title: Single channel function of inositol 1,4,5-trisphosphate receptor type-1 and -2 isoform domain-swap chimeras.
abstract: The InsP3R proteins have three recognized domains, the InsP3-binding, regulatory/coupling, and channel domains (Mignery, G.A., and T.C. Südhof. 1990. EMBO J. 9:3893-3898). The InsP3 binding domain and the channel-forming domain are at opposite ends of the protein. Ligand regulation of the channel must involve communication between these different regions of the protein. This communication likely involves the interceding sequence (i.e., the regulatory/coupling domain). The single channel functional attributes of the full-length recombinant type-1, -2, and -3 InsP3R channels have been defined. Here, two type-1/type-2 InsP3R regulatory/coupling domain chimeras were created and their single channel function defined. One chimera (1-2-1) contained the type-2 regulatory/coupling domain in a type-1 backbone. The other chimera (2-1-2) contained the type-1 regulatory/coupling domain in a type-2 backbone. These chimeric proteins were expressed in COS cells, isolated, and then reconstituted in proteoliposomes. The proteoliposomes were incorporated into artificial planar lipid bilayers and the single-channel function of the chimeras defined. The chimeras had permeation properties like that of wild-type channels. The ligand regulatory properties of the chimeras were altered. The InsP3 and Ca2+ regulation had some unique features but also had features in common with wild-type channels. These results suggest that different independent structural determinants govern InsP3R permeation and ligand regulation. It also suggests that ligand regulation is a multideterminant process that involves several different regions of the protein. This study also demonstrates that a chimera approach can be applied to define InsP3R structure-function.
authors.affiliation: Department of Physiology, Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153, USA.
authors.forename: Jorge
authors.initials: J
authors.lastname: Ramos
authors.forename: Wonyong
authors.initials: W
authors.lastname: Jung
authors.forename: Josefina
authors.initials: J
authors.lastname: Ramos-Franco
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Mignery
authors.forename: Michael
authors.initials: M
authors.lastname: Fill
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000737:chemistry; D015220:Calcium Channels / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D015151:Immunoblotting; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000737:chemistry; D053496:Inositol 1,4,5-Trisphosphate Receptors; D015640:Ion Channel Gating; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010539:Permeability; D020033:Protein Isoforms; D011510:Proteolipids / Q000737:chemistry; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014162:Transfection
chemical_list: D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D008051:Lipid Bilayers; D020033:Protein Isoforms; D011510:Proteolipids; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; C020485:proteoliposomes; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium