Measurement of electric current evoked by substrate transport via bi-directional H+/oligopeptide transporter over-expressed in HeLa cells: electrogenic efflux and existence of a newly observed channel-like state.
In the present study, we measured an electric current induced by substrate transport in a HeLa cell over-expressing a human intestinal di/tri-peptide transporter using the whole-cell patch-clamp technique. Gly-Sar, a typical substrate, induced an inward current associated with its uptake, which showed concentration-dependency following Michaelis-Menten-type kinetics with an apparent K(0.5) of 1.3mM as well as voltage-dependency. An outward current accompanying the efflux of Gly-Sar was also observed after washing out the cell. This outward current was voltage-dependent and was reduced by the inward proton gradient. In the case of hydrophobic dipeptides such as Gly-Phe and Gly-Leu, a distinctive current was observed: after washing out the cells, no outward current was observed, but rather, an 'inward leak' current was sustained in spite of the absence of transportable substrate. This leaky current was abolished by the perfusion of Gly-Sar and subsequent washing. It is considered that the hydrophobic substrate sticks within the substrate-binding site and causes the newly observed state, or the 'inward leak' current.
Fujisawa
Yuki
Y
Laboratory of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Japan.
Kitagawa
Taku
T
Miyake
Michihisa
M
Nara
Toshifumi
T
Kamo
Naoki
N
Miyauchi
Seiji
S
D004151:Dipeptides / Q000378:metabolism*; D004594:Electrophysiology; D006367:HeLa Cells; D006801:Humans; D017136:Ion Transport; D018408:Patch-Clamp Techniques; D000074011:Peptide Transporter 1; D027981:Symporters / Q000502:physiology*
D004151:Dipeptides; D000074011:Peptide Transporter 1; C082186:SLC15A1 protein, human; D027981:Symporters