Transmembrane Segment 6 of the Glut1 Glucose Transporter Is an Outer Helix and Contains Amino Acid Side Chains Essential for Transport Activity

Abstract

Experimental data and homology modeling suggest a structure for the exofacial configuration of the Glut1 glucose transporter in which 8 transmembrane helices form an aqueous cavity in the bilayer that is stabilized by four outer helices. The role of transmembrane segment 6, predicted to be an outer helix in this model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzene-sulfonate (pCMBS). A fully functional Glut1 molecule lacking all 6 native cysteine residues was used as a template to produce a series of 21 Glut1 point mutants in which each residue along helix 6 was individually changed to cysteine. These mutants were expressed in Xenopus oocytes, and their expression levels, functional activities, and sensitivities to inhibition by pCMBS were determined. moderately augmented specific transport activity relative to the control. These results were dramatically different from those previously reported for helix 12, the structural cognate of helix 6 in the pseudo-symmetrical structural model, for which none of the 21 single-cysteine mutants exhibited reduced activity. Only the substitution at Leu 188 conferred inhibition by pCMBS, suggesting that most of helix 6 is not exposed to the external solvent, consistent with its proposed role as an outer helix. These data suggest that helix 6 contains amino acid side chains that are critical for transport activity and that structurally analogous outer helices may play distinct roles in the function of membrane transporters.

A GSK-3/TSC2/mTOR pathway regulates glucose uptake and GLUT1 glucose transporter expression

“…In addition, a number of investigators have shown that cellular metabolism and ATP availability play a significant role in regulating GLUT1 expression and function through both direct and indirect mechanisms. Another report suggests that direct interaction of ATP with GLUT1-specific peptide sequences can lead to conformational changes that modulate transport of glucose and inhibit degradation of GLUT1 (18,35,36). Moreover, studies in skeletal muscle have shown that expression of a constitutively active form of AMP kinase (AMPK) results in increased GLUT1 and GLUT4 protein levels (11).…”

“…The signaling pathways involved in acute insulin-stimulated GLUT4 translocation have been the subject of many studies over the past two decades. However, relatively few studies have examined the signaling systems involved in long-term regulation of GLUT1 mediated glucose uptake in noninsulin responsive tissues (4,5,11,18,36), and none of these previous reports have investigated the role of a signaling pathway that is an important regulator of cellular growth and metabolism, namely, the pathway that includes glycogen synthase kinase-3 (GSK-3), tuberous sclerosis complex (TSC), and mammalian target of rapamycin (mTOR).…”

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

“…45,62,63 Cysteine scanning mutagenesis was subsequently employed to probe which residues lined this proposed central pore. [64][65][66][67][68][69][70][71][72][73][74] The resulting model indicated that TMs 2, 4, 5, 7, 8, 11, and possibly 1 and 10 formed a central aqueous transport channel for glucose, whereas TMs 3, 6, 9, and 12 formed a structural scaffold on the outside of the protein. 70 A possible substrate binding site was also proposed, involving Q161, Q282, and W412.…”

Sequence Determinants of GLUT1-mediated Accelerated-exchange Transport

“…V max /K m is obtained from measurements of 2-DG uptake and then normalized to cell surface GLUT expression to give k cat /K m . Although it is possible that TM6 mutants could alter the affinity (Ϸ1/K m ) of GLUT1 and GLUT4 for substrate, this seems unlikely because TM6 is a putative scaffold TM quite distant to the hypothesized GLUT1 substrate-binding cavity (25,26). Moreover, K m(app) for GLUT1-and GLUT4-mediated sugar uptake is similar for both 2-DG (9 -10 mM (63)) and 3-MG (ϳ6 mM (10)).…”

“…In the absence of GLUT crystal structures, our understanding of GLUT1 tertiary structure derives largely from scanning cysteine mutagenesis (22)(23)(24)(25) and modeling studies (26,27), which align and thread the GLUT1 sequence through the crystal structures of Major Facilitator Superfamily bacterial transporter homologs GlpT (28) and LacY (29). Although these homology-based threaded structures provide quite accurate descriptions of transporter topography and helix packing arrangements, they fail to accurately predict helix and amino acid side chain orientation within the active sites (30).…”