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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

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

Groupe d'étude des protéines membranaires (GÉPROM), and Département de physique, Université de Montréal, Montréal, Québec, Canada

Submitted 22 November 2007 ; accepted in final form 24 September 2008

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.

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

This article has been cited by other articles:

				M. D. Parker, R. Musa-Aziz, and W. F. Boron Letter to the editor: The use of extracellular, ion-selective microelectrodes to study the function of heterologously expressed transporters in Xenopus oocytes Am J Physiol Cell Physiol, May 1, 2009; 296(5): C1243 - C1243. [Full Text] [PDF]

				M. Blanchard, J.-P. Longpre, and J.-Y. Lapointe Reply to "Letter to the editor: 'The use of extracellular, ion-selective microelectrodes to study the function of heterologously expressed transporters in Xenopus oocytes'" Am J Physiol Cell Physiol, May 1, 2009; 296(5): C1244 - C1244. [Full Text] [PDF]