Replacement of Ala-166 with cysteine in the high affinity rabbit sodium/glucose transporter alters transport kinetics and allows methanethiosulfonate ethylamine to inhibit transporter function.
An alanine to cysteine mutation at position 166 has been introduced by site-directed mutagenesis into the rabbit sodium/glucose transporter (rSGLT1). When expressed in Xenopus laevis oocytes, this mutant transporter (A166C rSGLT1) demonstrates a significantly lower apparent affinity for alpha-methyl glucoside (alphaMG) compared with the wild-type transporter (apparent Km = 0.8 versus 0.15 mM). Using the two-electrode voltage clamp technique, transient currents have also been measured, and for the mutant transporter, the transients induced by large depolarizations exhibit longer time constants than those for wild type. Moreover, the substitution of Ala-166 with a cysteine allows the sulfydryl specific reagent, methanethiosulfonate ethylamine (MTSEA), to react with and alter the function of the transporter. Whereas the wild-type transporter is unaffected by reaction with MTSEA, A166C rSGLT1 has its steady-state currents induced by 1 mM alphaMG inhibited 83% within a minute of exposure to MTSEA. Furthermore, the pre-steady-state transients of the A166C mutant after MTSEA exposure demonstrate much shorter time constants than before while the total amount of charge transferred is only slightly diminished. These results together provide evidence that position 166 is situated in a region critical to the functioning of rSGLT1.
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Medical Research Council Group in Membrane Biology, Department of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Silverman
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D000409:Alanine / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1; D014982:Xenopus laevis
D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; D051269:Sodium-Glucose Transporter 1; C093171:methanethiosulfonate ethylammonium; D005020:Ethyl Methanesulfonate; D003545:Cysteine; D000409:Alanine