Allostery in Ca2+ channel modulation by calcium-binding proteins

Abstract

Distinguishing between allostery and competition among modulating ligands is challenging for large target molecules. 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). Since CaBPs resemble CaM, CaBPs are believed to competitively replace CaM on targets. Yet, brain CaM expression far surpasses that of CaBPs, so how can CaBPs 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 FRET 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.

Localization and expression of CaBP1/caldendrin in the mouse brain

“…Other CaBPs may also play a role in shaping cerebellar Ca 2+ signaling. While CaBP2 and CaBP5 show no expression in the brain (Haeseleer et al, 2000), a recent study demonstrates the presence of CaBP4 transcripts in the cerebellum (Yang et al, 2014). Further studies will be necessary to define the cellular localization and potential function of CaBP4 in the cerebellum.…”

CaBP1 regulates Cav1 L-type Ca2+ channels and their coupling to neurite growth and gene transcription in mouse spiral ganglion neurons

“…In particular, members of a family of Ca 2+ binding proteins (CaBPs) related to CaM are highly expressed in neural tissues (Haeseleer et al, 2000) and suppress CDI in heterologous expression systems (Hardie and Lee, 2016). The mechanism is thought to involve competitive displacement of CaM from the channel complex (Findeisen et al, 2013; Zhou et al, 2004) as well as allosteric modulation (Oz et al, 2013; Yang et al, 2014). …”

Detecting stoichiometry of macromolecular complexes in live cells using FRET

“…Second, unlike super-resolution approaches, fluorescence measurements for our method are obtained from freely diffusing complexes, and determined across a broad expression profile of donor- and acceptor-tagged binding partners. Accordingly, our assay is only minimally sensitive to unlabelled endogenous proteins and errors introduced by variable cellular expression of relevant binding partners, thereby permitting the study of molecules like CaM that are ubiquitous in all eukaryotic cells58. Third, our method does not require a priori knowledge of the spatial arrangement of individual donor-acceptor pairs.…”

“…In fact, to determine stoichiometry, we overexpress the CFP- or YFP-tagged binding partners to obtain the saturating values E A,max and E D,max . Under these conditions, the effect of endogenous protein is minimal58. A second confounding factor for determination of maximal apparent FRET efficiencies is slow or incomplete maturation of many fluorescent proteins that yield molecules with little fluorescence output3561.…”