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.

Position 170 of Rabbit Na+/glucose cotransporter (rSGLT1) lies in the Na+ pathway; modulation of polarity/charge at this site regulates charge transfer and carrier turnover.

Positions 163, 166, and 173, within the putative external loop joining transmembrane segments IV and V of rabbit Na(+)/glucose cotransporter, form part of its Na(+) interaction and voltage-sensing domain. Since a Q170C mutation within this region exhibits anomalous behavior, its function was further investigated. We used Xenopus oocytes coinjected with mouse T-antigen to enhance Q170C expression, and the two-microelectrode voltage-clamp technique.

Transition states of the high-affinity rabbit Na(+)/glucose cotransporter SGLT1 as determined from measurement and analysis of voltage-dependent charge movements.

The charge-membrane voltage (Q-V) distribution of wild-type rabbit Na(+)/glucose transporter (rSGLT1) expressed in Xenopus oocytes was investigated in the absence of glucose, using the two-electrode voltage-clamp technique. Although this distribution is generally believed to be well represented by a two-state Boltzmann equation, we recently provided evidence for the existence of at least four states (Krofchick D and Silverman M. Biophys J 84: 3690-3702, 2003), confirming an earlier finding for human SGLT1 (Chen XZ, Coady MJ, and Lapointe JY.