SMCT1 is a Na-coupled cotransporter of short chain monocarboxylates, which is expressed in the apical membrane of diverse epithelia such as colon, renal cortex, and thyroid. We previously reported that SMCT1 cotransport was reduced by extracellular Cl(-) replacement with cyclamate(-) and that the protein exhibited an ostensible anionic leak current. In this paper, we have revisited the interaction between small monovalent anions and SMCT cotransport and leak currents. We found that the apparent Cl(-) dependence of cotransport was due to inhibition of this protein by the replacement anion cyclamate, whereas several other replacement anions function as substrates for SMCT1; a suitable replacement anion (MES(-)) was identified. The observed outward leak currents represented anionic influx and favored larger anions (NO(3)(-)>I(-)>Br(-)>Cl(-)); currents in excess of 1 muA (at +50 mV) could be observed and exhibited a quasilinear relationship with anion concentrations up to 100 mM. Application of 25 mM bicarbonate did not produce measurable leak currents. The leak current displayed outward rectification, which disappeared when external Na(+) was replaced by N-methyl-d-glucamine(+). More precisely, external Na(+) blocked the leak current in both directions, but its K(i) value rose rapidly when membrane potential became positive. Thus SMCT1 possesses a anionic leak current that becomes significant whenever external Na(+) concentration is reduced. The presence of this leak current may represent a second function for SMCT1 in addition to cotransporting short chain fatty acids, and future experiments will determine whether this function serves a physiological role in tissues where SMCT1 is expressed.

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.

SMCT1 is encoded by the SLC5A8 gene, which is inactivated by methylation in colon, brain and thyroid cancer. SMCT1 cotransports Na+ with different monocarboxylates containing 3 to 8 carbon atoms but the transport properties discovered so far are insufficient to explain the putative role played by SMCT1 in cell proliferation. Three different stoichioimetries have been published for the cotransport passing through SMCT1, including a claim for different stoichiometries when lactate and propionate are cotransported. Electrophysiological analysis of this cotransporter is impeded by the presence of an anionic leak with the following selectivity (NO3−> I−>Br−>Cl−). We have established a 2:1 stoichiometry for lactate cotransport by use of three independent techniques: one method combined radiolabel uptake with current measurements, another combined radiolabel uptake with volume measurements and a third compared membrane potential changes in response to altered external substrate concentrations. We showed that the first two methods are unsuitable for measuring stoichiometry of propionate, which diffuses readily across the membrane. The third method is unaffected by passive diffusion of propionate and established a constant, 2:1 stoichiometry through this protein for both lactate and propionate. Volume measurement during the stimulation of a NO3− leak indicates a simple influx of inorganic anions which appears unaffected by the presence of substrate. In conclusion, SMCT1 exhibits a 2:1 stoichiometry which coexists with an anionic leak current.

The accessibility of the hydrophilic loop between putative transmembrane segments XIII and XIV of the Na+/glucose cotransporter (SGLT1) was studied in Xenopus oocytes, using the substituted cysteine accessibility method (SCAM) and fluorescent labelling. Fifteen cysteine mutants between positions 565 and 664 yielded cotransport currents of similar amplitude than the wild-type SGLT1 (wtSGLT1). Extracellular, membrane-impermeant MTSES(-) and MTSET(+) had no effect on either cotransport or Na+ leak currents of wtSGLT1 but 9 mutants were affected by MTSES and/or MTSET. We also performed fluorescent labelling on SGLT1 mutants, using tetramethylrhodamine-5-maleimide and showed that positions 586, 588 and 624 were accessible. As amino acids 604 to 610 in SGLT1 have been proposed to form part of a phlorizin (Pz) binding site, we measured the K(i)(Pz) and K(m)(alphaMG) for wtSGLT1 and for cysteine mutants at positions 588, 605-608 and 625. Although mutants A605C, Y606C and D607C had slightly higher K(i)(Pz) values than wtSGLT1 with minimal changes in K(m)((alpha)MG), the effects were modest and do not support the original hypothesis. We conclude that the large, hydrophilic loop near the carboxyl terminus of SGLT1 is thus accessible to the external solution but does not appear to play a major part in the binding of phlorizin.

Three different mammalian myo‐inositol cotransporters are currently known; two are Na+‐coupled (SMIT1 and SMIT2) and one is proton‐coupled (HMIT). Although their transport stoichiometries have not been directly determined, significant cooperativities in the Na+ activation of SMIT1 and SMIT2 suggest that more than one Na+ ion drives the transport of each myo‐inositol. The two techniques used here to determine transport stoichiometry take advantage of the electrogenicity of both SMIT2 and HMIT expressed in Xenopus oocytes. The first method compares the measurement of charge transferred into voltage‐clamped oocytes with the simultaneous uptake of radiolabelled substrate. The second approach uses high accuracy volume measurements to determine the transport‐dependent osmolyte uptake and compares it to the amount of charge transported. This method was calibrated using a potassium channel (ROMK2) and was validated with the Na+/glucose cotransporter SGLT1, which has a known stoichiometry of 2 : 1. Volume measurements indicated a stoichiometric ratio of 1.78 ± 0.27 ion per α‐methyl‐glucose (αMG) for SGLT1 whereas the radiotracer uptake method indicated 2.14 ± 0.05. The two methods yielded a SMIT2 stoichiometry measurement of 1.75 ± 0.30 and 1.82 ± 0.10, both in agreement with a 2 Na+:1 myo‐inositol stoichiometry. For HMIT, the flux ratio was 1.02 ± 0.04 charge per myo‐inositol, but the volumetric method suggested 0.67 ± 0.05 charge per myo‐inositol molecule. This last value is presumed to be an underestimate of the true stoichiometry of one proton for one myo‐inositol molecule due to some proton exchange for osmotically active species. This hypothesis was confirmed by using SGLT1 as a proton‐driven glucose cotransporter. In conclusion, despite the inherent difficulty in estimating the osmotic effect of a proton influx, the volumetric method was found valuable as it has the unique capacity of detecting unidentified transported substrates.