No abstract

Calmodulin regulation of CaV channels is a prominent Ca2+ feedback mechanism orchestrating vital adjustments of Ca2+ entry. The long-held structural correlate of this regulation has been Ca2+-bound calmodulin complexed alone with an IQ domain on the channel carboxy terminus. Here, however, systematic alanine mutagenesis of the entire carboxyl tail of an L-type CaV1.3 channel casts doubt on this paradigm. To identify the actual molecular states underlying channel regulation, we develop a structure-function approach relating the strength of regulation to the affinity of underlying calmodulin/channel interactions, by a Langmuir relation (iTL analysis). Accordingly, we uncover frank exchange of Ca2+-calmodulin to interfaces beyond the IQ domain, initiating substantial rearrangements of the calmodulin/channel complex. The N-lobe of Ca2+-calmodulin binds an NSCaTE module on the channel amino terminus, while the C-lobe binds an EF-hand region upstream of the IQ domain. This system of structural plasticity furnishes a next-generation blueprint for CaV channel modulation.

SUMMARY Voltage-gated Na and Ca2+channels comprise distinct ion-channel superfamilies, yet the carboxy tails of these channels exhibit high homology hinting at a long-shared and purposeful module. For different Ca2+ channels, carboxyl-tail inter actions with calmodulin do elaborate robust and similar forms of Ca2+ regulation. However, Na channels have only shown subtler Ca2+modulation that differs among reports, challenging attempts at unified understanding. Here, by rapid Ca2+photoreleaseon to Na channels, we reset this view of Na channel regulation. For cardiac muscle channels (NaV1.5), reported effects from which most mechanistic proposals derive, we observe no Ca2+modulation. Conversely, for skeletal-muscle channels (NaV1.4), we uncover fast Ca2+ regulation eerily similar to that of Ca2+ channels. Channel opathic myotonia mutations halve NaV1.4 Ca2+ regulation, and transplanting the NaV1.4 carboxy tail onto Ca2+ channels recapitulates Ca2+ regulation. Thus we argue for the persistence and physiological relevance of an ancient Ca2+ regulatory module across Na and Ca2+ channels.

Ca2+ channels and calmodulin are two prominent signaling hubs1 that synergistically impact functions as diverse as cardiac excitability2, synaptic plasticity3, and gene transcription4. It is thereby fitting that these hubs are in some sense coordinated, as the opening of CaV1-2 Ca2+ channels are regulated by a single calmodulin (CaM) constitutively complexed with channels5. The Ca2+-free form of CaM (apoCaM) is already preassociated with the IQ domain on the channel carboxy terminus, and subsequent Ca2+ binding to this ‘resident’ CaM drives conformational changes that then trigger regulation of channel opening6. Another potential avenue for channel-CaM coordination could arise from the absence of Ca2+ regulation in channels lacking a preassociated CaM6,7. Natural fluctuations in CaM levels might then influence the fraction of regulatable channels, and thereby the overall strength of Ca2+ feedback. However, the prevailing view has been that the ultra-strong affinity of channels for apoCaM ensures their saturation with CaM8, yielding a significant form of concentration independence between Ca2+ channels and CaM. Here, we reveal significant exceptions to this autonomy, by combining electrophysiology to characterize channel regulation, with optical FRET sensor determination of free apoCaM concentration in live cells9. This approach translates quantitative CaM biochemistry from the traditional test-tube context, into the realm of functioning holochannels within intact cells. From this perspective, we find that long splice forms of CaV1.3 and CaV1.4 channels include a distal carboxy tail10-12 that resembles an enzyme competitive inhibitor, which retunes channel affinity for apoCaM so that natural CaM variations affect the strength of Ca2+ feedback modulation. Given the ubiquity of these channels13,14, the connection between ambient CaM levels and Ca2+ entry via channels is broadly significant for Ca2+ homeostasis. Strategies like ours promise key advances for the in situ analysis of signaling molecules resistant to in vitro reconstitution, such as Ca2+ channels.

Voltage-gated Na and Ca2+ channels represent two major ion channel families that enable myriad biological functions including the generation of action potentials and the coupling of electrical and chemical signaling in cells. Calmodulin regulation (calmodulation) of these ion channels comprises a vital feedback mechanism with distinct physiological implications. Though long-sought, a shared understanding of the channel families remained elusive for two decades as the functional manifestations and the structural underpinnings of this modulation often appeared to diverge. Here, we review recent advancements in the understanding of calmodulation of Ca2+ and Na channels that suggest a remarkable similarity in their regulatory scheme. This interrelation between the two channel families now paves the way towards a unified mechanistic framework to understand vital calmodulin-dependent feedback and offers shared principles to approach related channelopathic diseases. An exciting era of synergistic study now looms.

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.

Förster resonance energy transfer (FRET) is a versatile method for analyzing protein-protein interactions within living cells. This protocol describes a nondestructive live-cell FRET assay for robust quantification of relative binding affinities for protein-protein interactions. Unlike other approaches, our method correlates the measured FRET efficiencies to relative concentration of interacting proteins to determine binding isotherms while including collisional FRET corrections. We detail how to assemble and calibrate the equipment using experimental and theoretical procedures. A step-by-step protocol is given for sample preparation, data acquisition and analysis. The method uses relatively inexpensive and widely available equipment and can be performed with minimal training. Implementation of the imaging setup requires up to 1 week, and sample preparation takes ∼1-3 d. An individual FRET experiment, including control measurements, can be completed within 4-6 h, with data analysis requiring an additional 1-3 h.

Calmodulin (CaM) regulation of voltage-gated calcium (CaV1-2) channels is a powerful Ca2+-feedback mechanism to adjust channel activity in response to Ca2+ influx. Despite progress in resolving mechanisms of CaM-CaV feedback, the stoichiometry of CaM interaction with CaV channels remains ambiguous. Functional studies that tethered CaM to CaV1.2 suggested that a single CaM sufficed for Ca2+ feedback. Yet, biochemical, FRET, and structural studies showed that multiple CaM molecules interact with distinct interfaces within channel cytosolic segments suggesting that functional Ca2+-regulation may be more nuanced. Resolving this ambiguity is critical as CaM is enriched in subcellular domains where CaV channels reside, such as the cardiac dyad. We here localized multiple CaM to the CaV nanodomain by tethering either wild-type or mutant CaM that lack Ca2+-binding capacity to the pore-forming α-subunit of CaV1.2, CaV1.3, and CaV2.1 and/or the auxiliary β2A subunit. We observed that a single CaM tethered to either the α or β2A subunit tunes Ca2+-regulation of CaV channels. However, when multiple CaMs are localized concurrently, CaV channels preferentially respond to signaling from the α-subunit tethered CaM. Mechanistically, the introduction of a second IQ domain to the CaV1.3 carboxy-tail switched the apparent functional stoichiometry permitting two CaMs to mediate functional regulation. In all, Ca2+-feedback of CaV channels depends exquisitely on a single CaM pre-associated to the α-subunit carboxy-tail. Additional CaMs that colocalize with the channel complex are unable to trigger Ca2+-depended feedback of channel gating but may support alternate regulatory functions.