The functional properties of nicotinic ACh receptors on vertebrate skeletal muscle are regulated by both muscle type and the state of innervation. Embryonic muscle, denervated muscle, and slow tonic muscle are characterized by a family of 'slow' receptor types, whereas adult twitch muscle expresses two families of 'fast' Ach receptor types. Within these two families there is additional diversity in kinetics and channel conductances, giving rise to 5 or more distinct receptor types. 1) The first aim of the proposed studies is to identify the structural bases for these differences in receptor function through a multi-pronged approach. The structure of each functionally-defined receptor type in muscle will be identified through engineering of receptors with known subunit composition and functional characterization at the signal channel level. Structural regions responsible for conferring developmentally relevant changes in muscle receptor function will be studied using chimeric receptors and subunit concatamers. 2) In aim 2 the mechanisms underlying nerve-dependent alterations in receptor function on muscle will be examined through the use of two animal systems uniquely amenable to study of muscle development. The first is developing Xenopus embryonic muscle where synaptic development is accessible at all stages of neuromuscular junction formation. The second is a cell line of mammalian muscle which has retained the capacity to recapitulate the developmental changes in receptor function in vitro. We will determine which of the developmental changes in muscle receptor function require nerve contact and which occur through intrinsic programmed changes in muscle. 3) in aim 3 a newly discovered 'mode shift' in receptor open time will be examined in order to establish whether developmentally relevant changes in receptor subunit composition alter channel open time through stabilization of the receptor in a particular mode. Also, the structural regions of gamma and epsilon subunits which are responsible for selecting between gating modes will be identified by gamma-epsilon chimeric receptors. 4) In aim 4 the single channel bases for the rapid component of receptor desensitization will be disclosed by the use of a fast perfusion system. Comparisons of epsilon and gamma type receptors will determine whether these subunits affect desensitization rates via altered affinity to Ach or by affecting the gating mode of the receptor. As with aims 1 and 3, the structural regions conferring subunit specificity on desensitization rates will be identified by construction of gamma-epsilon chimeric receptors. Studies of this ligand-gated receptor channel provide a model for understanding how functional diversity within receptor families is achieved and how appropriate nerve- muscle contact is able to select the receptor type to be expressed.
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