Little research has been carried out into the acute regulation of the epithelial H+/oligopeptide transporter, PepT1. However, one earlier study has shown downregulation of PepT1 activity in Caco-2 cells by activation of protein kinase C, but the exact mechanism by which it occurs remains unresolved (Brandsch et al. 1994). In this study we have investigated electrophysiologically the regulation of PepT1 using the activator of protein kinase C, PMA (phorbol 12-myristate, 13-acetate) and the inactive phorbol ester 4αPDD (4α-phorbol 12, 13-didecanoate) in PepT1-expressing Xenopus laevis oocytes.
The two-electrode voltage clamp was employed in PepT1-expressing oocytes to record peptide-induced currents in response to the neutral peptide Gly-Gln before and after treatment with PMA and 4αPDD.
Following incubation in PMA the Km for Gly-Gln decreased 2-fold, 0.6 ± 0.08 mM compared with 0.3 ± 0.05 mM (mean ± S.E.M., n = 4). Vmax decreased 2.2-fold upon treatment with PMA, 920.2 ± 39.3 nA compared with 422.7 ± 16.8 nA (mean ± S.E.M., n = 4). Values are statistically significant using Student’s t test with P < 0.05 for Km values and P < 0.001 for Vmax values. Control experiments using 4αPDD showed no change in Km or Vmax (see Fig. 1B).
We conclude that PepT1 is under the regulatory control of protein kinase C. PMA downregulates PepT1 activity under conditions when driving forces are not altered, suggesting that phosphorylation of the PepT1 protein itself may alter the Km and Vmax.
We thank M.A. Hediger for the PepT1 clone and The Wellcome Trust for financial support.
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| Figure 1. Steady-state current-voltage (I-V) relationships were measured in the presence of Gly-Gln in oocytes expressing rabbit PepT1 before and after (a) PMA or (b) 4αPDD treatment. Membrane potential was held at -50 mV and stepped to test voltages between +50 and -150 mV in 10 mV increments. Imax for the peptide-induced currents at pHo 7.5 were plotted as a function of substrate concentration (n = 4, mean ± S.E.M.). Solutions contained (mM): NaCl 95, KCl 2, CaCl2 1, MgCl2 1 and Hepes 20, pH adjusted with Tris, plus the appropriate concentration of Gly-Gln. Oocytes were incubated with 10 nM PMA or 4αPDD for 30 min. |
- Brandsch, M., Miyamoto, Y., Ganapathy, V. & Leibach, F. (1994). Biochem. J. 299, 253-260.