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Biophysical Journal 87:295-310 (2004)
© 2004 The Biophysical Society

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

Steven A. Huntley, Daniel Krofchick and Mel Silverman

Department of Medicine, University of Toronto, Toronto, Ontario, Canada

Correspondence: Address reprint requests to Mel Silverman, Medical Sciences Building, Room 7205, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada. Tel.: 416-978-7189; Fax: 416-971-2132; E-mail: melvin.silverman{at}utoronto.ca.

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. For Q170C, {alpha}-methyl D-glucopyranoside, phloridzin, and Na+ affinity values are equivalent to those of wild-type; but turnover is reduced ~50%. Decreased [Na+] reduces Q170C, but not wild-type, charge transfer. Q170C presteady-state currents exhibit three time constants, {tau}, identical to wild-type. MTSES decreases maximal {alpha}-methyl D-glucopyranoside-induced currents by ~64% and Na+ leak by ~55%; phloridzin and Na+ affinity are unchanged. MTSES also reduces charge transfer (dithiothreitol-reversible) and Q170C turnover by ~60–70%. MTSEA and MTSET protect against MTSES, but neither affect Q170C function. MTSES has no obvious effect on the {tau}-values. Q170A behaves the same as Q170C. The mutation Q170E affects voltage sensitivity and reduces turnover, but also appears to influence Na+ interaction. We conclude that 1), glutamine 170 lies in the Na+ pathway in rabbit Na+/glucose cotransporter and 2), altered polarity and charge at position 170 affect a cotransporter conformational state and transition, which is rate-limiting, but probably not associated with empty carrier reorientation.




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 Am. J. Physiol. Cell Physiol.Home page
T. Liu, B. Lo, P. Speight, and M. Silverman
Transmembrane IV of the high-affinity sodium-glucose cotransporter participates in sugar binding
Am J Physiol Cell Physiol, July 1, 2008; 295(1): C64 - C72.
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