Formation of the Ca2+ channel complex in the skeletal muscle triad: Identification of the targeting signal of the L-type Ca2+ channel alpha1 subunit

The cellular function of Ca2+ channels heavily depends on their specific localization in functional membrane domains. However, little is known about the signals and mechanisms responsible for the targeting and immobilization of Ca2+ channels in excitable cells. In this study we will take advantage of different targeting properties of two Ca2+ channel isoforms when expressed in mutant myotubes, to determine the molecular domain of the Ca2+ channel responsible for its specific localization in the skeletal muscle triad. We have previously developed mutant muscle expression systems for the study of interactions between Ca2+ channels and their subunits during the development of the triad. In the present study plasmids encoding chimeras of the skeletal muscle and a non-muscle isoform of the voltage-gated Ca2+ channels hall be generated and expressed in dysgenic myotubes; their targeting properties will be analyzed with specific antibodies and by localization with the green fluorescent protein. The triad targeting motif will be further characterized with point mutations within the putative targeting domain and its specificity will be tested by transferring it into heterologius membrane proteins. Excitation- contraction coupling and channel properties of chimeric channels targeted to the triad will be analyzed with patch- clamp and microfluorometric Ca2+ measurements., The targeting signal is an important functional domain of the voltage-gated CA 2+ channel and knowing it is crucial for understanding the molecular mechanisms underlying triad formation as well as the formation of specialized membrane domains in excitable cells. In addition, the molecular and cellular tools developed in this and the previous project will be used for the localization and expression of the type 3 ryanodine receptor and of new putative triad proteins, and for the analysis of their roles in development and function of the excitation-contraction coupling apparatus in skeletal muscle.

Ca2+ channels play a central role in physiological processes like the muscle contraction, learning and memory, or secretion. In order to fulfill this multitude of functions, Ca2+ channels need to be precisely targeted to and inserted in specific structures of the respective cell types. The mechanisms underlying these targeting processes are still elusive. In this project we described for the first time an amino-acid sequence in the skeletal muscle Ca2+ channel that contains the information necessary for the functional targeting of the channel into the skeletal muscle triad. A defect in the genetic material of the dysgenic mouse leads to the lack of a Ca2+ channel in its skeletal muscles and consequently the animals die from respiratory failure at birth. However, the muscle cells of this mouse mutant can be maintained and differentiated in cell culture. If we insert a normal correlate of the defect gene, using modern cell biological techniques, the dysgenic muscle cells begin to contract. A normal Ca2+ channel is synthesized from the recombinant gene, the gene product is incorporated into the correct cellular structure, and thus the normal function of the muscle cell is reconstituted. This approach corresponds to gene-therapy in culture and can be exploited for structure-function studies of the Ca2+ channel. If, for example, we substitute the defect muscle gene with that of a neuronal Ca2+ channel, this channel isoforms is synthesized in the muscle cell but not targeted into the triad junction, the structure normally containing the skeletal muscle Ca2+ channel. Molecular biology techniques allow us to generate channel chimeras, composed of parts of the two channel types, or channels with other alterations. These recombinant channels can then be expressed and analyzed in the dysgenic muscle cells. Using this approach we characterized the function and mode of action of several molecular domains of the Ca2+ channel: (1) We showed that a domain, responsible for the association of the Ca2+ channel subunit is also important for the modulation of the drug binding site in the channel pore. (2) The region responsible for the activation of intracellular Ca2+ release and consequently for the activation of contraction was further characterized. (3) We localized a signal important for the targeting of the Ca2+ channel into the skeletal muscle triad to a 55-amino acid sequence near the C-terminus of the channel protein. Transferring this sequence to the corresponding position of the neuronal Ca2+ channel was sufficient to redirect this channel isoforms into skeletal muscle triads and thus enable it to activate muscle contraction. These results are important for our understanding of this prominent class of membrane proteins, the Ca2+ channels, and for understanding the mechanism of excitation-contraction coupling. Furthermore, this experimental system enables us to simulate genetically caused muscle diseases (e.g. malignant hyperthermia), and to study their pathogenesis on a cellular level. The cell systems developed in Innsbruck are meanwhile in use in bio-medical research in numerous laboratories world wide.