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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.