Calcium binding to the EF hand motif regulates polycystin 2 function

I.Y. Kuo,¹ C. Keeler,² M.E. Hodsdon² and B.E. Ehrlich,¹ ¹Department of Pharmacology, School of Medicine, Yale University. 333 Cedar St, New Haven, CT 06520, USA and ²Department of Laboratory Medicine, School of Medicine, Yale University, 333 Cedar St, New Haven, CT 06520, USA.

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.