Centrin controls the activity of the ciliary reversal-coupled voltage-gated Ca channels Ca-dependently

In Paramecium, ciliary reversal is coupled with voltage-gated Ca 2+ channels on the ciliary membrane. We previously isolated a P. caudatum mutant, cnrC, with a malfunction of the Ca 2+ channels and discovered that the channel activity of cnrC was restored by transfection of the P. caudatum centrin (Pccentrin1p) gene, which encodes a member of the Ca 2+-binding EF-hand protein family. In this study, we injected various mutated Pccentrin1p genes into cnrC and investigated whether these genes restore the Ca 2+ channel activity of cnrC. A Pccentrin1p mutant gene lacking Ca 2+ sensitivity of the third and fourth EF-hands lost the ability to restore the channel function of cnrC, and mutation of the fourth EF-hand caused more serious impairment than mutation of the third EF-hand. Moreover, a Pccentrin1p gene lacking the N-terminal 34-amino acid sequence also lost the ability to restore the channel activity. Native-PAGE analysis demonstrated that the N-terminal sequence is important for the Ca 2+-dependent structural change of Pccentrin1p. These results demonstrate that Pccentrin1p Ca 2+-dependently regulates the Ca 2+ channel activity in vivo.