TY - JOUR
T1 - Allostery in Ca 2+ channel modulation by calcium-binding proteins
AU - Yang, Philemon S.
AU - Johny, Manu Ben
AU - Yue, David T.
N1 - Funding Information:
We thank W. Yang for dedicated technical support and other members of the Ca2+ signals lab for valuable comments. H. Bazzazi generously made available the β2a–CaMWT construct. Y. Gao contributed the data analysis showing CaM, CaBP4 and CaBP1 gene expression from human prefrontal cortex. This work is supported by grants from the US National Institutes of Health (NIH) Heart, Lung, and Blood Institute MERIT Award (to D.T.Y.), NIH National Institute on Deafness and Other Communication Disorders (P.S.Y. and P. Fuchs), NIH National Institute of General Medical Sciences (GM08752 to P.S.Y.) and the NIH National Institute of Mental Health (M.B.J.).
PY - 2014/3
Y1 - 2014/3
N2 - Distinguishing between allostery and competition among modulating ligands is challenging for large target molecules. Out of practical necessity, inferences are often drawn from in vitro assays on target fragments, but such inferences may belie actual mechanisms. One key example of such ambiguity concerns calcium-binding proteins (CaBPs) that tune signaling molecules regulated by calmodulin (CaM). As CaBPs resemble CaM, CaBPs are believed to competitively replace CaM on targets. Yet, brain CaM expression far surpasses that of CaBPs, raising questions as to whether CaBPs can exert appreciable biological actions. Here, we devise a live-cell, holomolecule approach that reveals an allosteric mechanism for calcium channels whose CaM-mediated inactivation is eliminated by CaBP4. Our strategy is to covalently link CaM and/or CaBP to holochannels, enabling live-cell fluorescence resonance energy transfer assays to resolve a cyclical allosteric binding scheme for CaM and CaBP4 to channels, thus explaining how trace CaBPs prevail. This approach may apply generally for discerning allostery in live cells.
AB - Distinguishing between allostery and competition among modulating ligands is challenging for large target molecules. Out of practical necessity, inferences are often drawn from in vitro assays on target fragments, but such inferences may belie actual mechanisms. One key example of such ambiguity concerns calcium-binding proteins (CaBPs) that tune signaling molecules regulated by calmodulin (CaM). As CaBPs resemble CaM, CaBPs are believed to competitively replace CaM on targets. Yet, brain CaM expression far surpasses that of CaBPs, raising questions as to whether CaBPs can exert appreciable biological actions. Here, we devise a live-cell, holomolecule approach that reveals an allosteric mechanism for calcium channels whose CaM-mediated inactivation is eliminated by CaBP4. Our strategy is to covalently link CaM and/or CaBP to holochannels, enabling live-cell fluorescence resonance energy transfer assays to resolve a cyclical allosteric binding scheme for CaM and CaBP4 to channels, thus explaining how trace CaBPs prevail. This approach may apply generally for discerning allostery in live cells.
UR - http://www.scopus.com/inward/record.url?scp=84894049891&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84894049891&partnerID=8YFLogxK
U2 - 10.1038/nchembio.1436
DO - 10.1038/nchembio.1436
M3 - Article
C2 - 24441587
AN - SCOPUS:84894049891
SN - 1552-4450
VL - 10
SP - 231
EP - 238
JO - Nature chemical biology
JF - Nature chemical biology
IS - 3
ER -