Three surface subdomains form the vestibule of the Na+/glucose cotransporter SGLT1.
A combination of biophysical and biochemical approaches was employed to probe the topology, arrangement, and function of the large surface subdomains of SGLT1 in living cells. Using atomic force microscopy on the single molecule level, Chinese hamster ovary cells overexpressing SGLT1 were probed with atomic force microscopy tips carrying antibodies against epitopes of different subdomains. Specific single molecule recognition events were observed with antibodies against loop 6-7, loop 8-9, and loop 13-14, demonstrating the extracellular orientation of these subdomains. The addition of D-glucose in Na+-containing medium decreased the binding probability of the loop 8-9 antibody, suggesting a transport-related conformational change in the region between amino acids 339 and 356. Transport studies with mutants C345A, C351A, C355A, or C361S supported a role for these amino acids in determining the affinity of SGLT1 for D-glucose. MTSET, [2-(trimethylammonium)ethyl] methanethiosulfonate and dithiothreitol inhibition patterns on alpha-methyl-glucoside uptake by COS-7 cells expressing C255A, C560A, or C608A suggested the presence of a disulfide bridge between Cys255 and Cys608. This assumption was corroborated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry showing mass differences in peptides derived from transporters biotinylated in the absence and presence of dithiothreitol. These results indicate that loop 6-7 and loop 13-14 are connected by a disulfide bridge. This bridge brings also loop 8-9 into close vicinity with the former subdomains to create a vestibule for sugar binding.
Puntheeranurak
Theeraporn
T
Department of Biology, Faculty of Science, Mahidol University, and Center of Excellence, National Nanotechnology Center at Mahidol University, Bangkok, 10400, Thailand.
Kasch
Myriam
M
Xia
Xiaobing
X
Hinterdorfer
Peter
P
Kinne
Rolf K H
RK
D019943:Amino Acid Substitution; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001693:Biological Transport, Active / Q000502:physiology; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D004220:Disulfides / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D018625:Microscopy, Atomic Force; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000737:chemistry / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D004220:Disulfides; D011994:Recombinant Proteins; D051269:Sodium-Glucose Transporter 1; D012964:Sodium; D005947:Glucose