Measuring ion transport activities in Xenopus oocytes using the ion-trap technique

作者：Blanchard Maxime G; Longpre Jean Philippe; Wallendorff Bernadette; Lapointe Jean Yves^*

来源：American Journal of Physiology - Cell Physiology, 2008, 295(5): C1464-C1472.

DOI：10.1152/ajpcell.00560.2007

• ion-selective electrode
• cotransporter
• SGLT1
• MCT1
• electrogenic
• electroneutral
• transporters

Blanchard MG, Longpre JP, Wallendorff B, Lapointe JY. Measuring ion transport activities in Xenopus oocytes using the ion-trap technique. Am J Physiol Cell Physiol 295: C1464-C1472, 2008. First published October 1, 2008; doi: 10.1152/ajpcell.00560.2007.-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.

• 出版日期2008-11