Centrin controls the activity of the ciliary reversal-coupled voltage-gated Ca2+ channels Ca2+-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.
Gonda
Kohsuke
K
Biomedical and Engineering Research Organization, Tohoku University, Sendai, Miyagi 980-8579, Japan; Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan. Electronic address: gonda@tubero.tohoku.ac.jp.
Oami
Kazunori
K
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.
Takahashi
Mihoko
M
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.
D000200:Action Potentials; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D004594:Electrophysiology; D008969:Molecular Sequence Data; D009154:Mutation; D010247:Paramecium / Q000378:metabolism; D011188:Potassium / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015662:Trimethoprim, Sulfamethoxazole Drug Combination / Q000737:chemistry* / Q000378:metabolism
D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; D015662:Trimethoprim, Sulfamethoxazole Drug Combination; D011188:Potassium; D002118:Calcium