Gorraitz E et al. (2017), Active site voltage clamp fluorometry of the so...

XB-ART-54225

Proc Natl Acad Sci U S A 2017 Nov 14;11446:E9980-E9988. doi: 10.1073/pnas.1713899114.

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Active site voltage clamp fluorometry of the sodium glucose cotransporter hSGLT1.

Gorraitz E , Hirayama BA , Paz A , Wright EM , Loo DDF .

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In the human sodium glucose cotransporter (hSGLT1) cycle, the protein undergoes conformational changes where the sugar-binding site alternatively faces the external and internal surfaces. Functional site-directed fluorometry was used to probe the conformational changes at the sugar-binding site. Residues (Y290, T287, H83, and N78) were mutated to cysteines. The mutants were expressed in Xenopus laevis oocytes and tagged with environmentally sensitive fluorescent rhodamines [e.g., tetramethylrhodamine (TMR)-thiols]. The fluorescence intensity was recorded as the mutants were driven into different conformations using voltage jumps. Sugar binding and transport by the fluorophore-tagged mutants were blocked, but Na+ binding and the voltage-dependent conformational transitions were unaffected. Structural models indicated that external Na+ binding opened a large aqueous vestibule (600 Å3) leading to the sugar-binding site. The fluorescence of TMR covalently linked to Y290C, T287C, and H83C decreased as the mutant proteins were driven from the inward to the outward open Na+-bound conformation. The time courses of fluorescence changes (milliseconds) were close to the SGLT1 capacitive charge movements. The quench in rhodamine fluorescence indicated that the environment of the chromophores became more polar with opening of the external gates as the protein transitioned from the inward to outward facing state. Structural analyses showed an increase in polar side chains and a decrease in hydrophobic side chains lining the vestibule, and this was reflected in solvation of the chromophore. The results demonstrate the opening and closing of external gates in real time, with the accompanying changes of polarity of the sugar vestibule.

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Species referenced: Xenopus laevis
Genes referenced: slc5a1.2 tpm3

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	Fig. 1. (A) Kinetic model of Na+/glucose cotransport by hSGLT1 emphasizing the five states shown in the absence of glucose (C6, A/B, C1, and C2Na2). External Na+ binds to the Na2 and Na1 sites in C1 to form the outward-facing conformation C2Na2. SGLT1 has an apparent valence of 1, and in the absence of Na+, Vm drives the protein between outward-facing C1 and inward-facing C6 through two intermediate states A and B. The rate of Na+ binding is also voltage-dependent. At hyperpolarizing potentials (â150 mV), the protein is driven to C2Na2, and at depolarizing potentials (+50 mV), it is driven to C6. The distribution of hSGLT1 between C6, C1, and C2Na2 can be manipulated experimentally in a predictable manner by external Na+ and Vm (e.g., at high external Na+ at â150 mV, 100% of the protein is in C2Na2, and at +50 mV, 100% of the protein is in C6). (B) Cartoon of hSGLT1 showing the location of the glucose-binding site and the Na1 and Na2 sites in an occluded inward-facing conformation showing the position of residues N78, H83, T287, Y290, and W291. The structural model was based on the X-ray structure of the bacterial homolog vSGLT in the inward-facing occluded conformation (8). T287, Y290, and W291 are on TM6. H83 and N78 are on TM1.
	Fig. 2. External vestibule of hSGLT1. The space predicted to be occupied by MTS-TAMRA in the outward open conformation of hSGLT1 (C2Na2) when covalently linked to Y290C, T287C, H83C, and N78C is shown. The volume of the space is 600 Ã3, and it is bounded by TM1, TM2, TM3, TM6, TM8, TM9, and TM10 with 57 side chains. Over 50% are aliphatic and small polar chains (Fig. 6 and SI Appendix, Figs. S6âS9). A side view (A) and a top view (B) are shown.
	Fig. 3. Comparison between charge movement (Q) and fluorescence (âF) in the presence and absence of Na+ for SGLT1-Y290C labeled with MTS-TAMRA. (A and C) Time courses of MTS-TAMRA âF for voltage jumps from the Vh (â50 mV) and to test voltages of +50, +10, â30, â90, â130, and â150 mV in the presence and absence of external 100 mM NaCl. (B and D) Simultaneous Q and âF changes for voltage steps from â50 mV to +50 and â150 mV. The preâsteady-state currents were isolated from the total current by subtraction of the capacitive and steady-state currents, and integrated to obtain Q at each voltage (SI Appendix, Fig. S1). The time constants for the hyper- and depolarizing pulses were similar in the presence and absence of Na+ (7â10 ms). In external sodium, Y290C-hSGLT1 is in the C2Na2 conformation at â150 mV and in the C6 conformation at +50 mV. In the absence of sodium Y290C-hSGLT1 is in the C1 conformation at â150 mV and in the C6 conformation at +50 mV (SI Appendix, Fig. S1). All of the data in this figure and SI Appendix, Fig. S1 were from a single oocyte. In B and D, Q and âF records are normalized (norm) to agree at the end of the pulse.
	Fig. 4. Time course of the fluorescence changes for MTS-TAMRA and TMR6M attached to Y290C. Oocytes expressing hSGLT1 mutant Y290C were labeled with either MTS-TAMRA or TMR6M. The Vm was held at â90 mV, and the change in fluorescence (âF) was recorded on stepping Vm to +50 mV. After the fluorescence in 100 mM NaCl was recorded, the experiment was repeated in 100 mM choline-Cl (0 mM NaCl). The traces (black, MTS-TAMRA; red, TMR6M) have been normalized (norm) to agree at the end of the voltage pulse. The half-times for the TMR6M changes were faster in the presence and absence of Na+ (4 and 10 ms) than those for MTS-TAMRA (10 ms and 8 and 57 ms). The âF/F signals were 0.39% for TMR6M and 4.74% for MTS-TAMRA. In 100 mM Na+, 95% of the proteins were in the C2Na2 conformation at â90 mV and 100% were in the C6 conformation at +50 mV, and in the absence of sodium, 80% of the proteins were in the C1 conformation at â90 mV and 100% were in the C6 conformation at +50 mV (SI Appendix, Fig. S1).
	Fig. 5. Simultaneous recordings of voltage-induced charge movement (Q) and fluorescence (âF) for SGLT1-T287C labeled with MTS-TAMRA. A single oocyte expressing SGLT1-T287C labeled with MTS-TAMRA was held at â50 mV, and changes in ÎF were recorded in response to 100-ms step depolarizations (to +50 mV) and hyperpolarization (to â150 mV) in the presence of external Na+ (100 mM NaCl). (A) Time course of the MTS-TAMRA ÎF changes. (C) For clarity, only two ÎF records are shown (at steps of +50 and â150 mV). (B) Time courses of simultaneous ÎF and Q for voltage steps of +50 and â150 mV. The traces have been normalized (norm) to agree at the end of the voltage pulse (100 ms). The time constants (Ï) for the +50 mV pulse were 5 ms for Q and 10 and 75 ms for âF, and for the â150 mV pulses, they were 5 ms for Q and 4.5 and 75 ms for âF. (D) Time course of ÎF for voltage jumps to +50 and â150 mV in the absence of Na+. Complete ÎF and Q/V curves are given in SI Appendix, Fig. S2.
	Fig. 6. Location of MTS-TAMRA linked to Y290C in the outward- and inward-facing conformations of hSGLT1. (A and B) Only TM1, TM2, TM3, TM6, TM8, TM9, and TM10 surround the dye in the C2Na2 and C6 conformations. (C) Major difference between the C2Na2 and C6 states is the position of the outer half of TM10, which is clearly indicated. (D and E) Side chains within 6 Ã of the chromophore in the outward open and inward occluded conformations are given. In the ligand interaction plots, residues in green are hydrophobic, those in cyan are polar, and those in white are glycine; chromophore atoms accessible to solvent are shown by a cloud. The green lines between F101 and the dye represent Ï-Ï stacking. The major difference is the accessibility of the chromophore to solvent upon closing of the outer gate (F453 shown in C).

References [+] :

Abramson, Structure and function of Na(+)-symporters with inverted repeats. 2009, Pubmed