Calmodulin regulates TRPV5 intracellular trafficking and plasma membrane abundance

As a member of the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a unique Ca²⁺-selective channel important for active reabsorption of Ca²⁺ in the kidney. TRPV5-mediated Ca²⁺ entry into the cell is controlled by a negative feedback mechanism, in which calmodulin (CaM) blocks the TRPV5 pore upon Ca²⁺ binding. Combining microscopy techniques and biochemical assays, the present study uncovered an auxiliary role for CaM in the regulation of human (h)TRPV5 intracellular trafficking. Overexpressed hTRPV5 was mainly localised to the endoplasmic reticulum (ER) and associated with peripheral ER tubules. Limiting expression using the HEK293 TET-off system revealed that hTRPV5 trafficked through the endocytic recycling pathway. CaM co-localised with hTRPV5 at intracellular sites and overexpression of CaM slowed hTRPV5 exit from the ER. In accordance, CaM binding-disrupting truncations of the TRPV5 C-terminus (698X) or knockdown of endogenous CaM by small interfering RNA resulted in an increased fraction of TRPV5 that localised to the plasma membrane. hTRPV5 expressing cells had an increased intracellular Ca²⁺ concentration upon knockdown of CaM. The protein abundance of the Ca²⁺ impermeable hTRPV5-D542 mutant is also regulated by CaM, which suggests that the mode of action is independent of disrupted intracellular calcium concentrations. In conclusion, our study reveals a novel role for CaM in Ca²⁺-dependent TRPV5 regulation, modulating TRPV5 intracellular trafficking. KEY POINTS: The renal Ca²⁺ channel TRPV5 is a crucial player in maintenance of the body's Ca²⁺ homeostasis. Ca²⁺ transport through TRPV5 is controlled by single channel activity, as well as TRPV5 plasma membrane abundance. Calmodulin (CaM) co-localised with TRPV5 at intracellular sites and retained TRPV5 in the endoplasmic reticulum. Disrupted CaM-TRPV5 binding or knockdown of endogenous CaM by small interfering RNA (siRNA) resulted in an increased TRPV5 plasma membrane abundance. Knockdown of endogenous CaM by siRNA resulted in increased intracellular Ca²⁺ concentrations. The regulation of TRPV5 trafficking by CaM is independent of the effect of CaM on intracellular Ca²⁺ concentrations. This study reveals a novel role for CaM in Ca²⁺-dependent TRPV5 regulation, next to its ability to directly block the TRPV5 channel pore, by modulating TRPV5 trafficking in the secretory pathway.