A glutamine to glutamate mutation at position 170 (Q170E) in the rabbit Na+/glucose cotransporter, rSGLT1, enhances binding affinity for Na+ |
| |
Authors: | Huntley Steven A Krofchick Daniel Silverman Mel |
| |
Affiliation: | Department of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8. |
| |
Abstract: | 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. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|