Effect of extracellular pH on the myo-inositol transporter SMIT expressed in Xenopus oocytes.
The myo-inositol transporter SMIT is expressed particularly at high extracellular osmolarity and serves to accumulate the osmolyte myo-inositol. Transport of myo-inositol is coupled to the cotransport of Na+ and is electrogenic. In Xenopus oocytes injected with mRNA encoding SMIT but not in water-injected oocytes, myo-inositol creates an inward current that is dependent on the ambient Na+ concentration. The present study has been performed to elucidate the pH dependence of myo-inositol-induced currents. Therefore, Xenopus oocytes were injected with mRNA encoding SMIT and two-electrode voltage-clamp studies were performed. The myo-inositol-induced currents in oocytes expressing SMIT were found to have a sigmoidal dependence on the ambient pH between pH 5.5 and 8.5 with an apparent Ki of 0.21+/-001 microM H+ and a Hill coefficient of 1.80+/-0.16. Kinetic analysis of the myo-inositol-induced currents at pH 8.0 and -90 mV holding potential revealed a Hill coefficient of 0.93+/-0.07 and an apparent Km for myo-inositol of 0.031+/-0.003 mM as well as a Hill coefficient of 1. 64+/-0.24 and an apparent Km of 38.8+/-4.1 mM for Na+. A decrease of the Na+ concentra-tion from 150 mM to 50 mM significantly altered the maximal observed current and increased the apparent Km for myo-inositol. Acidification to pH 6.5 significantly increased the apparent Km for myo-inositol and for Na+ to 0.057+/-0.005 mM and 73. 9+/-4.8 mM, respectively. The Hill coefficients for myo-inositol and Na+ were not affected and remained close to 1 for myo-inositol and 2 for Na+. In summary, acidification impedes SMIT-mediated myo-inositol transport at least partially by decreasing the affinity of the carrier for Na+. The impaired Na+ binding subsequently decreases binding and transport of myo-inositol.
Matskevitch
J
J
Institute for Physiology, University of Tübingen, Gmelinstrasse 5, D-72076 Tübingen, Germany.
Wagner
C A
CA
Risler
T
T
Kwon
H M
HM
Handler
J S
JS
Waldegger
S
S
Busch
A E
AE
Lang
F
F
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D004553:Electric Conductivity; D005260:Female; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D012964:Sodium / Q000494:pharmacology; D027981:Symporters; D014981:Xenopus
D002352:Carrier Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D027981:Symporters; C073884:SLC5A3 protein, human; D007294:Inositol; D012964:Sodium