Douglas Fambrough - US grants

Gap junctions consist of ensembles of intercellular channels that mediate electrotonic, metabolic, and dye coupling among cells of many tissues. Conductance through gap junction channels of fish and amphibian embryos is gated by transjunctional voltage, cytoplasmic pH, and to a lesser extent, by Ca ions. By the combined use of electrophysiological and optical techniques we intend to determine whether these gating mechanisms are common to other cell types, localize gating regions of the channel macromolecule, correlate junctional conductance with permeability and determine biophysical properties of individual gap junction channels. Although no disease state is now attributable to alterations in gap junctions, role in pathological conditions have been postulated (e.g., cataract formation, tumorigenesis, cardiac arrhythmias). Detailed analysis of permeation of gap junction channels may indicate the plausibility of a role in pathology and also provide information generalizable to other smaller ion channels.

The broad objective of this research project is to explore the mechanisms which regulate the level of expression of specific membrane proteins and their spatial distribution in nerve and muscle. The focus of current research is upon the sodium- and potassium-ion stimulated ATPase. Our exploration of Na/K ATPase regulation is subdivided into six projects. First, the genetic basis of the Na/K ATPase is being described through cloning and sequencing the genes and gene transcripts encoding each of the subunits. Second, the expression of each gene in various cell types of nerve and muscle is being described through use of isoform-specific DNA probes and isoform-specific monoclonal antibodies. Third, up- and down-regulation of the Na/K ATPase in skeletal muscle is being examined in detail at both nucleic acid and protein levels. In this study, four mechanisms of regulation have been identified: these occur at the level of transcription, subunit assembly, incorporation into plasma membrane, and protein turnover. The underlying mechanisms are being investigated. Fourth, a number of structure/function and structure/bioregulation relationships are being examined through expression of avian Na/K ATPase subunits in mouse L cell and myogenic cell lines, including studies on the nature of the cardiac glycoside binding site, the requirements for subunit assembly, and targeting to plasma membrane. Fifth, plasma membrane dynamics during nerve growth are being studied in tissue cultured sensory neurons, with focus upon determining the major sites of insertion of newly synthesized Na/K ATPase molecules in relation to the growth cone and extending axons. Sixth, the Na/K ATPase of Drosophila is being studied with particular attention to roles of the Na/K ATPase in early development and possible behavioral consequences of Na/K ATPase mutations.

A symposium on Molecular Evolution of Physiological Processes will be held in September, 1993 at the Marine Biological Laboratory in Woods Hole, Massachusetts, under the auspices of the Society of General Physiologists. This Symposium will bring together scientists who study cytoskeletal and membrane functions with scientists whose research is focused upon molecular evolution or on mechanisms that generate genetic diversity. Four lecture sessions will set the intellectual framework of the symposium: (1) approaches to analyzing nucleic acid and protein sequences and applications to phylogenetic problems; (2) recently appreciated genetic mechanisms that may underlie the evolution of increasingly complex biological systems; (3) proteins of the actin/myosin cytoskeleton: the variety of isoforms and the correlated functions these play in cell structure and behavior; and (4) structure, function and evolution of plasma membrane proteins. Sessions 3 and 4 emphasize that molecular-level studies of physiological processes are very profitably informed by ideas developed in the fields of molecular evolution and genetics, and that conserved structural motifs characterize superfamilies of proteins that may function through homologous mechanisms. There will also be poster sessions and open discussion/workshops on key topics. Keynote addresses will highlight perspectives from immunology and integrative biology. %%% A symposium on Molecular Evolution of Physiological Processes will be held in September, 1993 at the Marine Biological Laboratory in Woods Hole, Massachusetts, under the auspices of the Society of General Physiologists. This Symposium will bring together scientists who study cytoskeletal and membrane functions with scientists whose research is focused upon molecular evolution or on mechanisms that generate genetic diversity. A major emphasis of the conference is that molecular-level studies of physiological processes can be illuminated by ideas developed in the fields of molecular evolution and genetics.