This proposal serves the broad, long-term objective of providing insight into common molecular mechanisms underlying i) excitation-contraction (E-C) coupling in muscle, ii) voltage controlled gating of ion channels in nerve and muscle cells, and iii) cell biological control of functional diversity in adult and developing neuromuscular systems. An integrated experimental approach will utilize three model systems: 1. Calcium channel gating in arthropod muscle. Ionic currents through Ca channels and gating currents controlling channel activation will be studied with voltage clamp techniques. Kinetic and pharmacologic analysis will advance understanding for Ca channel gating and its functional and molecular relationships to E- C coupling in skeletal muscle and to NA channel gating in neurons and muscle cells. 2. E-C coupling, charge movement and ion channels in developing vertebrate muscle. Myogenic cells form two waves (embryonic vs fetal) of avian muscle developemnt will be studied in vitro. Myotubes formed from cells of these time frames differ in morphological and biochemical parameters, but physiological differences remain unknown. Patch voltage clamp and video microscopy techniques will be used to characterize ion channels, voltage-dependent charge movement E-C coupling, and contractile properties in myotubes of each type. Embryonic myotubes which express distinct myosing heavy chains (fast and slow) will also be studied with this methodology and later identified immunocytochemically to correlate myosin-type and physiological properties. A smiilar approach will be applied to fast fetal myotubes which cna be induced to express slow myosin and to satellite cell-derived myotubes from injured adult muscle. This project will fill in several outstanding gaps in our knowledge of muscle cell developmental biology. 3. Cell biology of Na and Ca channels in neurons. Knowledge of biosynthesis and processing of membrane proteins classified as Na channels, Ca channels and voltage-sensors for E-C coupling is very limited, but primary sequence homologies in these proteins show that some relation between them exists. Proposed experiments on cultured neurons will explore the possibility that cell biological control processes can alter properties of these proteins in physiologically definable ways. Such processes must be vital in maintaining normal cellular properties and in establishing these during development (e.g., in myotubes) or recovery from injury. Studies of Na channels in cultured squid neurons following axotomy suggest that newly synthesized NA channels may have an unusual set of properties that makes the labels of Na and Ca channel ambiguous. Patch clamp studies in this system will extend these observations which are relevant to fundamental problems in neurobiology.
Showing the most recent 10 out of 26 publications
