Serine-294 and threonine-295 in the exofacial loop domain between helices 7 and 8 of glucose transporters (GLUT) are involved in the conformational alterations during the transport process

Abstract

The role of a conserved polar motif (STS) in the exofacial loop between helices 7 and 8 of GLUT4 for transporter function was investigated by site-directed mutagenesis and expression of the constructs in COS-7 cells. Reconstituted glucose-transport activity, cytochalasin B binding and photolabelling with the exofacial label 2-N4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1, 3-bis-(d-mannosyloxy)-2-propylamine (ATB-BMPA) were assayed in membranes from transfected cells and corrected for immunoreactivity of expressed transporters. Replacement of Ser-294 with Ala or Thr suppressed transport activity and cytochalasin B binding. ATB-BMPA photolabelling was normal in S294A mutants, and even increased in S294T mutants. Replacement of Thr-295 with Ala suppressed transport activity and cytochalasin B binding, whereas ATB-BMPA photolabelling was normal; substitution of Ser failed to alter the investigated parameters. Similarly, exchanging Ser-296 for Ala generated a normally functioning protein. The data suggest that Ser-294 and Thr-295 are involved in the conformational change in GLUT during the transport process, and that their substitution may arrest the transporter in an outward-facing conformation.

Functional Studies of the T295M Mutation Causing Glut1 Deficiency: Glucose Efflux Preferentially Affected by T295M

“…These observations reveal that both steric and polar influences are required at position T295. The steric influence predominates as the T295A markedly affects transport function whereas the T295G, T295S, and T295C mutations are associated with normal transport activity (36,38). Recently, a T295M mutagenesis study in CHO cells correlated with a significant decrease in the 2-DOG uptake under zero-trans influx conditions (39).…”

“…T295 is located in the STS polar motif of the exofacial loop between helix 7 and 8 and is conserved in the mammalian glucose transporters 1 to 4 (36). Substitutions at Y293 in this loop impair transport activity by reducing accessibility to the cytoplasmic substratebinding site, effectively locking the transporter into an outward facing conformation (11).…”

Tryptophan 388 in Putative Transmembrane Segment 10 of the Rat Glucose Transporter Glut1 Is Essential for Glucose Transport

“…In this regard, Seatter et al (27) have proposed that TM7 contributes to substrate selection and that a conserved QLS motif in TM7 interacts with the C-1 position of D-glucose, based on their observations after exchanging this motif between Glut2 (glucose and fructose transporter) and Glut3 (glucose transporter). Doege et al (14) have also proposed that a structure close to TM7 is important for substrate recognition.…”

“…The molecular mechanism of transport has been extensively studied with these transporters, especially with Glut1. (14) in Glut4 or Val 165 (15) in Glut2 also reduced or abolished glucose transport activity (amino acid residues are numbered according to the corresponding residues in Glut1). Many of these replacements also affected sensitivity to specific inhibitors, indicating that these sites are important for transport function.…”

Structure of, and functional insight into the GLUT family of membrane transporters

“…53 The serinethreonine-serine motif in extracellular Loop7 between TM7 and TM8 is also conserved in Class I GLUTs. 54 Mutation of these serine or threonine residues locks the protein conformation, suggesting this is a critical site for conformation change. Another highly conserved motif is GPXXXP in TM10, where a tryptophan also appears immediately after this sequence.…”