Determination of the Na +/Glucose Cotransporter (SGLT1) Turnover Rate using the Ion-Trap Technique

Abstract

The Na+/glucose cotransporter (SGLT1) is a membrane protein that couples the transport of 2 Na+ ions and 1 glucose molecule using the “alternating access mechanism”. This mechanism involves the cotransporter oscillating between two conformations characterized by intracellular versus extracellular accessibility to the binding sites. The turnover rate (TOR) is the number of complete cycles that each cotransporter performs per second. It is a crucial parameter to establish since it can be used to estimate the number of cotransporters present and it sets a lower limit for the slowest rate constant involved in the cotransport mechanism (conformational changes, binding and de-binding reactions). In this study, we obtained an independent estimate of the TOR for human SGLT1 expressed in Xenopus laevis oocytes using a new approach: the Ion-Trap technique (ITT, Blanchard MG. et al. AJP 2008;295(5):C1464-72.) where an extracellular ion-selective electrode of large diameter is used to detect the quantity of ions that are rapidly taken up by the cotransporter (within 20 ms) when the binding sites are suddenly exposed to the extracellular solution by application of a negative membrane potential. Taking advantage of the fact that hSGLT1, in the absence of Na+, can cotransport glucose with protons, we used a pH electrode to determine a TOR of 8.00 ± 1.3 s−1 in the presence of 35 mM α-methyl-glucose (αMG) at −150 mV (pH 5.5). This can be used to calculate a TOR of 13.3 ± 2.4 s−1, for the same group of oocytes, under near Vmax conditions i.e. in the presence of 90 mM Na+ and 5 mM αMG. Under these circumstances, the average cotransport current was −1.08 ± 0.61 μA (n=14) and this activity was generated by an average of 3.6 ± 0.7 ×1011 cotransporter molecules per oocyte.