Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1

Abstract

PAT1 is a recently identified member of the PAT family of proton/amino acid co-transporters with predominant expression in the plasma membrane of enterocytes and in lysosomal membranes of neurons. Previous studies in Xenopus oocytes expressing PAT1 established proton/substrate co-transport associated with positive inward currents for a variety of small neutral amino acids. Here we provide a detailed analysis of the transport mode of the murine PAT1 in oocytes using the two-electrode voltage-clamp technique to measure steady-state and pre-steady-state currents. The GPC (giant patch clamp) technique and efflux studies were employed to characterize the reversed transport mode. Kinetic parameters [K(m) (Michaelis constant) and I(max) (maximum current)] for transport of various substrates revealed a dependence on membrane potential: hyperpolarization increases the substrate affinity and maximal transport velocity. Proton affinity for interaction with PAT1 is almost 100 nM, corresponding to a pH of 7.0 and is independent of substrate. Kinetic analysis revealed that binding of proton most likely occurs before substrate binding and that the proton and substrate are translocated in a simultaneous step. No evidence for a substrate-uncoupled proton shunt was observed. As shown by efflux studies and current measurements by the GPC technique, PAT1 allows bidirectional amino acid transport. Surprisingly, PAT1 exhibits no pre-steady-state currents in the absence of substrate, even at low temperatures, and therefore PAT1 takes an exceptional position among the ion-coupled co-transporters.

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.…”

Characterization of a blood-meal-responsive proton-dependent amino acid transporter in the disease vector, Aedes aegypti

“…In previously identified PATs from mice and humans (Boll et al, 2002), it was hypothesized that VATPases and sodium-hydrogen exchangers (NHEs) may create a proton gradient (Boll et al, 2003a;Foltz et al, 2004;Foltz et al, 2005). The resulting proton gradient developed can then be used as a driving force for amino acid transport.…”

Mechanisms of Net Chloride Secretion During Rotavirus Diarrhea in Young Rabbits : Do Intestinal Villi Secrete Chloride?

“…4), strongly indicating that Cl -influx and efflux both involve the same carrier system across these membranes. The capability for bidirectional transport, in both normal (outside to inside) and reversed (inside to outside) modes, has been recently demonstrated for SGLT1 [42,43], the H + /peptide carrier PEPT1 [44] and the H + /amino acid symporter PAT1 [45], each expressed in Xenopus laevis oocytes. While all these symporters are known to function as absorptive carriers in small intestinal epithelial cells, the physiological significance of their reverse transport mode is currently not known.…”