Abstract
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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF438203
We have identified and characterized a novel SK3 transcript, SK3-1C, that is generated by alternative utilization of a novel exon 1C instead of exon 1A used by SK3
When expressed heterologously in mammalian cells, SK3-1C suppressed whole cell Kϩ currents produced by SK1, SK2, and SK3 channels, and to a lesser extent IKCa1 channels, but did not affect the iberiotoxin-sensitive BKCa channel or KV channels
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF438203. Expression of SK3 has been reported in the brain, skeletal muscle, endothelium, uterus, and hepatobiliary cells [6, 7, 13,14,15]. SK3-1C, a Dominant-negative Suppressor of SKCa/IKCa Channels behavior in the brain should have consequences similar to pharmacological blockade of SKCa channels, namely enhanced firing frequency and increased neurotransmitter release (3, 4, 7, 19 –21). We describe a new SK3 transcript, SK3-1C, that is expressed in peripheral human tissues but not the brain. When expressed heterologously in mammalian cells, SK3-1C suppressed SK1, SK2, SK3, and IKCa1 channels but not KV or BKCa channels. Variations in the levels of functional SKCa and IKCa channels versus SK3-1C subunits in peripheral tissues may provide an endogenous mechanism to regulate membrane potential and Ca2ϩ signaling
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