Di and tripeptide (di/tripeptide) absorption in the intestine occurs via PepT1, an electrogenic transporter that uses an inwardly-directed proton electrochemical gradient to drive the transport of more than 8,000 different molecules inside the cells. Studies in mammals indicate that PepT1 also exhibits peptide sensing function(s). In teleost fish, two genes, namely PepT1b and more recently PepT1a, have been identified. The two paralogues – the result of (at least one) round of whole-genome duplication during the early evolution of the ray-finned fish lineage – encode proteins that share 64-67% similarity at the amino acid level. While PepT1b has been widely studied in several teleost fish species, including S. salar, PepT1a function is fully unknown to date. The aim of this work was to characterize and compare S. salar PepT1a and Pept1b after heterologous expression in Xenopus laevis oocytes, and study their role(s) in peptide transport and/or sensing. Injection of cRNAs into X. laevis oocytes allowed high functional “in membrane” expression of both proteins. Function was verified by Two Electrode Voltage Clamp (TEVC), as currents elicited by perfusion of di/tripeptides were regularly recorded for both proteins. Notably, ours is the first experimental evidence that PepT1a is an electrogenic transporter of di/tripeptides. Measuring the transport currents at two different pH (6.5 and 7.6) and in the presence of increasing concentrations of glycine-L-glutamine (GQ) (from 0.01 to 30 mM) allowed to investigate kinetic values, as the maximal current (Imax) and the substrate apparent affinity (K0.5), for each transporter and pH. Results suggest that the two transporters interact differently with the substrate and that the external pH influence the substrate affinity and consequently the transport efficiency, which is definitely lower for PepT1a with respect to PepT1b. Moreover, preliminary results suggest that L-lysine-containing substrates elicit transport-associated currents of different amplitude in the two isoforms. Large currents are recorded from PepT1b in the presence of L-lysine-glycine (KG), and currents similar to GQ are recorded for glycine-L-lysine (GK); conversely, small responses for both substrates are obtained in PepT1a. Overall, these data support the idea of a different role of the two isoforms, opening the possibility to investigate on PepT1a as a “transceptor” involved in nutrient sensing. Daniel, H. & Zietek, T. Experimental physiology 100, 1441-1450, (2015). Diakogiannaki, E. et al. Diabetologia 56, 2688-2696, (2013). Romano, A., Barca, A., Storelli, C. & Verri, T. J.Physiol 592, 881-897 (2014). Ronnestad, I. et al. J.Nutr. 140, 893-900 (2010). Sangaletti, R. et al. Pflugers Arch. (2009).
Comparison of the electrophysiological properties of the two paralogues of Salmo salar oligopeptide transporter PepT1: new insights from PepT1a vs. PepT1b
Vacca F;Barca A;Verri T;
2017-01-01
Abstract
Di and tripeptide (di/tripeptide) absorption in the intestine occurs via PepT1, an electrogenic transporter that uses an inwardly-directed proton electrochemical gradient to drive the transport of more than 8,000 different molecules inside the cells. Studies in mammals indicate that PepT1 also exhibits peptide sensing function(s). In teleost fish, two genes, namely PepT1b and more recently PepT1a, have been identified. The two paralogues – the result of (at least one) round of whole-genome duplication during the early evolution of the ray-finned fish lineage – encode proteins that share 64-67% similarity at the amino acid level. While PepT1b has been widely studied in several teleost fish species, including S. salar, PepT1a function is fully unknown to date. The aim of this work was to characterize and compare S. salar PepT1a and Pept1b after heterologous expression in Xenopus laevis oocytes, and study their role(s) in peptide transport and/or sensing. Injection of cRNAs into X. laevis oocytes allowed high functional “in membrane” expression of both proteins. Function was verified by Two Electrode Voltage Clamp (TEVC), as currents elicited by perfusion of di/tripeptides were regularly recorded for both proteins. Notably, ours is the first experimental evidence that PepT1a is an electrogenic transporter of di/tripeptides. Measuring the transport currents at two different pH (6.5 and 7.6) and in the presence of increasing concentrations of glycine-L-glutamine (GQ) (from 0.01 to 30 mM) allowed to investigate kinetic values, as the maximal current (Imax) and the substrate apparent affinity (K0.5), for each transporter and pH. Results suggest that the two transporters interact differently with the substrate and that the external pH influence the substrate affinity and consequently the transport efficiency, which is definitely lower for PepT1a with respect to PepT1b. Moreover, preliminary results suggest that L-lysine-containing substrates elicit transport-associated currents of different amplitude in the two isoforms. Large currents are recorded from PepT1b in the presence of L-lysine-glycine (KG), and currents similar to GQ are recorded for glycine-L-lysine (GK); conversely, small responses for both substrates are obtained in PepT1a. Overall, these data support the idea of a different role of the two isoforms, opening the possibility to investigate on PepT1a as a “transceptor” involved in nutrient sensing. Daniel, H. & Zietek, T. Experimental physiology 100, 1441-1450, (2015). Diakogiannaki, E. et al. Diabetologia 56, 2688-2696, (2013). Romano, A., Barca, A., Storelli, C. & Verri, T. J.Physiol 592, 881-897 (2014). Ronnestad, I. et al. J.Nutr. 140, 893-900 (2010). Sangaletti, R. et al. Pflugers Arch. (2009).I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
