SYMMETRY OF H-CELLULAR AND EXTRACELLULAR SIDE OF THE H+-COUPLED OLIGOPEPTIDE COTRANSPORTER PEPT1( BINDING TO THE INTRA)

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 i on near the extracellular transporter surface, Little attention was gi ven to analogous events that may occur at the intracellular surface, T o address this issue, we performed voltage clamp studies of Xenopus oo cytes expressing the intestinal H+-coupled peptide cotransporter PepT1 and recorded the dependence of transient charge movements in the abse nce 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 movemen ts were mostly inwardly directed, whereas reduction of pH(o) to below 7.0 resulted in outwardly directed charge movements, When pH(i) was re duced, inwardly directed charge movements were observed, The data on t he 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 (V-0.5) of the charge distribution shif ted linearly as a function of pH(i) and pH(o). Our results indicate th at, as a first approximation, the magnitude and polarity of the transi ent charge movements depend upon the prevailing H+ electro-chemical gr adient, We propose that PepT1 has a single proton binding site that is symmetrically accessible from both sides of the membrane and that dec reasing the H+ chemical potential (Delta mu(H)) or increasing the memb rane 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 transporter s by including a more detailed description of intracellular events.