Expression of 5-HT1C receptors in transfected MDCK cells is functionally and anatomically asymmetric.
Madin-Darby canine kidney (MDCK) cells were transfected with the cDNA for the rat 5-HT1C receptor (pMV7-SR1c) using electroporation. Cells that survived G418 selection medium were loaded with indo-1 and run through a fluorescence-activated cell sorter (FACS); 10% responded to serotonin (5-HT) with an increase in intracellular Ca2+ concentration ([Ca2+]i). Responding cells were separated with the FACS, grown to confluence, and resorted two more times until a clone of 100% respondents was obtained (SR-MDCK). In SR-MDCK cells grown on porous filters, [Ca2+]i increased only when 5-HT was applied to the basolateral membrane (change in [Ca2+]i = 190 +/- 43 nM); there was no response of [Ca2+]i to apical application of 5-HT. The asymmetric response to 5-HT was likely due to targeting of 5-HT1C receptors exclusively to the basolateral membrane of SR-MDCK cells; 125I-labeled lysergic acid diethylamide binding sites, a marker of high-affinity 5-HT receptors, were located only in the basolateral membrane. These experiments demonstrate that epithelial cells can be stably transfected to express G protein-linked, calcium-mobilizing receptors and that the receptors may be targeted asymmetrically to specific domains of the plasma membrane.
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Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032.
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D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D016764:Cell Polarity; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D011985:Receptors, Serotonin / Q000235:genetics* / Q000378:metabolism*; D014018:Tissue Distribution; D014162:Transfection
D011985:Receptors, Serotonin