This R21 exploratory research grant responds to the NIGMS call for the development of novel eukaryotic expression systems capable of generating pure membrane proteins in sufficient quantities to afford crystallization trials. The lack of such a system has critically limited the number of structural studies of integral membrane proteins, such as ion channels and transporters, with atomic level resolution. The current application aims to cope with this problem by investigating the potential use of mammalian skeletal muscle to produce large quantities of recombinant membrane proteins when transfected in vivo with genetically engineered DNA plasmids. We propose that skeletal muscle may be an ideal preparation not only for the massive synthesis of cytosolic proteins, but most importantly, for the production and exportation of large quantities of transmembrane proteins. We plan to pursue these ideas experimentally in two separate, but intrinsically connected specific aims.
Specific Aim 1 will test whether in vivo electroporation of muscle tissue with piasmids engineered for mammalian expression of six-histidine (6His) tagged EGFP and ECFP allows us to purify them to homogeneity (using standard chromatographic methods) in quantities compatible with crystallization protocols. With this knowledge, we will attempt to purify a very relevant cytosolic muscle protein, the beta-1a subunit of the skeletal muscle dihydropyridine receptor (DHPR), whose structure has not yet being determined with atomic resolution because of limitations in its synthesis.
In Specific Aim 2, we will first electroporate muscle with plasmids encoding fluorescently-tagged transmembrane proteins and invest gate which of the observed expression patterns for these proteins leads to an efficient membrane protein extraction protocol. We will focus on the expression of the following integral membrane proteins (ionic channels): the alpha-15 subunit of the skeletal muscle DHPR, the Shaker K channel, and the mammalian skeletal ryanodine receptor channel. Later on, plasmids will be engineered for the expression of 6His-tagged membrane proteins in order to ensure that each of them can be isolated and purified to homogeneity in sufficient amount to permit their crystallization. ? ?
| Capote, Joana; DiFranco, Marino; Vergara, Julio L (2010) Excitation-contraction coupling alterations in mdx and utrophin/dystrophin double knockout mice: a comparative study. Am J Physiol Cell Physiol 298:C1077-86 |
| DiFranco, Marino; Capote, Joana; Quinonez, Marbella et al. (2007) Voltage-dependent dynamic FRET signals from the transverse tubules in mammalian skeletal muscle fibers. J Gen Physiol 130:581-600 |
| Vergara, Julio L; Difranco, Marino (2006) Modulation by caffeine of calcium-release microdomains in frog skeletal muscle fibers. Biol Res 39:567-81 |
| DiFranco, M; Capote, J; Vergara, J L (2005) Optical imaging and functional characterization of the transverse tubular system of mammalian muscle fibers using the potentiometric indicator di-8-ANEPPS. J Membr Biol 208:141-53 |
