Shreyasi Sarkar - November 4, 2019
Trace amine transport in human intestinal cells involves both passive and active processes
Trace amines are a class of endogenous compounds that regulate various physiological processes through selective activation of trace amine-associated receptor 1 (TAAR1). Recently, TAAR1 has been validated as a viable, safe target for human pharmacotherapy. TAAR1 has an intracellular localization, requiring that trace amines cross cell membranes to access the receptor. The Berry Lab has previously shown that trace amines readily diffuse across synthetic lipid bilayers, while passage across native neuronal membranes involved a transporter exhibiting the pharmacological profile of Organic Cation Transporter 2 (OCT2; Slc22A2). Since trace amines are also constituents of foodstuffs and can be produced by the intestinal microbiota, the Caco-2 human intestinal epithelial cell line was selected to determine if similar transport processes control diet and microbiota derived trace amines access across the intestinal cells. Using the endogenous TAAR1 ligand p-tyramine, it was shown that there are two distinct p-tyramine transport processes in Caco-2 cells. A facilitated diffusion-mediated process with the pharmacological characteristics of OCT2 is present in apical membranes, while the basolateral membrane contained an unidentified active transporter. Further characterization of this active transporter by replacing Na+ with choline on an equimolar basis, revealed the active transport was Na+-dependent. Kinetic analysis of this Na+-dependent active transport revealed Vmax = 43.0 nM/sec with a Kt = 33.1 nM. In order to identify this transporter, p-tyramine affinity columns have been prepared by crosslinking p-tyramine to N-hydroxysuccinimide activated sepharose columns. This has enabled successful isolation of p-tyramine binding proteins. Ongoing studies are validating these findings and scaling up procedures to facilitate sequencing of isolated proteins. Overall, the results of this study will identify transporters that may provide novel targets through which trace amine function can be regulated pharmacologically and provide an additional molecular basis for host-diet-microbiota interactions, and how the intestinal microbiota can affect human health.