A glutamine to glutamate mutation at position 170 (Q170E) in the rabbit Na+/glucose cotransporter, rSGLT1, enhances binding affinity for Na+.
Using cysteine mutagenesis and chemical modification by methanethiosulfonate derivatives, it was demonstrated that the external putative loop, joining transmembrane segments (TM's) IV-V of rabbit Na+/glucose cotransporter, rSGLT1, forms part of a Na+ binding and voltage sensing domain. Within this region, exposure to cationic (2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) inhibited F163C, A166C, and L173C, but anionic sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) had no effect. Unexpectedly, MTSEA had no effect on Q170C; however, MTSES profoundly altered Q170C charge transfer and turnover, leaving Na+ and sugar binding affinity unchanged, but mutation of glutamine to anionic glutamate (Q170E) shifted V(0.5) to positive potentials, suggesting enhanced Na+ affinity. To clarify the role of glutamine 170 in Na+ interaction, we embarked on a more detailed investigation of Q170E using the two-microelectrode voltage clamping in Xenopus oocytes. Compared to wild-type (wt) rSGLT1, Q170E exhibits (i) a 2-fold decrease in methyl alpha-D-glucopyranoside affinity (-150 to -90 mV), (ii) a 5-fold increase in Na+ affinity (-150 to -100 mV) with less voltage dependency, (iii) reduced Na+ leak, and (iv) two transient current decay constants (tau(fast), tau(slow)) compared to three (tau(fast), tau(medium), tau(slow)) for wt, and computer simulation of Q170E pre-steady-state currents with a four-state kinetic model yields parameters similar to wt SGLT1, except for a reduced Na+ debinding rate constant compared to wt. Taken together, the data strengthen the conclusion that residue 170 lies in the Na+ pathway and provide the first evidence that it participates in determining Na+ binding.
Department of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Steven A
SA
Huntley
Daniel
D
Krofchick
Mel
M
Silverman
D000595:Amino Acid Sequence; D000818:Animals; D003198:Computer Simulation; D018698:Glutamic Acid / Q000235:genetics*; D005973:Glutamine / Q000235:genetics*; D008564:Membrane Potentials / Q000187:drug effects; D008757:Methylglucosides / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D011817:Rabbits; D012964:Sodium / Q000378:metabolism*; D051269:Sodium-Glucose Transporter 1 / Q000235:genetics*; D014982:Xenopus laevis
D008757:Methylglucosides; D051269:Sodium-Glucose Transporter 1; D005973:Glutamine; D018698:Glutamic Acid; C027020:methylglucoside; D012964:Sodium