Effects on Conformational States of the Rabbit Sodium/Glucose Cotransporter through Modulation of Polarity and Charge at Glutamine 457
| Autor: | Tiemin Liu, Daniel Krofchick, Mel Silverman |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
Patch-Clamp Techniques
Alkylation Stereochemistry Protein Conformation Sodium Glutamine Mutant Mutation Missense Biophysics chemistry.chemical_element Models Biological Membrane Potentials 03 medical and health sciences chemistry.chemical_compound Xenopus laevis 0302 clinical medicine Sodium-Glucose Transporter 1 Bromide Animals Computer Simulation Sugar 030304 developmental biology Ions Mesylates 0303 health sciences Chemistry Chemical modification Electrophysiology Kinetics Phlorhizin Oocytes Carbohydrate Metabolism Rabbits Cotransporter 030217 neurology & neurosurgery |
| Zdroj: | Biophysical Journal. 96(2):748-760 |
| ISSN: | 0006-3495 |
| DOI: | 10.1016/j.bpj.2008.09.045 |
| Popis: | The high affinity sodium/glucose cotransporter (SGLT1) couples transport of Na(+) and glucose. Previous studies established that mutant Q457C human SGLT1 retains full activity, and sugar translocation is abolished in mutant Q457R or in mutant Q457C after reaction with methanethiosulfonate derivatives, but Na(+) and sugar binding remain intact. To explore the mechanism by which modulation of Q457 abolishes transport, Q457C and Q457R of rabbit SGLT1 were studied using chemical modification and the two-electrode voltage-clamp technique. Compared to wild-type SGLT1, Q457C exhibits approximately 20-fold reduction in phloridzin affinity and preferential occupancy of an inward-facing state. Alkylation of Q457C by [(2-trimethylammonium) ethyl] methanethiosulphonate bromide, (MTSET), reverses these changes while blocking transport. Analysis of pre-steady-state currents in the absence of sugar yields three decay constants for each of Q457C, Q457C-MTSET and Q457R. Comparison of Q457C-MTSET and Q457R with Q457C and wild-type, reveals that inhibition of transport is accompanied by a decrease in magnitude and voltage-independence of the slow decay constant at negative potentials. But fast and medium decays remain unchanged. Computer simulation of transient currents suggests that introduction of positive charge at position 457 leads to a predominant outward rather than inward-facing conformational state. Taken together, the results suggest that glutamine 457, in addition to being involved in sugar binding, is a residue that is sensitive to conformational changes of the carrier. |
| Databáze: | OpenAIRE |
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