Stable expression of heterologous multisubunit protein complexes established by calcium phosphate- or lipid-mediated cotransfection.
For most of the studies conducted in our laboratory, we were interested in expressing the AChR in a nonmuscle cell background, in part, to distinguish inherent AChR properties from those contributed by its environment. The fibroblast host cells we used do not express endogenous AChRs, and although there can be considerable daily variability, for most of the studies, approximately 80% of the cells did not express any type of endogenous channel (S. Sine, personal observations, 1988). Thus, characterization of Torpedo AChRs in fibroblasts was simplified by not having endogenous channel expression. We have shown that the pharmacological and electrophysiological properties of AChRs appear fully correct, only alpha 2 beta gamma delta pentamers are expressed on the cell surface, AChRs expressed in fibroblasts can be regulated as they are in muscle cells by agents that increase intracellular levels of cAMP and the AChRs cluster in fibroblasts as they do in muscle cells in response to extracellularly added clustering factors. We have stably expressed Torpedo AChRs in NIH 3T3 and L fibroblasts as well as rat muscle L6 cells. Different transfectants express different levels of AChRs, with the numbers varying between about 2.5 x 10(4) and 1.5 x 10(6) surface AChRs per cell. In choosing a host cell for expressing a protein of interest, it is always prudent to characterize the line prior to transfection for expression of the homolog of the protein being introduced. When transfecting channel proteins, one might also wish to characterize the host cell for endogenous channel expression. Other considerations for selecting a host cell depend on the types of experiments that will be performed. For example, if fluorescent microscopic experiments will be performed, the ability of the cells to adhere to glass coverslips can be a very important consideration.
Claudio
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D000818:Animals; D002130:Calcium Phosphates / Q000494:pharmacology; D002478:Cells, Cultured; D008055:Lipids / Q000494:pharmacology; D011950:Receptors, Cholinergic / Q000096:biosynthesis* / Q000235:genetics; D014101:Torpedo / Q000502:physiology*; D014162:Transfection / Q000187:drug effects
D002130:Calcium Phosphates; D008055:Lipids; D011950:Receptors, Cholinergic; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous