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¹ Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095
² Department of Anesthesiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095

Correspondence to Donald Loo: dloo{at}mednet.ucla.edu

Conformational changes of the human Na⁺/glucose cotransporter (hSGLT1) were studied using voltage-jump methods. The cotransporter was expressed in Xenopus laevis oocytes, and SGLT1 charge movements were measured in the micro- to millisecond time scale using the cut-open oocyte preparation and in the millisecond to second time scale using the two-electrode voltage clamp method. Simultaneous charge and fluorescence changes were studied using tetramethylrhodamine-6-maleimide-labeled hSGLT1 Q457C. In 100 mM external [Na⁺], depolarizing voltage steps evoked a charge movement that rose initially to a peak (with time constant = 0.17 ms) before decaying to steady state with two time constants ( = 2–30 and 25–150 ms). The time to peak (0.9 ms) decreased with [Na⁺], and was not observed in 0 mM [Na⁺]. In absence of Na⁺, charge movement decayed monotonically to steady state with three time constants (0.2, 2, and 150 ms). Charge movement was accompanied by fluorescence changes with similar time courses, indicating that global conformational changes monitored by charge movement are reflected by local environmental changes at or near Q457C. Our results indicate that the major voltage-dependent step of the Na⁺/glucose transport cycle is the return of the empty carrier from inward to outward facing conformations. Finally, we observed subtle differences between time constants for charge movement and for optical changes, suggesting that optical recordings can be used to monitor local conformational changes that underlie the global conformational changes of cotransporters.

Key Words: Na⁺/glucose cotransport • presteady-state kinetics • charge movement • fluorescence • conformational changes

¹ Since the V_0.5 for charge is very negative in the absence of Na (–200 mV), some SGLT charge is being subtracted using the P/4 protocol. However, it does not invalidate the conclusions of the present study because the time course of the fast charge movement was not altered since the time constants were independent of voltage (Fig. 6 C).

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