Cloned Catecholamine Transporters Expressed in Polarized Epithelial Cells: Sorting, Drug Sensitivity, and Ion-Coupling Stoichiometry

Abstract

Publisher Summary This chapter discusses a project in which researchers generated nine cell lines. In the pig kidney cell line (LLC-PK 1 ), rGAT-1, hNET, rSERT, and rDAT were transfected. In Modin-Darby Canine Kidney (MDCK) cells, transfecting those same cDNAs and also hDAT was possible. The transport characteristics and inhibitor sensitivities for the cell lines were consistent with what was known for each transporter in vivo and in other heterologous expression systems. The advantage of stably transfecting the three biogenic amine transporters into the same cellular background was that it allowed us to compare in detail the catalytic and pharmacological properties of these proteins. A detailed understanding of biogenic amine transport mechanism requires knowing how many Na + and Cl - ions are cotransported, and K + ions counter-transported, with each molecule of substrate. This ion coupling stoichiometry is difficult to determine using rate measurements in intact cells. Stably transfected LLC-PK1 cells also were useful for characterizing the amphetamine specificity of SERT, NET, and DAT. In these cell lines, amphetamine and its derivatives inhibited biogenic amine influx and caused efflux of previously accumulated amine. These effects are likely to underlie amphetamine-induced biogenic amine release in vivo .