Perturbation analysis of the voltage-sensitive conformational changes of the [Na.sup.+]/glucose cotransporter

Conformational changes of the human [Na.sup.+]/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.sup.+]], depolarizing voltage steps evoked a charge movement that rose initially to a peak (with time constant [tau] = 0.17 ms) before decaying to steady state with two time constants ([tau] = 2-30 and 25-150 ms). The time to peak (0.9 ms) decreased with [[Na.sup.+]], and was not observed in 0 mM [[Na.sup.+]]. In absence of [Na.sup.+], 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.sup.+]/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.sup.+]/glucose cotransport * presteady-state kinetics * charge movement * fluorescence * conformational changes