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 Cl-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 Cl 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 Cl channels, very little is known about single Cl 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 Cl pump. The technique involves the use of a Cl-sensitive fluorescent dye which, when loaded into a muscle fiber, provides a measure of internal Cl activity. We will measure regional differences in Cl conductance and pump activity with Cl-sensitive dyes. We predict from previous work that Cl channels are restricted from the end plate region. For example, with the Cl pump blocked with furosemide, we will measure the rate of decline of internal Cl concentration in end plate and extrajunctional regions when external Cl is removed; we predict the fall in internal Cl 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 Cl channels and pump activity. We will test specific hypotheses concerning factors which govern the expression of Cl transport pathways; factors to be studied include muscle activity calcium ions, and second messenger systems.
