Approximately 15% of autosomal dominant polycystic kidney disease (ADPKD) is caused by variants in PKD2 PKD2 encodes polycystin-2, which forms an ion channel in primary cilia and endoplasmic reticulum (ER) membranes of renal collecting duct cells. Elevated internal Ca2+ modulates polycystin-2 voltage dependent gating and subsequent desensitization- two biophysical regulatory mechanisms that control its function at physiological membrane potentials. Here, we refute the hypothesis that Ca2+ occupancy of the polycytsin-2 intracellular EF hand is responsible for these forms of channel regulation, and if disrupted, results in ADPKD. We identify and introduce mutations that attenuate Ca2+-EF hand affinity but find channel function is unaltered in the primary cilia and ER membranes. We generated two new mouse strains that harbor distinct mutations that abolish Ca2+-EF hand association but do not result in a PKD phenotype. Our findings suggest additional Ca2+ binding sites within polycystin-2 or Ca2+-dependent modifiers are responsible for regulating channel activity. © 2020. Published by The Company of Biologists Ltd.