Measuring ion transport activities in Xenopus oocytes using the ion-trap technique.
The ion-trap technique is an experimental approach allowing measurement of changes in ionic concentrations within a restricted space (the trap) comprised of a large-diameter ion-selective electrode apposed to a voltage-clamped Xenopus laevis oocyte. The technique is demonstrated with oocytes expressing the Na(+)/glucose cotransporter (SGLT1) using Na(+)- and H(+)-selective electrodes and with the electroneutral H(+)/monocarboxylate transporter (MCT1). In SGLT1-expressing oocytes, bath substrate diffused into the trap within 20 s, stimulating Na(+)/glucose influx, which generated a measurable decrease in the trap Na(+) concentration ([Na(+)](T)) by 0.080 +/- 0.009 mM. Membrane hyperpolarization produced a further decrease in [Na(+)](T), which was proportional to the increased cotransport current. In a Na(+)-free, weakly buffered solution (pH 5.5), H(+) drives glucose transport through SGLT1, and this was monitored with a H(+)-selective electrode. Proton movements can also be clearly detected on adding lactate to an oocyte expressing MCT1 (pH 6.5). For SGLT1, time-dependent changes in [Na(+)](T) or [H(+)](T) were also detected during a membrane potential pulse (150 ms) in the presence of substrate. In the absence of substrate, hyperpolarization triggered rapid reorientation of SGLT1 cation binding sites, accompanied by cation capture from the trap. The resulting change in [Na(+)](T) or [H(+)](T) is proportional to the pre-steady-state charge movement. The ion-trap technique can thus be used to measure steady-state and pre-steady-state transport activities and provides new opportunities for studying electrogenic and electroneutral ion transport mechanisms.
Blanchard
Maxime G
MG
Groupe d'étude des protéines membranaires (GEPROM Université de Montréal, C.P. 6128, Succ. "Centre-ville," Montréal, Québec, Canada H3C 1J7.
Longpré
Jean-Philippe
JP
Wallendorff
Bernadette
B
Lapointe
Jean-Yves
JY
D000818:Animals; D018014:Gene Transfer Techniques; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017736:Ion-Selective Electrodes; D007700:Kinetics; D019344:Lactic Acid / Q000378:metabolism; D008564:Membrane Potentials; D008757:Methylglucosides / Q000378:metabolism; D008954:Models, Biological; D027501:Monocarboxylic Acid Transporters / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism*; D018408:Patch-Clamp Techniques / Q000295:instrumentation; D051381:Rats; D015203:Reproducibility of Results; D012964:Sodium / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1 / Q000235:genetics / Q000378:metabolism*; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism*; D000099233:Monocarboxylate Transport Protein 1
D019344:Lactic Acid; D008757:Methylglucosides; D027501:Monocarboxylic Acid Transporters; D012964:Sodium; D051269:Sodium-Glucose Transporter 1; D027981:Symporters; D000099233:Monocarboxylate Transport Protein 1; C495674:SLC5A1 protein, human; C027020:methylglucoside