Skeletal muscle excitation is normally controlled by the influence of innervating nerve. However, prior to innervation, upon denervation, in patients with myotonic muscular dystrophy (DM), or myotubes cultured in the absence of nerve, skeletal muscle is hyperexcitable, in that a train of action potentials is often induced following an evoked contraction. The cellular hallmark of these conditions is the appearance of receptors for the peptide toxin apamin, a potent blocker of small conductance calcium-activated potassium (SK) channels. Indeed, application of apamin to denervated or myotonic dystrophic skeletal muscle dramatically repress the hyperexcitability, demonstrating that SK channels are central to the hyperexcitable state. We have cloned the apamin sensitive SK channels from skeletal muscle, SK3, and found that upon denervation or after differentiation of the muscle cell line, L6, the SK3 gene is expressed while in normally innervated muscle it is not expressed. Neither the physiological role of SK channels in hyperexcitable skeletal muscle nor the molecular cues controlling SK3 gene expression are yet understood. In this proposal, we will test the hypothesis that: (1). SK3 channels reside in the transverse tubules of denervated skeletal muscle cells. Patch clamp measurements will be performed using denervated normal and detubulated cultured myotubes. Immunohistochemistry using SK3 channel-specific antibodies, and I125-apamin binding studies will be performed. (2). SK channel activity induces hyperexcitability. Skeletal muscle myotubes and nerve cells will be co-cultured. SK channels will be heterologously expressed by infection with recombinant retroviruses and the cells electrophysiologically assayed. (3). The SK3 promotor is activated following differentiation of cultured L6 myoblasts. a) SK3 promotor/luciferase constructs will be introduced into L6 myoblasts, and luciferase activity assessed before and after differentiation; b) gel-shift and footprint assays will be performed with nuclear extracts from pre- and post-differentiated L6 cells; c) previously uncharacterized sequences in the SK3 promotor shown to be necessary for activation following myoblast differentiation will be used to screen a differentiated L6 skeletal muscle cDNA expression library. (4). DMAHP (myotonic dystrophy associated homeodomain protein or DMPK (myotonic dystrophy protein kinase) regulates SK channel expression. a) DMAHP and/or DMPK will be ectopically expressed in L6 myoblasts and SK3 mRNA and channel activity assessed before and after differentiation. b) gel-shift assays and footprints will be performed with the SK gene promotor and recombinant DMAHP; c) SK3 promoter/luciferase constructs will be introduced with or without DMAHP and/or DMPK into L6 myoblasts and the promoter elements responsible for regulation will be determined. These studies will establish a framework for understanding the molecular, cellular and physiological abnormalities of hyperexcitable skeletal muscle as well as the coordinate regulation of SK gene expression in muscle tissue.
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| Litt, M; LaMorticella, D; Bond, C T et al. (1999) Gene structure and chromosome mapping of the human small-conductance calcium-activated potassium channel SK1 gene (KCNN1). Cytogenet Cell Genet 86:70-3 |
