Expression and function of a new skeletal muscle calcium channel splice variant

In skeletal muscle the voltage-gated calcium channel (Ca V 1.1) functions as voltage-sensor for excitation- contraction coupling. Its primary role is to activate calcium release from the sarcoplasmic reticulum, which in turn triggers contraction. However calcium influx through CaV 1.1 activates slowly, with poor voltage-sensitivity, and is not essential for EC coupling in mature muscle fibers. Recently we isolated the transcript of a new CaV 1.1 splice variant lacking exon 29 [Tuluc et al,. 2009]. Contrary to the current properties of the full-length channel, the CaV 1.1E29 isoform activates at 30 mV more negative potentials and conducts eightfold larger calcium currents. Whereas the CaV 1.1E29 channel isoform is expressed at low levels in adult muscle, it is the predominant splice variant in human and mouse cultured myotubes and in embryonic muscle. Therefore we hypothesize that CaV 1.1E29 represents the embryonic calcium channel isoform in skeletal muscle. Moreover, its biophysical properties and developmental expression patterns suggest that it may be responsible for excitation-coupled calcium entry (ECCE), a phenomenon implicated in the pathophysiology of malignant hyperthermia and central core disease. Therefore, we propose here to study the expression of CaV 1.1E29 in muscles at different developmental and physiological states, and to investigate its functions in normal and injured muscle. Quantitative RT-PCR and in situ hybridization will be applied to determine expression levels of CaV 1.1E29 in developing, ageing, denervated, damaged and regenerating muscles. An antibody specific to the exon 28/30 border will be generated and used to analyze CaV 1.1E29 protein expression in extracts and sections of developing muscle. Whole-cell and single channel patch-clamp recordings, fluorescent calcium indicators and pharmacological tools will be used to provide evidence for the functional expression of the channel in muscle and to further characterize its biophysical and pharmacological properties. To ultimately elucidate the physiological importance of the CaV 1.1E29 to CaV 1.1 isoform switch in vivo and to analyze the role of CaV1.1 E29 in EC coupling and ECCE in isolated muscle fibers an exon 29 knock-out mouse will be generated. Finally, our previous findings showing that the characteristic current properties of the skeletal muscle CaV 1.1 channel depend on the presence of the nineteen amino acid exon 29 evoked new predictions about the structural bases of the channel`s activation mechanism, which now shall be tested using chimeric and mutated channel constructs expressed in dysgenic myotubes. Answering these questions will be important for our understanding of skeletal muscle physiology and can be expected to reveal a fundamentally new role of the skeletal muscle calcium channel during development.

Muscle contractions are regulated in response to an electrical signal from the nerve by a voltage-activated calcium channel. Surprisingly, in mature skeletal muscle this channel does not function as a channel, but instead triggers the release of calcium from stores within the muscle fibers. This is different from the situation in embryonic muscle, where we recently discovered a channel variant that functions both as calcium channel and as trigger for intracellular calcium release. Apparently, during development skeletal muscle actively turns off calcium influx by exchange of the embryonic, calcium conducting, for the adult, virtually non-conducting calcium channel variant. Why this might be important is not known. However, calcium influx in adult muscle may be harmful, as the embryonic isoform was found aberrantly expressed in muscles of patients with a form of muscle weakness called myotonic dystrophy. Here we addressed this problem by generating and analyzing a genetically modified mouse, which after birth does not switch to the adult channel variant but expresses the embryonic channel variant throughout life. We reasoned that if the switch to the non-conducting channel is of physiological importance, failure of this switch will result in altered muscle function. Conversely, if the calcium influx through the embryonic channel by itself is sufficient to cause muscle disease this would show up in the mutant mouse model. The overall behavior of the mutant mice did not reveals muscle disease, although muscle force was weaker and the mice ran less than normal controls. However, experiments on isolated muscle fibers revealed significantly reduced contractile force and increased muscle endurance. These changed muscle properties were accompanied and explained by an altered muscle composition with a higher proportion of slow muscle fiber types. Analysis of calcium signals and intracellular signaling pathways revealed that the increased calcium influx during contraction activated signaling cascades responsible for the specification of muscle fiber types. This indicated that the developmental shift from the calcium-conducting to the non-conducting channel is important to maintain the secondary role of the calcium signal in regulating activity-dependent control of muscle fiber composition. Moreover, our experiments revealed that with age the increased calcium influx caused mitochondrial damage, which may contribute to the symptoms of dystrophic muscle. Finally, biophysical and pharmacological analysis allowed us to determine the molecular mechanism underlying the distinct gating properties of the two channel variants and the sensitivity of the calcium-conducting variant to channel blockers. Together our results suggest that already licensed calcium channel drugs could potentially be applied in the treatment of patients with myotonic dystrophy.