David H. MacLennan, Ph.D. - US grants

calcium transporting ATPase; adenosinetriphosphatase; point mutation;

The long term objective is to understand the mechanism whereby the Ca2+ - ATPase of sarcoplasmic reticulum transports Ca2+ to luminal spaces. The expression and mutagenesis of full length cDNA encoding the Ca2+ -ATPase, followed by assay of the function of the product, is being used to address questions of structure/function relationships in the protein. Further research is proposed under four headings: I. Development of an expression system for large scale production of normal and mutant forms of the Ca2+ -pump: In order to complement the present expression/mutagenesis system in which Ca-ATPase cDNA is expressed in COS-1 cells, development of a Baculovirus expression system that will allow production of large amounts of protein is proposed. Purification of the expressed mutant Ca2+-ATPase protein in one step using a monoclonal antibody affinity column with the IgM antibody A20 is also proposed. II. Determination of the topology of the transmembrane domain of the Ca2+ -ATPase. Luminal and cytoplasmic epitopes in the Ca2+-ATPase have been distinguished with monoclonal antibodies against defined sequences. This analysis will be extended to other sequences predicted to lie on lumenal or cytoplasmic sides. Alternatively, a specific epitope from the fast-twitch Ca2+-ATPase will be inserted at various sites in the sequence of the slow- twitch Ca2+-ATPase in order to determine topology. The boundaries of membrane spanning sequences will be determined by insertion of basic clusters at each end. III. Site-specific mutagenesis to define amino acids critical to Ca2+ transport. Mutation is proposed of: (a) residues in transmembrane sequences 4,5,6 and 8 to determine how they relate to Ca2+ binding and Ca2+ transport; (b) residues throughout the proposed nucleotide binding domain to identify those involved in ATP binding; (c) residues potentially involved in conformational changes in the molecule; (d) residues that will permit measurement of spatial arrangements in the Ca2+-ATPase; (e) Gly, Pro, Cys and Trp residues throughout the molecule. IV. Kinetic analysis of mutant function. Since many mutants retain partial reactions of Ca2+ transport, equilibrium and kinetic analyses of these partial reactions will be carried out. Ca2+ and ATP binding constants, rates of formation and breakdown of phosphorylated intermediates from ATP and Pi, and rates for overall Ca2+ transport will be measured where possible.

The long term objective is to understand the mechanism of Ca2+ transport by the Ca2+ ATPase of sarcoplasmic reticulum. Expression and mutagenesis of full length or partial cDNAs encoding the Ca2+ ATPase, followed by assay of the function of the product, is being used to address questions of structure/function relationships in the protein. Research is proposed under six headings: 1. Determination of the roles of transmembrane amino acids in Ca2+ binding. The mutation, E309Q, abolishes one of two Ca2+ binding sites and this defective site is 'stacked' on the cytoplasmic surface of the membrane. The demonstration that other Ca2+ binding and Ca2+ affinity mutations also abolish one of two Ca2+ binding sites and are 'stacked' on either cytoplasmic or lumenal surfaces is proposed. 2. Analysis of Ca2+ binding 'patches' in transmembrane sequences. Analysis of functional consequences of mutations throughout all of M4 has revealed an active Ca2+ binding patch. The demonstration of similar active 'patches" in each of M5, M6 and M8 and use of the information to refine models for the Ca2+ binding sites is proposed. 3. Determination of the topology of Mg/M10. The hypothesis that Mg and M10 are transmembrane sequences will be tested by investigation of their location and function. 4. Expression, purification and crystallization of cytoplasmic domains. The cytoplasmic sequence between N330 and D738, which can expressed stably in E. coli and which binds TNP-ATP, will be purified in sufficient quantity to initiate attempts at crystallization for high resolution X- ray diffraction analysis of the structures of domains contained in the sequence. 5. Identification of ATP binding residues by measurement of TNP-ATP binding. TNP-ATP binding to wild-type and mutant ATPases, will be developed as a more definitive test for the involvement of specific residues in ATP binding. The hypothesis that the B-strand domain plays a functional role in energy transduction will be tested using mutants and chimeras. 6. Determination of a potential regulatory role for acidic residues in stalk sector 2. Chimera formation reveals regions in the nucleotide binding/hinge domain that affect Ca2+ affinity, while mutation of a group of acidic residues in stalk sector 2 also results in lowered Ca2+ affinity. The hypothesis that this acidic stalk sequence interacts with sequences in other domains to control Ca2+ affinity will be tested.

DESCRIPTION (Taken from the applicants abstract): This is a proposal for partial funding of a Gordon Research Conference on Muscle: Excitation-Contraction (E-C) Coupling to be held in June, 2000. E-C coupling is the process that translates a muscle action potential into an increase in [Ca2+]i and contraction. Recent advances have brought into sharp focus the necessity of understanding the molecular events that underlie E-C coupling. In particular, the interaction between the two sarcotubular molecules that are key to E-C coupling. The Ca release channel ryanodine receptor (RyR) and the voltage sensor or dihydropyridine receptor (DHPR). The meeting will bring together specialists in membrane protein structure, human genetics, molecular biology, biochemistry, biophysics and physiology to discuss the approaches that will advance molecular understanding of Ca signaling in skeletal and cardiac muscle, with implications for other tissues. The problems dicussed by this diverse group will become more sharply defined as high resolution structures of the molecules under study become available. The meeting will focus discussion on several key Ca signaling molecules in skeletal and cardiac muscle in nine platform sessions: session 1, molecular structures of key proteins; session 2, molecular basis and pathophysiology of inherited muscle diseases caused by defects in E-C coupling proteins; session 3, structure-function relationships in RyR; session 4, interactions between RyR and DHPR; sessions 5 and 6, modulation of RyR activity by other proteins and transmembrane gradients and pharmacological agents: session 7, determination of elementary events of Ca release; session 8, global aspects of Ca release; and session 9, diversity among Ca release channels and novel proteins. Posters will be shown continuously. The Gordon Conference on Muscle: E-C coupling, held every three years for several decades, has consistently been the sole forum in which active investigators from all over the world are brought together for discussion and dissemination of ideas that have led to advances in this field. The format encourages the exchange of ideas and unpublished data. The organizing committee, with representatives of varying ages from five continents, will strive to achieve appropriate representation of women. minorities and disabled.