allostery in Ca 2 + channel modulation by calcium-binding proteins

Distinct modulatory ligands frequently act upon common target molecules1–3, supporting molecular computations important to biological signaling networks3,4. These ligands may act by competition, wherein only one ligand can bind at a time, or by allostery, allowing simultaneous interaction5. This contrast in classic biochemical mechanism holds crucial implications for signaling behavior but can be notoriously difficult to establish6. This is particularly true for large target molecules resistant to reconstitution outside of live cells. If studied piecemeal as subdomain peptides, such targets often exhibit multiple potential ligand-binding sites, but the relevance of these within the holomolecule often remains uncertain7. One prime example of such mechanistic ambiguity concerns the interaction of voltage-gated Ca2+ channels (CaV) with two modulatory ligands: CaM and CaBPs. To start, CaM regulation of CaV channels has a major role in the intracellular Ca2+ feedback orchestrating many biological processes8. This modulation frequently manifests as a Ca2+-dependent inactivation (CDI) of channel opening9,10, which is important for Ca2+ homeostasis11. Additionally, exciting recent discoveries identify CaBPs, a family of CaM-like brain molecules12–17, that may also bind CaV channels and other targets18,19. CaBPs are bilobed like CaM, with each lobe containing two EF-hand Ca2+-binding motifs12. However, though all four EF hands bind Ca2+ in CaM, one of them is nonfunctional in CaBPs. Coexpressing CaBPs with CaV1 channels then eliminates their CDI14,20. Indeed, modulation like this diversifies biological responses to Ca2+ signals13,14,18,21. Given the similarity of CaM and CaBPs, the prevailing hypothesis is that CaBPs competitively replace CaM on channels14,22,23. Indeed, these ligands exhibit competitive binding to an isolated channel IQ domain (Fig. 1a), a principal element for CDI. The interaction of CaM and CaBPs may thus be formulated as in Figure 1b. State 1 portrays an ‘empty’ channel without ligand that is thus incapable of CaM-mediated Ca2+ regulation24,25. This empty channel may bind CaM at the IQ domain to form a complex (state 2) that can then undergo Ca2+ regulation (for example, inactivation). Alternatively, CaBP may bind at the IQ domain, yielding a state 3 with altered Ca2+ regulatory behavior (for example, being noninactivatable). Importantly, both CaM and CaBP cannot simultaneously bind (state 4 excluded) under competition. Hypothetical dispositions of CaBP and CaM are shown in Figure 1b. As with many large molecules, studies of isolated channel peptides raise the possibility of more complex scenarios. Specifically, peptide assays hint at multiple potential binding sites for CaM and CaBPs. Thus, these two ligands need not compete at the IQ domain, and the possibility of an allosteric mechanism arises (Fig. 1b). For CaM regulation itself, interaction sites extend beyond the IQ domain (Fig. 1a). In fact, CaM regulation starts with a single apoCaM (Ca2+-free CaM) preassociated with the C-lobe of apoCaM embracing the IQ domain24–27 (Fig. 1a) and with the N-lobe engaging upstream vestigial EF hands24 (Fig. 1a), all within an encompassing Ca2+-inactivating (CI) region9,28,29. Ca2+ binding to this ‘resident’ CaM alters channel opening by driving rearrangements that may include the binding of the N-lobe of Ca2+-bound CaM (Ca2+-CaM) to the channel amino terminus30 (Fig. 1a). Regarding CaBPs, peptide assays also hint at interactions beyond the IQ domain31,32, but the relevance of these assays to holochannels remains unsettled. One study argues for the functional impact of such sites32, whereas another argues otherwise31. In all, the scheme of CaM and CaBP interaction remains ambiguous (Fig. 1b), and clear tests for concurrent ligand binding to holochannels (i.e., state 4) have proven infeasible. This state of impasse is common to many large molecules33,34. Importantly, the distinctions between competitive versus allosteric mechanism concern more than academic interest; they seem to be critical to the biological activity of CaBPs. In particular, the affinity of CaV channels for apoCaM may be greater than that for CaBPs14,25,26, as the present study substantiates. Moreover, this study highlights a predominance of CaM over CaBP expression throughout the brain. Under competition, then, how could CaBPs exert functional effects? By contrast, an allosteric mechanism with the right mix of parameters would readily explain how naturally occurring CaBPs could still exert modulation. Here, we establish an allosteric mechanism for CaV1.3 Ca2+ channels, a robust prototype system where strong CaM-mediated CDI can nonetheless be completely suppressed by CaBPs. Our approach is to exploit covalent linkage of holochannels to CaM and/or CaBP, along with live-cell fluorescence resonance energy