Serotonin, l‐tryptophan, and tryptamine are effective inhibitors of the amino acid transport system PAT1

Abstract

The proton-coupled amino acid transporter PAT1, cloned recently from brain and intestine, mediates the uphill transport of l- and d-proline, l-alanine, glycine, taurine, d-serine, GABA, and many other related compounds and drugs. Here we describe the novel finding that l-tryptophan and its derivatives tryptamine, 5-hydroxy-l-tryptophan, serotonin, and indole-3-propionic acid strongly inhibit H+-dependent l-[3H]proline uptake into Caco-2 cells with inhibition constants (K(i)) of 0.9 to 6.1 mM. Uptake of l-[3H]tryptophan into Caco-2 cells on the other hand was not inhibited by l-proline. Whereas PAT1 substrates produced significant changes in a membrane potential assay for electrogenic transport in Caco-2 cells, l-tryptophan, tryptamine, and 5-hydroxy-l-tryptophan failed to alter membrane voltage. When PAT1 was expressed in Xenopus laevis oocytes and analyzed by the two-electrode voltage clamp technique, glycine elicited high inward currents that were dependent on membrane potential but no currents were observed with l-tryptophan, tryptamine, 5-hydroxy-l-tryptophan, or serotonin. Although not transported electrogenically by PAT1, l-tryptophan and its derivatives inhibited glycine-evoked currents dose-dependently. We conclude that serotonin, l-tryptophan, and tryptamine bind to PAT1 with potencies similar to the prototype substrates, inhibit transport function but are not transported by this carrier protein. They may be considered as the carriers' naturally occurring inhibitors that may alter the transport function of PAT1.

Identification of a Disulfide Bridge Essential for Transport Function of the Human Proton-coupled Amino Acid Transporter hPAT1

“…The Michaelis constants obtained for the glycine transport were 11.4 Ϯ 1.2 mM for hPAT1 and 12.4 Ϯ 1.7 mM for HA-hPAT1. The transport characteristics of hPAT1 and HAhPAT1 expressed in X. laevis oocytes correspond very well to previously published data on mouse and human PAT1 transport function (3,(33)(34)(35). Furthermore, these data show that the introduction of an HA tag at the N terminus of hPAT1 does neither alter the transport activity nor the apparent affinity of hPAT1-mediated transport.…”

PAT1 (SLC36A1) shows nuclear localization and affects growth of smooth muscle cells from rats

“…The cross-sectional N-PAT staining illustrates the localization of N-PAT1 reactivity throughout the nucleus. The immunocytochemical stainings were repeated with three different passages (17,19, and 20) of A7r5 cells and showed similar results when they were grown on permeable polycarbonate filters. No morphological changes or redistribution of proteins of interest were observed when cells were grown on collagen-coated coverslips compared with polycarbonate filters (not shown).…”

“…The last exon encodes the COOH terminus of the protein as well as a large 3=-untranslated region (7). Transport of proline, alanine, glycine, and GABA via PAT1 is proton coupled and sodium independent (30 -32) and may be inhibited by tryptophan and 5-hydroxytryptophan (5-HTP) and some dipeptides (13,19). Attempts to map modifications and amino acids in PAT1 with influence on transport activity has led to identification of an extracellular disulfide bridge (10) and a histidine residue probably localized within the protein binding site involved in the proton-coupled PAT1-mediated transport (20).…”

Transport of the Photodynamic Therapy Agent 5-Aminolevulinic Acid by Distinct H+-Coupled Nutrient Carriers Coexpressed in the Small Intestine

“…5-Hydroxy-L-tryptophan (OH-Trp) has been proposed as a nontransported inhibitor of PAT1 (Metzner et al, 2005). Uptake of [ 3 H]-␤-alanine by PAT1 expressed in oocytes was inhibited in a concentration-dependent manner by OH-Trp (IC 50 ϭ 1.2 Ϯ 0.3 mM) ( Fig.…”