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Expression of 5-HT1C receptors in transfected MDCK cells is functionally and anatomically asymmetric
1Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Abstract:
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.
Insights
Madin-Darby canine kidney cells stably express rat 5-HT1C receptors, showing an asymmetric response to serotonin (5-HT). These receptors are targeted to the basolateral membrane, demonstrating polarized expression in epithelial cells.
Area of Science:
- Cell biology
- Molecular pharmacology
- Epithelial cell biology
Background:
- G protein-linked receptors mediate cellular responses to signaling molecules.
- Intracellular calcium ([Ca2+]i) is a critical second messenger in cellular signaling.
- Madin-Darby canine kidney (MDCK) cells are a widely used model for studying epithelial cell biology.
Purpose of the Study:
- To establish a stable cell line expressing the rat 5-HT1C receptor in MDCK cells.
- To investigate the functional expression and membrane targeting of the 5-HT1C receptor in epithelial cells.
- To determine if G protein-linked receptors can be asymmetrically targeted in transfected epithelial cells.
Main Methods:
- Transfection of MDCK cells with rat 5-HT1C receptor cDNA.
- Fluorescence-activated cell sorting (FACS) for selection of responsive cells.
- Measurement of intracellular calcium ([Ca2+]i) changes using indo-1 fluorescence.
- Assessment of receptor localization using radioligand binding assays.
Main Results:
- A stable clone (SR-MDCK) expressing 100% serotonin (5-HT)-responsive 5-HT1C receptors was generated.
- SR-MDCK cells exhibited an increase in [Ca2+]i only upon basolateral, not apical, application of 5-HT.
- High-affinity 5-HT receptor binding sites were localized exclusively to the basolateral membrane.
Conclusions:
- Epithelial cells can be stably transfected to express functional G protein-linked, calcium-mobilizing receptors.
- The 5-HT1C receptor is asymmetrically targeted to the basolateral membrane domain in SR-MDCK cells.
- This study demonstrates the feasibility of generating epithelial cell models with polarized expression of specific membrane receptors.
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