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, alpha-methyl D-glucopyranoside, phloridzin, and Na(+) affinity values are equivalent to those of wild-type; but turnover is reduced approximately 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 approximately 64% and Na(+) leak by approximately 55%; phloridzin and Na(+) affinity are unchanged. MTSES also reduces charge transfer (dithiothreitol-reversible) and Q170C turnover by approximately 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.
Huntley
Steven A
SA
Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
Krofchick
Daniel
D
Silverman
Mel
M
D000818:Animals; D000957:Antigens, Viral, Tumor / Q000378:metabolism; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D005973:Glutamine / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects* / Q000502:physiology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008698:Mesylates / Q000494:pharmacology; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010695:Phlorhizin / Q000494:pharmacology*; D011817:Rabbits; D012964:Sodium / Q000378:metabolism*; D051269:Sodium-Glucose Transporter 1; D014981:Xenopus
D000957:Antigens, Viral, Tumor; D008562:Membrane Glycoproteins; D008698:Mesylates; D009004:Monosaccharide Transport Proteins; D051269:Sodium-Glucose Transporter 1; C093171:methanethiosulfonate ethylammonium; D005973:Glutamine; C109153:(2-sulfonatoethyl)methanethiosulfonate; D005020:Ethyl Methanesulfonate; D012964:Sodium; D010695:Phlorhizin