The long-term objective of this research is to elucidate how the genome, development, and cell-cell interactions govern the emergence and maintenance of the types and subcellular localization of ion channels in a cell. The loose patch clamp technique will be used on developing and denervated muscle cells to determine whether Na channel distribution is affected by altered acetylcholine receptor distribution. A relationship between the distribution of these two channels is suggested by our earlier studies showing that Na channels in adult muscle are concentrated near the neuromuscular junction. The loose patch clamp will also be used on identified snail neurons to determine whether the kinds of ionic channels in the cell body differ from those in processes, and to determine the relationship between ionic currents and the neuron's growth state. The tight-seal patch clamp technique will be used to measure whole cell currents in single muscle, nerve and cardiac cells from both normal mice and mice with muscular dysgenesis, a fatal, recessive mutation that is expressed in skeletal muscle as a failure of E-C coupling. Normal developing muscle cells have been found to express two distinct voltage-dependent calcium currents, the slower one of which is lacking in dysgenic skeletal muscle. Dysgenic nerve and cardiac cells will be examined to determine whether the mutation also affects slow calcium currents in these cells. It will be determined if the slow calcium current and/or E-C coupling of dysgenic muscle can be restored by injection of cytosolic proteins from normal muscle. IP3 has been proposed as an internal second mesenger in E-C coupling. It will be determined if IP3 injection fails to cause dysgenic muscle cells to contract. The experiments with dysgenic mice should yield important information on both E-C coupling and the genetic regulation of calcium channels. Muscle mRNA will be injected in Xenopus oocytes in order to obtain the in vitro synthesis of muscle membrane proteins. Voltage clamp measurements will be used to establish the presence of calcium channels and the voltage-dependent charge movement thought to be involved in E-C coupling. In parallel, biochemical methods will be used to establish the presence or absence of individual proteins. This combined approach promises a new technique for identifying calcium channels and other membrane proteins important in E-C coupling.
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