Regulation of Mg2+ Reabsorption and Transient Receptor Potential Melastatin Type 6 Activity by cAMP Signaling

The transient receptor potential melastatin type 6 (TRPM6) epithelial Mg²⁺ channels participate in transcellular Mg²⁺ transport in the kidney and intestine. Previous reports suggested a hormonal cAMP-dependent regulation of Mg²⁺ reabsorption in the kidney. The molecular details of this process are, however, unknown. Adenylate cyclase 3 (Adcy3) has been shown to colocalize with the Na⁺/Cl⁻ cotransporter, a marker of the distal convoluted segment of the kidney, the principal site of TRPM6 expression. Given the critical role of TRPM6 in Mg²⁺ reabsorption, an inducible kidney-specific Adcy3 deletion mouse model was characterized for blood and urinary electrolyte disturbances under a normal—and low—Mg²⁺ diet. Increased urinary Mg²⁺ wasting and Trpm6 mRNA levels were observed in the urine and kidney of Adcy3-deleted animals compared with wild-type controls. Serum Mg²⁺ concentration was significantly lower in Adcy3-deleted animals at day 7 on the low Mg²⁺ diet. Using patch clamp electrophysiology, cell surface biotinylation, and total internal reflection fluorescence live cell imaging of transfected HEK293 cells, we demonstrated that cAMP signaling rapidly potentiates TRPM6 activity by promoting TRPM6 accumulation at the plasma membrane and increasing its single-channel conductance. Comparison of electrophysiological data from cells expressing the phosphorylation-deficient S1252A or phosphomimetic S1252D TRPM6 mutants suggests that phosphorylation at this intracellular residue participates in the observed stimulation of channel activity. Altogether, these data support a physiologically relevant magnesiotropic role of cAMP signaling in the kidney by a direct stimulatory action of protein kinase A on the plasma membrane trafficking and function of TRPM6 ion channels.