Abstract
Voltage clamp recording was used to measure steady-state and presteady-state currents mediated by a myo-inositol transporter cloned from Leishmania donovani and expressed in Xenopus oocytes. Application of myo-inositol resulted in inward currents, which did not require external sodium and which were increased by increasing the extracellular proton concentration and by membrane hyperpolarization. Alkalinization of the extracellular space occurred concomitantly with myo-inositol influx. Correlation of membrane currents with radiolabeled myo-inositol flux revealed that one positive charge is translocated with each molecule of myo-inositol, consistent with cotransport of one proton. The transport concentration dependence on both species suggested ordered binding of a proton followed by a molecule of myo-inositol. In the absence of myo-inositol, a voltage-dependent capacitance was observed that correlated with the transporter expression level. This charge movement obeyed a Boltzmann function, which was used to estimate a turnover of 0.70 +/- 0.06 s-1 at -60 mV. The pH and voltage dependence of the charge movements were simulated with a model involving alternating access of internal and external protons to sites within an occluded pore.
Highlights
Secondary transporters drive concentrative solute flux by utilizing the energy stored in the electrochemical gradients of cotransported ions (Crane, 1977)
Measurement of flux mediated by a cloned L. donovani myo-inositol transporter revealed that a steady-state flow of electrical current occurs during transport such that one charge is translocated with each molecule of myo-inositol
The transport kinetic parameters Km and Imax revealed an interaction between myo-inositol and protons, which was in turn influenced by the membrane potential
Summary
Secondary transporters drive concentrative solute flux by utilizing the energy stored in the electrochemical gradients of cotransported ions (Crane, 1977). Some transporters are capable of utilizing multiple ion gradients, suggesting the possibility of common functional mechanisms that may be conserved among different transporter types. The amino acid transporter, CAT-1, is capable of either Naϩ-dependent or Naϩ-independent facilitated uptake, depending on the charge on the amino acid transported (Christensen et al, 1969; Wang et al, 1991). A genomic DNA clone (D1) related to a family of genes encoding facilitated and proton-coupled hexose transporters was cloned from the parasitic protozoan Leishmania donovani and demonstrated to encode a transporter for myo-inositol (Drew et al, 1995). Evidence is presented for ordered binding of a proton followed by myo-inositol binding and translocation, and charge movements are observed in the absence of myo-inositol that are suggested to reflect this proton binding
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