Kidney Week

Abstract: TH-OR58

Mucin 1 Stimulates TRPM6 Single-Channel Activity and Cell Surface Abundance by Impairing Dynamin-Dependent TRPM6 Endocytosis

Session Information

• Fluid, Electrolyte, and Acid-Base Disorders: Back to the Basics
  October 24, 2024 | Location: Room 4, Convention Center
  Abstract Time: 05:00 PM - 05:10 PM

Category: Fluid, Electrolytes, and Acid-Base Disorders

• 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

• Wolf, Matthias Tilmann, University of Michigan Michigan Medicine, Ann Arbor, Michigan, United States

Matthias Tilmann Wolf, MD, PhD, FASN

• Zhang, Jing, The University of Texas Southwestern Medical Center, Dallas, Texas, United States

Jing Zhang, MD, PhD

• Kozlitina, Julia, The University of Texas Southwestern Medical Center, Dallas, Texas, United States

Julia Kozlitina, PhD

• Hoenderop, Joost, Radboud Universitair Medisch Centrum, Nijmegen, Netherlands

Joost Hoenderop, PhD

• An, Sung Wan, University of Michigan Michigan Medicine, Ann Arbor, Michigan, United States

Sung Wan An, PhD

Background

Genome-wide association (GWAS) studies revealed an association of Mucin 1 (MUC1) variants with hypomagnesemia. The role of MUC1 in Mg²⁺ homeostasis remains unclear. Therefore, we evaluated if MUC1 enhances the activity of the Mg²⁺ channel TRPM6.

Methods

We analyzed the association of MUC1 variants and hypomagnesemia in the Dallas Heart Study (DHS), a cohort containing over 3,500 individuals. In a more focused study, 24 h urine samples of 12 control and 12 calcium-nephrolithiasis patients were analyzed for urinary electrolytes and MUC1 protein content. MUC1 and TRPM6 plasmids were co-expressed in HEK293 cells to investigate the effect of MUC1 on TRPM6 single channel and whole-cell current density by patch-clamp recording. Results were confirmed applying cell surface biotinylation and protein co-immunoprecipitation.

Results

We found that the MUC1 variant rs4072037 associated with hypomagnesemia. Urine samples from control and calcium-nephrolithiasis patients showed significantly lower urinary MUC1 levels and hypermagnesuria in nephrolithiasis patients pointing to a stimulatory role of MUC1 towards Mg²⁺ absorption. In vitro, co-expression of MUC1 with TRPM6 stimulated TRPM6 single channel current and whole-cell current density. In biotinylation experiments, MUC1 enhanced TRPM6 cell surface abundance. Co-immunoprecipitation confirmed physical interaction between MUC1 and TRPM6. We found that MUC1 interferes with TRPM6 endocytosis by examining the effect of dominant-negative dynamin and dynasore with co-transfected MUC1 and TRPM6. We tested TRPM6 endocytosis more specifically by knocking down clathrin heavy chain or caveolin-1 applying siRNA. Knockdown of both proteins abolished the stimulation of TRPM6 by MUC1. Furthermore, the N-glycan mutant TRPM6 (N787A) was not activated by MUC1. Finally, knockdown of the endogenous urinary lectins Galectin-1 and Galectin-3 also impaired MUC1 activation of TRPM6.

Conclusion

The MUC1 variant rs4072037 associates with hypomagnesemia. In vitro, MUC1 stimulates TRPM6 single channel activity, TRPM6 whole-cell current density and TRPM6 cell surface abundance by impairing dynamin-dependent TRPM6 endocytosis. TRPM6 and MUC1 physically interact and form lattice with the TRPM6 N-glycan N787. Urinary Galectin-1 and Galectin-3 stabilize this complex.

Funding

• Other U.S. Government Support