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(Received for publication, June 26, 1996, and in revised form, January 15, 1997)
From the ‡ Renal Division, Department of Medicine,
Brigham and Women's Hospital and Harvard Medical School, and
Department of Biological Chemistry and Molecular Pharmacology, Harvard
Medical School, Boston, Massachusetts 02115 and the ¶ Yale
University, Department of Cellular and Molecular Physiology, School of
Medicine, New Haven, Connecticut 06510
Ion-coupled solute transporters exhibit
pre-steady-tate currents that resemble those of
voltage-dependent ion channels. These currents were assumed
to be mostly due to binding and dissociation of the coupling ion near
the extracellular transporter surface. Little attention was given to
analogous events that may occur at the intracellular surface. To
address this issue, we performed voltage clamp studies of
Xenopus oocytes expressing the intestinal H+-coupled peptide cotransporter PepT1 and recorded the
dependence of transient charge movements in the absence of peptide
substrate on changing intra- (pHi) and extracellular pH
(pHo). Rapid steps in membrane potential induced transient
charge movements that showed a marked dependence on pHi and
pHo. At a pHo of 7.0 and a holding potential
(Vh) of
Volume 272, Number 12,
Issue of March 21, 1997
pp. 7777-7785
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
50 mV, the charge movements were mostly inwardly
directed, whereas reduction of pHo to below 7.0 resulted in
outwardly directed charge movements. When pHi was reduced,
inwardly directed charge movements were observed. The data on the
voltage dependence of the transient charge movements were fitted by the
Boltzmann equation, yielding an apparent valence of 0.65 ± 0.03 (n = 7). The midpoint voltage (V0.5) of
the charge distribution shifted linearly as a function of
pHi and pHo. Our results indicate that, as a
first approximation, the magnitude and polarity of the transient charge
movements depend upon the prevailing H+ electrochemical
gradient. We propose that PepT1 has a single proton binding site that
is symmetrically accessible from both sides of the membrane and that
decreasing the H+ chemical potential (
µH)
or increasing the membrane potential (Vm)
shifts this binding site from an outwardly to an inwardly facing
occluded state. This concept constitutes an important extension of
previous kinetic models of ion-coupled solute transporters by including
a more detailed description of intracellular events.
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