Mechanisms of the human intestinal H+-coupled oligopeptide transporter hPEPT1.
The hPEPT1 cDNA cloned from human intestine (Liang, R., Fei, Y.-J., Prasad, P. D., Ramamoorthy, S., Han, H., Yang-Feng, T. L., Hediger, M. A., Ganapathy, V., and Leibach, F. H. (1995) J. Biol. Chem. 270, 6456-6463) encodes a H+/oligopeptide cotransporter. Using two-microelectrode voltage-clamp in Xenopus oocytes expressing hPEPT1, we have investigated the transport mechanisms of hPEPT1 with regard to voltage dependence, steady-state kinetics, and transient charge movements. The currents evoked by 20 mM glycyl-sarcosine (Gly-Sar) at pH 5.0 were dependent upon membrane potential (Vm) between -150 mV and +50 mV. Gly-Sar-evoked currents increased hyperbolically with increasing extracellular [H+], with Hill coefficient approximately 1, and the apparent affinity constant (K0.5H) for H+ was in the range of 0.05 1 microM. K0.5 for Gly-Sar (K0.5GS) was dependent upon Vm and pH; at -50 mV, K0.5H was minimal (approximately 0.7 mM) at pH 6.0. Following step-changes in Vm, in the absence of Gly-Sar, hPEPT1 exhibited H+-dependent transient currents with characteristics similar to those of Na+-coupled transporters. These charge movements (which relaxed with time constants of 2-10 ms) were fitted to Boltzmann relations with maximal charge (Qmax) of up to 12 nC; the apparent valence was determined to be approximately 1. Qmax is an index of the level of transporter expression which for hPEPT1 was in the order of 1011/oocyte. In general our data are consistent with an ordered, simultaneous transport model for hPEPT1 in which H+ binds first.
Mackenzie
B
B
Department of Physiology, UCLA School of Medicine, Los Angeles, California 90095-1751, USA.
Loo
D D
DD
Fei
Y
Y
Liu
W J
WJ
Ganapathy
V
V
Leibach
F H
FH
Wright
E M
EM
D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004151:Dipeptides / Q000494:pharmacology; D005071:Evoked Potentials / Q000187:drug effects; D005260:Female; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008564:Membrane Potentials / Q000502:physiology; D008962:Models, Theoretical; D009842:Oligopeptides / Q000378:metabolism*; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D000074011:Peptide Transporter 1; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D027981:Symporters; D014982:Xenopus laevis
D002352:Carrier Proteins; D018076:DNA, Complementary; D004151:Dipeptides; D009842:Oligopeptides; D000074011:Peptide Transporter 1; D011994:Recombinant Proteins; C082186:SLC15A1 protein, human; D027981:Symporters; C004194:glycylsarcosine