RESUMEN
Many neurons, including pyramidal cells of the cortex, express a slow afterhyperpolarization (sAHP) that regulates their firing. Although initial findings suggested that the current underlying the sAHP could be carried through SK(Ca) channels, recent work has uncovered anomalies that are not congruent with this idea. Here, we used overexpression and dominant-negative strategies to assess the involvement of SK(Ca) channels in mediating the current underlying the sAHP in pyramidal cells of the cerebral cortex. Pyramidal cells of layer V exhibit robust AHP currents composed of two kinetically and pharmacologically distinguishable currents known as the medium AHP current (I(mAHP)) and the slow AHP current (I(sAHP)). I(mAHP) is blocked by the SK(Ca) channel blockers apamin and bicuculline, whereas I(sAHP) is resistant to these agents but is inhibited by activation of muscarinic receptors. To test for a role for SK(Ca) channels, we overexpressed K(Ca)2.1 (SK1) and K(Ca)2.2 (SK2), the predominant SK(Ca) subunits expressed in the cortex, in pyramidal cells of cultured brain slices. Overexpression of K(Ca)2.1 and K(Ca)2.2 resulted in a fourfold to fivefold increase in the amplitude of I(mAHP) but had no detectable effect on I(sAHP). As an additional test, we examined I(sAHP) in a transgenic mouse expressing a truncated SK(Ca) subunit (SK3-1B) capable of acting as a dominant negative for the entire family of SK(Ca)-IK(Ca) channels. Expression of SK3-1B profoundly inhibited I(mAHP) but again had no discernable effect on I(sAHP). These results are inconsistent with the proposal that SK(Ca) channels mediate I(sAHP) in pyramidal cells and indicate that a different potassium channel mediates this current.
Asunto(s)
Calcio/fisiología , Corteza Cerebral/fisiología , Neuronas/fisiología , Canales de Potasio/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Electrofisiología , Lóbulo Frontal/citología , Lóbulo Frontal/metabolismo , Lóbulo Frontal/fisiología , Expresión Génica , Proteínas Fluorescentes Verdes , Humanos , Técnicas In Vitro , Canales de Potasio de Gran Conductancia Activados por el Calcio , Proteínas Luminiscentes/genética , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/efectos de los fármacos , Canales de Potasio/genética , Canales de Potasio Calcio-Activados/antagonistas & inhibidores , Canales de Potasio Calcio-Activados/genética , Canales de Potasio Calcio-Activados/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Células Piramidales/fisiología , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , Corteza Somatosensorial/citología , Corteza Somatosensorial/metabolismo , Corteza Somatosensorial/fisiologíaRESUMEN
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.