Determination of transport stoichiometry for two cation-coupled myo-inositol cotransporters: SMIT2 and HMIT.

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