title: The transport mechanism of the human sodium/myo-inositol transporter 2 (SMIT2/SGLT6), a member of the LeuT structural family.
abstract: The sodium/myo-inositol transporter 2 (SMIT2) is a member of the SLC5A gene family, which is believed to share the five-transmembrane segment inverted repeat of the LeuT structural family. The two-electrode voltage-clamp (TEVC) technique was used to measure the steady-state and the pre-steady-state currents mediated by human SMIT2 after expression in Xenopus laevis oocytes. Phlorizin is first shown to be a poor inhibitor of pre-steady-state currents for depolarizing voltage pulse. From an up to threefold difference between the apparent ON and OFF transferred charges during a voltage pulse, we also show that a fraction of the transient current recorded for very negative potentials is not a true pre-steady-state current coming from the cotransporter conformational changes. We suggest that this transient current comes from a time-dependent leak current that can reach large amplitudes when external Na(+) concentration is reduced. A kinetic model was generated through a simulated annealing algorithm. This algorithm was used to identify the optimal connectivity among 19 different kinetic models and obtain the numerical values of the associated parameters. The proposed 5-state model includes cooperative binding of Na(+) ions, strong apparent asymmetry of the energy barriers, a rate-limiting step that is likely associated with the translocation of the empty transporter, and a turnover rate of 21 s(-1). The proposed model is a proof of concept for a novel approach to kinetic modeling of electrogenic transporters and allows insight into the transport mechanism of members of the LeuT structural family at the millisecond timescale.
authors.affiliation: Groupe d'étude des protéines membranaires (GÉPROM) and Département de Physique, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Louis J
authors.initials: LJ
authors.lastname: Sasseville
authors.affiliation: Groupe d'étude des protéines membranaires (GÉPROM) and Département de Physique, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Longpré
authors.affiliation: Groupe d'étude des protéines membranaires (GÉPROM) and Département de Physique, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Bernadette
authors.initials: B
authors.lastname: Wallendorff
authors.affiliation: Groupe d'étude des protéines membranaires (GÉPROM) and Département de Physique, Université de Montréal, Montreal, Quebec, Canada jean-yves.lapointe@umontreal.ca.
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Lapointe
mesh_terms: D000818:Animals; D005260:Female; D006360:Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D005810:Multigene Family; D010695:Phlorhizin / Q000494:pharmacology; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D027981:Symporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D006360:Heat-Shock Proteins; D018398:Homeodomain Proteins; C000590731:LEUTX protein, human; D050480:Plasma Membrane Neurotransmitter Transport Proteins; D027981:Symporters; C073884:SLC5A3 protein, human; D010695:Phlorhizin
keywords: LeuT structural family; Na-glucose cotransporters (SGLTs); cotransporter; kinetic modeling; myo-inositol