WNK4 kinase negatively regulates the surface expression of Muscarinic M3 receptor

With-No-Lysine [K] 4 (WNK4) kinase regulates the surface expression of various ion transporters. Not only ion transporters but G-protein coupled receptors (GPCR) can function properly when their expression level is appropriate at the plasma membrane. In this study, we examined the role of WNK4 kinase in the regulation of muscarinic receptor 3 (M₃R) using physiological and biochemical experiments. Measurement of the pilocarpine-responsive [Ca²⁺]_i change demonstrated that WNK4 kinase decreased the activity of M₃R through its reduced surface expression. Kinase domain of WNK4 bound with the third intracellular region of M₃R whereas its negative regulation was independent on the kinase activity. Comparable to wild-type WNK4, kinase-inactive WNK4^D318A mutant also reduced the surface expression of M₃R, whereas the kinase domain of WNK4_1-441 failed to reduce the surface expression of M₃R. In accordance with surface biotinylation experiments, non-permeable immunostaining of M₃R also showed that M₃R surface expression is independent on the kinase activity of WNK4. Interestingly, comparison of the half life of total and surface M₃R revealed that only the half life of total M₃R, but not surface M₃R was decreased by WNK4 kinase. Nevertheless, the rate of decrease in surface M₃R always exceeded that of total M₃R. Taken together, these results suggest that WNK4 kinase negatively regulates the anterograde trafficking of M₃R through kinase-independent mechanism.