Polycystin 2 (PC2) is one of the two proteins that when mutated result in polycystic kidney disease. PC2 is a calcium (Ca2+) release channel that opens and closes in response to increasing intracellular Ca2+. We have previously identified an EF hand motif on PC2 that controls the Ca2+ sensitivity of the protein. The EF hand motif is a well-conserved region for Ca2+ binding and is a typically found as a pair (EF hand domain). Mammalian PC2 differs from most EF domain containing proteins in that the first EF hand motif is non-canonical (site 1), and cannot bind Ca2+, whereas the second EF hand motif (site 2) binds Ca2+. However, the EF domain of PC2 proteins in evolutionary earlier organisms can bind two Ca2+ molecules, albeit with different affinity.
In this study, we sought to determine if the two EF motifs in mammalian PC2 are interchangeable, or if Ca2+ binding to site 2 only is essential and necessary for PC2 function. We created a series of mutants in the mammalian EF hand domain with one or two functional Ca2+ binding sites. Expression of these mutants in mammalian kidney cells revealed that binding of Ca2+ to site 2 was essential for function. However, the Ca2+ signal could be enhanced by the introduction of an additional Ca2+ binding site at site 1. We conclude that the position and affinity of the Ca2+ binding site in PC2 is critical for maintaining function.