This proposal concerns the mechanisms by which chloride ions are transported across the surface membrane of rat skeletal muscle fibers. We will characterize single chloride channels using patch clamp technology and chloride pumps using C1-sensitive fluorescent dyes. We will study the spatial distribution of channels and pumps, focussing on differences between end plate and extrajunctional regions. Finally we will study the long term regulation of C1 channels and pumps using a preparation of isolated adult muscle fibers maintained in cell culture. C1 channels form the major resting conductance pathway in adult skeletal muscle. These channels appear rather late in postnatal development and disappear from adult muscle after denervation. While a considerable amount is known about macroscopic properties of C1 channels, very little is known about single C1 in adults fibers. We will study these channels with patch clamps technology, and compare them to neonatal and denervated channels. C1 ions are actively transported into skeletal muscle fibers. We will use a new technique to study this C1 pump. The technique involves the use of a C1-sensitive fluorescent dye which, when loaded into a muscle fiber, provides a measure of internal C1 activity. We will measure regional differences in C1 conductance and pump activity with C1-sensitive dyes. We predict from previous work that C1 channels are restricted from the end plate region. For example, with the C1 pump blocked with furosemide, we will measure the rate of decline of internal C1 concentration in end plate and extrajunctional regions when external C1 is removed; we predict the fall in internal C1 will be faster in extrajunctional regions than at the end plate. Finally, we will culture isolated adult muscle fibers for 1-2 weeks to study the long term regulation of C1 channels and pump activity. We will test specific hypotheses concerning factors which govern the expression of C1 transport pathways; factors to be studied include muscle activity calcium ions, and second messenger systems.
