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. 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.