Symmetry of H+ binding to the intra- and extracellular side of the H+- coupled oligopeptide cotransporter PepT1

Stephan Nussberger, Angela Steel, Davide Trottif, Michael F Romero, Walter F. Boron, Matthias A. Hediger

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

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- (pH(i)) and extracellular pH (pH(o)). Rapid steps in membrane potential induced transient charge movements that showed a marked dependence on pH(i) and pH(o). At a pH(o) of 7.0 and a holding potential (V(h)) of -50 mV, the charge movements were mostly inwardly directed, whereas reduction of pH(o) to below 7.0 resulted in outwardly directed charge movements. When pH(i) 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 pH(i) and pH(o). 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 (Δμ(H)) or increasing the membrane potential (V(m)) 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.

Original languageEnglish (US)
Pages (from-to)7777-7785
Number of pages9
JournalJournal of Biological Chemistry
Volume272
Issue number12
DOIs
StatePublished - 1997
Externally publishedYes

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Oligopeptides
Electric potential
Ions
Membranes
Binding Sites
Peptides
Boltzmann equation
Chemical potential
Charge distribution
Clamping devices
Ion Channels
Protons
Membrane Potentials
Kinetics
Substrates
Xenopus
Oocytes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Symmetry of H+ binding to the intra- and extracellular side of the H+- coupled oligopeptide cotransporter PepT1. / Nussberger, Stephan; Steel, Angela; Trottif, Davide; Romero, Michael F; Boron, Walter F.; Hediger, Matthias A.

In: Journal of Biological Chemistry, Vol. 272, No. 12, 1997, p. 7777-7785.

Research output: Contribution to journalArticle

Nussberger, Stephan ; Steel, Angela ; Trottif, Davide ; Romero, Michael F ; Boron, Walter F. ; Hediger, Matthias A. / Symmetry of H+ binding to the intra- and extracellular side of the H+- coupled oligopeptide cotransporter PepT1. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 12. pp. 7777-7785.
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AU - Trottif, Davide

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AU - Boron, Walter F.

AU - Hediger, Matthias A.

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