范围的显性负性抑制提供了一种强大的机制来调节膜兴奋性，并且是通过靶向沉默来确定这些通道在不同组织中的体内功能作用的有用方法。
