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XB-ART-2606
J Gen Physiol January 1, 2005; 125 (1): 13-36.
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Perturbation analysis of the voltage-sensitive conformational changes of the Na+/glucose cotransporter.

Loo DD , Hirayama BA , Cha A , Bezanilla F , Wright EM .


Abstract
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 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(+)], 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.

PubMed ID: 15596535
PMC ID: PMC2217483
Article link: J Gen Physiol
Grant support: [+]

Species referenced: Xenopus laevis
Genes referenced: mapt ppp3cb rela slc5a1.2 tbx2


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References [+] :
Abramson, Structure and mechanism of the lactose permease of Escherichia coli. 2003, Pubmed