Establishing a definitive stoichiometry for the Na+/monocarboxylate cotransporter SMCT1

Abstract

Several different stoichiometries have been proposed for the Na(+)/monocarboxylate cotransporter SMCT1, including variable Na(+)/substrate stoichiometry. In this work, we have definitively established an invariant 2:1 cotransport stoichiometry for SMCT1. By using two independent means of assay, we first showed that SMCT1 exhibits a 2:1 stoichiometry for Na(+)/lactate cotransport. Radiolabel uptake experiments proved that, unlike lactate, propionic acid diffuses passively through oocyte membranes and, consequently, propionate is a poor candidate for stoichiometric determination by these methods. Although we previously determined SMCT1 stoichiometry by measuring reversal potentials, this technique produced erroneous values, because SMCT1 simultaneously mediates both an inwardly rectifying cotransport current and an outwardly rectifying anionic leak current; the leak current predominates in the range where reversal potentials are observed. We therefore employed a method that compared the effect of halving the external Na(+) concentration to the effect of halving the external substrate concentration on zero-current potentials. Both lactate and propionate were cotransported through SMCT1 using 2:1 stoichiometries. The leak current passing through the protein has a 1 osmolyte/charge stoichiometry. Identification of cotransporter stoichiometry is not always a trivial task and it can lead to a much better understanding of the transport activity mediated by the protein in question.

FIGURE 1

Estimation of SMCT1 cotransport stoichiometry for lactate using uptake data and current measurements. Oocytes were exposed to radiolabeled lactic acid (at a variety of concentrations) while the cell membrane potentials were clamped at −50 mV; the substrate-dependent currents were measured, and the total amount of current was deduced by integrating the area bound by the increased current due to substrate superfusion. At the end of the experiment, the oocyte was superfused with a solution lacking lactic acid but containing 1 mM ibuprofen; the voltage clamp was simultaneously ended. (A) A typical experiment is shown. The gray bar indicates the period during which lactic acid was present in the superfusing solution. (B) Comparison of uptake/current measurements. The data points for uptake and current measurement were used for linear regression; the best fit is shown by the dashed line.

FIGURE 2

Estimation of SMCT1 Na⁺/lactate cotransport stoichiometry using volume measurements and current measurements. SMCT1 stoichiometry was determined by measuring the changes in oocyte volume and the substrate-induced current when the cells were exposed to 1 mM lactic acid. A typical experiment is shown; the upper trace indicates the transmembrane current (clamped at −50 mV), whereas the lower trace indicates the oocyte volume. Note dramatic changes in each tracing when 1 mM lactic acid was added to the superfusing solution. Coexpression of aquaporin was necessary to permit rapid volume change. Three lines obtained from modeling the volume changes are shown alongside the volume tracing; the numbers after each of these lines represent the numbers of osmolytes per unit charge that should yield that particular theoretical change in volume.

FIGURE 3

Uptakes of (control) propionate and lactate. Oocytes were injected with water or with mRNA encoding SMCT1 (SMCT1) and uptake of radiolabeled lactic acid or propionic acid into the oocytes was assessed at 5–7 days after injection. Uptake experiments were performed for 15 min either: 1), in the absence of Na⁺ (replaced by NMDG⁺), 2), in the presence of 96 mM Na⁺, or 3), in the presence of 96 mM Na⁺ and 1 mM ibuprofen. Data shown represent one experiment with six oocytes per value shown, and are representative of three separate experiments. Both ordinate axes are labeled with respect to the transported anion, although the radiolabel uptake value actually reflects both anion transport and free acid diffusion.

FIGURE 4

Current-clamp measurements of lactate and propionate cotransport stoichiometries. By preloading oocytes with Na⁺ and substrate via prior cotransport (not shown), rapid changes in the internal concentrations of these agents during subsequent current-clamp measurements were minimized. The lines at the bottom of the figure indicate where external superfusing solution contained Na⁺ at 96, 48, and 24 mM, and where the external monocarboxylic acid was present at 0.5 or 1 mM. The cotransport stoichiometry is estimated from the ratio between the changes in membrane voltage when either Na⁺ or substrate concentration was halved.

FIGURE 5

SMCT1 activity with different lactate concentrations. In an effort to determine why simple measurement of reversal potentials at different substrate concentrations did not accurately yield the stoichiometric ratio, oocytes expressing SMCT1 were superfused with different concentrations of lactic acid and the ibuprofen-sensitive currents were recorded at different membrane potentials. A typical experiment is shown. The ibuprofen-sensitive currents observed in the absence of external lactic acid represent the anionic leak current passing through SMCT1. Note that the ibuprofen-sensitive currents all merge into the leak current at positive potentials, where the reversal potentials would normally be observed.

FIGURE 6

Stoichiometry of the leak current. Current/volume measurements, similar to those of Fig. 2, were used to examine the stoichiometry of the SMCT1 leak current at −25 mV holding potential. The upper tracing indicates the current measurement during the experiment and the lower tracing indicates the oocyte volume measurement. Replacement of most of the Cl⁻ in the superfusing solution by resulted in a large outward current (i.e., inward flux of anions) and concomitant swelling of the oocyte (a typical result is shown). Dotted lines indicate the mean change in volume under conditions before, during, and after exposure to