The mechanisms underlying the ontogeny of voltage-gated ion channels in muscle are unknown. To determine whether expression of voltage-gated channels is dependent on mitogen withdrawal and growth arrest, as is generally true for the induction of muscle specific gene products, the BC3H1 muscle cell line is being used as an advantageous model system. Differentiated BC3H1 myocytes form functional calcium and sodium channels which, by patch clamp techniques were found to correspond to those in transverse tubules of skeletal muscle. Ca and Na channels were first detected about 4 days after mitogen withdrawal. Since cellular ras genes have been found to have a role in the transduction of growth factor signals, BC3H1 cells have been modified with an activated ras expression vector. Transfection with a valine-12 c-Ha-ras vector suppressed the formation of functional Ca channels and Na channels for more than 4 weeks. In contrast, potassium channels were affected neither by mitogenic medium nor by the transfected ras gene. Thus, the ras gene selectively blocked the expression of ion channels whose induction was contingent upon mitogen withdrawal. These studies will be extended to elucidate the mechanisms that control Ca channel number. First, Ca channels expressed by differentiated BC3H1 muscle cells will be thoroughly characterized. Second, growth factors that inhibit muscle-specific gene expression in BC3H1 cells will be used under conditions that uncouple direct effects from secondary effects of cell proliferation. Third, Ca channel expression will be investigated in BC3H1 myocytes modified with genes that affect myogenesis. These studies will focus on the control of Ca channel expression by cellular ras genes and will analyze the effects produced by a reversibly inducible ras expression vector, by a graded series of missense mutations at codon 12 of the c-Ha-ras gene, by microinjected ras proteins, and by blocking antibodies against p21 ras. This research represents one of the first major efforts to elucidate the genetic mechanisms regulating the expression of specific voltage-dependent ion channels in muscle membranes. The proposed experiments should clarify the role of the cellular Ha-ras gene in the control of calcium channel expression during muscle differentiation.
