Abstract: SA-PO434
Residues L667 and L658: Critical Players in the Functional Activation of SGLT2 by MAP17
Session Information
- Diabetic Kidney Disease: Basic - II
November 04, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Diabetic Kidney Disease
- 701 Diabetic Kidney Disease: Basic
Authors
- Zhou, He, Tulane University School of Medicine, New Orleans, Louisiana, United States
- Abdulnour-Nakhoul, Solange, Tulane University School of Medicine, New Orleans, Louisiana, United States
- Hamm, L. Lee, Tulane University School of Medicine, New Orleans, Louisiana, United States
- Nakhoul, Nazih L., Tulane University School of Medicine, New Orleans, Louisiana, United States
Background
Sodium-glucose cotransporter 2 (SGLT2) is crucial for renal glucose reabsorption. Unlike its family member SGLT1, SGLT2 requires an activator protein, MAP17, for its transport activity. However, the mechanism of this unique activation is unclear. A recent cryogenic electron microscope (cryo-EM) study predicted interactive residues on SGLT2-MAP17 complex, but their functional significance requires validation. This study aims to investigate the role of specific SGLT2 amino acid residues in the functional activation of SGLT2 by MAP17 and their effect on glucose transport.
Methods
Five SGLT2 residues predicted to interact with MAP17 were selected based on cryo-EM predictions and aligned sequences of SGLT2 homologues. Mutations (mostly to alanine or cysteine) were designed to assess the effects of side chain and polarity changes. Transport studies were conducted using Xenopus oocytes expressing wild-type SGLT2 (SGLT2-WT) proteins or mutants and co-expressing MAP17. Oocytes were microinjected with mRNA for SGLT2-WT or SGLT2 mutants, and MAP17 mRNA was co-injected. Negative controls included H2O-injected oocytes and SGLT2-only-injected oocytes. Glucose transport in each oocyte was assessed by perfusing solutions containing 86 mM Na+ and 20 mM glucose and measuring whole cell currents generated by glucose-activated Na+ transport using two-electrode voltage clamp.
Results
Three SGLT2 mutations (L667C, L667M, and L658C) significantly inhibited glucose transport compared to SGLT2-WT. Specifically, L667C inhibited transport by 88%, L667M by 72%, and L658C by 70% (p<0.01). Notably, mutations F666A, F666Y, M661A, M661C, V665A and V665C did not significantly inhibit SGLT2 activity.
Conclusion
L667C, L667M and L658C mutations inhibited glucose transport potentially by disrupting the interaction between SGLT2 and MAP17, likely through altered hydrophobicity or disulfide bond formation. While other predicted interacting sites have a marginal contribution, L658 and L667 emerge as essential residues, displaying conservation and divergence among species and family members. This study confirmed the effects of cryo-EM predicted residues on SGLT2-MAP17 interaction and identified novel critical amino acids (L667 & L658) for SGLT2 function and MAP17 interaction.
Funding
- Private Foundation Support