Calpastatin binds to a calmodulin-binding site of cardiac Cav1.2 Ca2+ channels.
Calpastatin is an endogenous inhibitor of calpain and composed of domain L (CS(L)), which interacts with the Cav1.2 channels, and four repetitive calpain inhibitory domains. We have previously found that CS(L) reprimes activity of the Cav1.2 channels in cell-free patches of cardiac myocytes [L.Y. Hao, A. Kameyama, S. Kuroki, J. Takano, E. Takano, M. Maki, M. Kameyama, Calpastatin domain L is involved in the regulation L-type of Ca2+ channels in guinea pig cardiac myocytes, Biochem. Biophys. Res. Commun. 279 (2000) 756-761; E. Minobe, L.Y. Hao, Z.A. Saud, J.J. Xu, A. Kameyama, M. Maki, K.K. Jewell, T. Parr, R.G. Bardsley, M. Kameyama, A region of calpastatin domain L that reprimes cardiac L-type Ca2+ channels, Biochem. Biophys. Res. Commun. 348 (2006) 288-294]. In this study, we explored the CS(L) interaction site in the Ca2+ channel by the pull-down method, using glutathione-S-transferase-fused fragment peptides of the Cav1.2 channel. CS(L) bound directly to a proximal region of the C-terminal tail of the channel, but not with the N-terminal tail, a distal region of the C-terminal tail or cytoplasmic loops between repeats I-II, II-III or III-IV. Furthermore IQ domain, but not EF-hand-like region or CB domain, in the C-terminal tail was found to bind with CS(L) in a partially Ca2+-dependent manner and in a probably competitive manner with calmodulin. These results suggest that CS(L) modulates Ca2+-channel activity through interacting with the calmodulin-binding site on the C-terminal tail of the Cav1.2 channel.
Saud
Zahangir A
ZA
Department of Physiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8544, Japan.
Minobe
Etsuko
E
Wang
Wu-yang
WY
Han
Dong-yun
DY
Horiuchi
Masahisa
M
Hao
Li-ying
LY
Kameyama
Masaki
M
D000818:Animals; D001665:Binding Sites; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics; D006168:Guinea Pigs; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
D020746:Calcium Channels, L-Type; D002135:Calcium-Binding Proteins; D002147:Calmodulin; C419385:L-type calcium channel alpha(1C); D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C033668:calpastatin; D005982:Glutathione Transferase