REGULATION OF ACETYLCHOLINE RECEPTOR FUNCTION

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Synaptic transmission at the vertebrate skeletal neuromuscular junction is mediated by functionally distinct classes of nicotinic acetylcholine (ACh) receptors. The expression of these different types of ACh receptors is regulated by a complex interplay between the developmental age of the animal and the state of innervation of the muscle. An extensive number of studies has made it clear that innervation plays a major role in regulating the expression of ACh receptors in developing and adult animals. Although the regulation of ACh receptor expression by nerve-induced activity is indisputable, it is equally clear that other humoral and "intracellular factors affect ACh receptor expression as well. However, the physiololical consequences of these factors on ACh receptor function have not been addressed. The objective of this proposal will be to examine two preparations in which functional changes in ACh receptors are induced by factors independent of the state of innervation of the muscle fiber. First, it has been shown in a number of electrically excitable cells that steroid hormones induce changes in the expression of neurotransmitter receptors. In hormone-sensitive muscles of the frog, exposure to androgenic steroids significantly increases the time course of synaptic current decay, suggesting that steroids induce the expression of functionally distinct synaptic ACh receptors. This hypothesis will be directly tested by single channel recording and analysis. It will be established by Northern blot analysis whether steroid effects on channel function are mediated by changes in the levels of transcripts encoding the different subunits of the ACh receptor. The functional consequences of changes in message levels will be confirmed by expressing mRNA from steroid-treated fibers in Xenopus oocytes. Second, single channel analysis indicates that ACh receptors in dystrophic mouse muscle have significantly different properties than receptors in control muscle and that these differences are not dependent on the state of innervation of the fiber. Experiments will be made to determine whether a defective association between specific cytoskeletal elements and the ACh receptor protein causes functional changes in channel properties or if the dystrophic phenotype leads to altered expression of ACh receptor genes. Combined physiological/molecular biological studies on muscle ACh receptors have laid the groundwork for elucidating the structural bases of neuronal ion channels and have clearly demonstrated the relevance of experiments on muscle ACh receptors to the broader field of neurobiology. By using this approach, the experiments presented here will increase our understanding of the regulation of ion channels in excitable membranes. More specifically, these studies will elucidate cellular mechanisms associated with pathological conditions induced by steroid abuse and by genetic dystrophies