Establishing a definitive stoichiometry for the Na+/monocarboxylate cotransporter SMCT1.
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.
Coady
Michael J
MJ
Groupe d'étude des protéines membranaires and Département de Physique, Université de Montréal, Montréal, Canada. coady@magellan.umontreal.ca
Wallendorff
Bernadette
B
Bourgeois
Francis
F
Charron
Francois
F
Lapointe
Jean-Yves
JY
D001692:Biological Transport; D001703:Biophysics / Q000379:methods*; D002264:Carboxylic Acids / Q000737:chemistry; D004563:Electrochemistry / Q000379:methods; D006801:Humans; D017136:Ion Transport; D007477:Ions; D007773:Lactates / Q000737:chemistry; D015233:Models, Statistical; D027501:Monocarboxylic Acid Transporters / Q000737:chemistry* / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D011422:Propionates / Q000737:chemistry; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism; D012964:Sodium / Q000737:chemistry; D027981:Symporters
D002264:Carboxylic Acids; D007477:Ions; D007773:Lactates; D027501:Monocarboxylic Acid Transporters; D011422:Propionates; D012333:RNA, Messenger; C519836:SLC5A12 protein, human; D027981:Symporters; D012964:Sodium