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Single Channel Function of Inositol 1,4,5-trisphosphate Receptor Type-1 and -2 Isoform Domain-Swap Chimeras
The InsP(3)R proteins have three recognized domains, the InsP(3)-binding, regulatory/coupling, and channel domains (Mignery, G.A., and T.C. Südhof. 1990. EMBO J. 9:3893–3898). The InsP(3) binding domain and the channel-forming domain are at opposite ends of the protein. Ligand regulation of the chan...
Autores principales: | , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
The Rockefeller University Press
2003
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2217376/ https://www.ncbi.nlm.nih.gov/pubmed/12695486 http://dx.doi.org/10.1085/jgp.200208718 |
Sumario: | The InsP(3)R proteins have three recognized domains, the InsP(3)-binding, regulatory/coupling, and channel domains (Mignery, G.A., and T.C. Südhof. 1990. EMBO J. 9:3893–3898). The InsP(3) 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 InsP(3)R channels have been defined. Here, two type-1/type-2 InsP(3)R 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 InsP(3) and Ca(2+) regulation had some unique features but also had features in common with wild-type channels. These results suggest that different independent structural determinants govern InsP(3)R 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 InsP(3)R structure-function. |
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