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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

Authors
Journal
Biophysical Journal
0006-3495
Publisher
Elsevier
Publication Date
Volume
87
Issue
1
Identifiers
DOI: 10.1529/biophysj.104.040253
Keywords
  • Channels
  • Receptors
  • And Transporters

Abstract

Abstract 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, α-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, τ, identical to wild-type. MTSES decreases maximal α-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 τ-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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