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In cardiomyocytes, Na V 1.5 channels mediate initiation and fast propagation of action potentials. The Ca 2+ -binding protein calmodulin (CaM) serves as a de facto subunit of Na V 1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the Na V 1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a "gain-of-function" defect, and Brugada syndrome, a "loss-of-function" phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on Na V 1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca 2+ -free CaM preassociation with Na V 1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent Na V openings. Mechanistically, these effects arise from a CaM-dependent switch in the Na V inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant Na V 1.5, local Ca 2+ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of Na V 1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of Na V 1.5 channelopathies., Competing Interests: The authors declare no competing interest .

Ca 2+ /calmodulin-dependent inactivation (CDI) of Ca V channels is a critical regulatory process that tunes the kinetics of Ca 2+ entry for different cell types and physiologic responses. CDI is mediated by calmodulin (CaM), which is bound to the IQ domain of the Ca V carboxy tail. This modulatory process is tailored by alternative splicing such that select splice variants of Ca V 1.3 and Ca V 1.4 contain a long distal carboxy tail (DCT). The DCT harbors an inhibitor of CDI (ICDI) module that competitively displaces CaM from the IQ domain, thereby diminishing CDI. While this overall mechanism is now well described, the detailed interactions required for ICDI binding to the IQ domain are yet to be elucidated. Here, we perform alanine-scanning mutagenesis of the IQ and ICDI domains and evaluate the contribution of neighboring regions to CDI inhibition. Through FRET binding analysis, we identify functionally relevant residues within the Ca V 1.3 IQ domain and the Ca V 1.4 ICDI and nearby A region, which are required for high-affinity IQ/ICDI binding. Importantly, patch-clamp recordings demonstrate that disruption of this interaction commensurately diminishes ICDI function resulting in the re-emergence of CDI in mutant channels. Furthermore, Ca V 1.2 channels harbor a homologous DCT; however, the ICDI region of this channel does not appear to appreciably modulate Ca V 1.2 CDI. Yet coexpression of Ca V 1.2 ICDI with select Ca V 1.3 splice variants significantly disrupts CDI, implicating a cross-channel modulatory scheme in cells expressing both channel subtypes. In all, these findings provide new insights into a molecular rheostat that fine-tunes Ca 2+ -entry and supports normal neuronal and cardiac function., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Calmodulin (CaM) regulation of voltage-gated calcium (Ca V 1-2) channels is a powerful Ca 2+ -feedback mechanism to adjust channel activity in response to Ca 2+ influx. Despite progress in resolving mechanisms of CaM-Ca V feedback, the stoichiometry of CaM interaction with Ca V channels remains ambiguous. Functional studies that tethered CaM to Ca V 1.2 suggested that a single CaM sufficed for Ca 2+ feedback, yet biochemical, FRET, and structural studies showed that multiple CaM molecules interact with distinct interfaces within channel cytosolic segments, suggesting that functional Ca 2+ regulation may be more nuanced. Resolving this ambiguity is critical as CaM is enriched in subcellular domains where Ca V channels reside, such as the cardiac dyad. We here localized multiple CaMs to the Ca V nanodomain by tethering either WT or mutant CaM that lack Ca 2+ -binding capacity to the pore-forming α-subunit of Ca V 1.2, Ca V 1.3, and Ca V 2.1 and/or the auxiliary β 2A subunit. We observed that a single CaM tethered to either the α or β 2A subunit tunes Ca 2+ regulation of Ca V channels. However, when multiple CaMs are localized concurrently, Ca V channels preferentially respond to signaling from the α-subunit-tethered CaM. Mechanistically, the introduction of a second IQ domain to the Ca V 1.3 carboxyl tail switched the apparent functional stoichiometry, permitting two CaMs to mediate functional regulation. In all, Ca 2+ feedback of Ca V channels depends exquisitely on a single CaM preassociated with the α-subunit carboxyl tail. Additional CaMs that colocalize with the channel complex are unable to trigger Ca 2+ -dependent feedback of channel gating but may support alternate regulatory functions., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Chakouri et al.)

The Ca2+-binding protein calmodulin has emerged as a pivotal player in tuning Na+ channel function, although its impact in vivo remains to be resolved. Here, we identify the role of calmodulin and the NaV1.5 interactome in regulating late Na+ current in cardiomyocytes. We created transgenic mice with cardiac-specific expression of human NaV1.5 channels with alanine substitutions for the IQ motif (IQ/AA). The mutations rendered the channels incapable of binding calmodulin to the C-terminus. The IQ/AA transgenic mice exhibited normal ventricular repolarization without arrhythmias and an absence of increased late Na+ current. In comparison, transgenic mice expressing a lidocaine-resistant (F1759A) human NaV1.5 demonstrated increased late Na+ current and prolonged repolarization in cardiomyocytes, with spontaneous arrhythmias. To determine regulatory factors that prevent late Na+ current for the IQ/AA mutant channel, we considered fibroblast growth factor homologous factors (FHFs), which are within the NaV1.5 proteomic subdomain shown by proximity labeling in transgenic mice expressing NaV1.5 conjugated to ascorbate peroxidase. We found that FGF13 diminished late current of the IQ/AA but not F1759A mutant cardiomyocytes, suggesting that endogenous FHFs may serve to prevent late Na+ current in mouse cardiomyocytes. Leveraging endogenous mechanisms may furnish an alternative avenue for developing novel pharmacology that selectively blunts late Na+ current.

Skeletal muscle voltage-gated Na + channel (Na V 1.4) activity is subject to calmodulin (CaM) mediated Ca 2+ -dependent inactivation; no such inactivation is observed in the cardiac Na + channel (Na V 1.5). Taken together, the crystal structures of the Na V 1.4 C-terminal domain relevant complexes and thermodynamic binding data presented here provide a rationale for this isoform difference. A Ca 2+ -dependent CaM N-lobe binding site previously identified in Na V 1.5 is not present in Na V 1.4 allowing the N-lobe to signal other regions of the Na V 1.4 channel. Consistent with this mechanism, removing this binding site in Na V 1.5 unveils robust Ca 2+ -dependent inactivation in the previously insensitive isoform. These findings suggest that Ca 2+ -dependent inactivation is effected by CaM's N-lobe binding outside the Na V C-terminal while CaM's C-lobe remains bound to the Na V C-terminal. As the N-lobe binding motif of Na V 1.5 is a mutational hotspot for inherited arrhythmias, the contributions of mutation-induced changes in CDI to arrhythmia generation is an intriguing possibility.

Calmodulin (CaM) regulation of voltage-gated calcium (Ca V ) channels is a powerful Ca 2+ feedback mechanism that adjusts Ca 2+ influx, affording rich mechanistic insights into Ca 2+ decoding. CaM possesses a dual-lobed architecture, a salient feature of the myriad Ca 2+ -sensing proteins, where two homologous lobes that recognize similar targets hint at redundant signaling mechanisms. Here, by tethering CaM lobes, we demonstrate that bilobal architecture is obligatory for signaling to Ca V channels. With one lobe bound, Ca V carboxy tail rearranges itself, resulting in a preinhibited configuration precluded from Ca 2+ feedback. Reconstitution of two lobes, even as separate molecules, relieves preinhibition and restores Ca 2+ feedback. Ca V channels thus detect the coincident binding of two Ca 2+ -free lobes to promote channel opening, a molecular implementation of a logical NOR operation that processes spatiotemporal Ca 2+ signals bifurcated by CaM lobes. Overall, a unified scheme of Ca V channel regulation by CaM now emerges, and our findings highlight the versatility of CaM to perform exquisite Ca 2+ computations., Competing Interests: The authors declare no conflict of interest.

Voltage-gated Na and Ca(2+) 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 Ca(2+) 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.

The Ca(2+)-free form of calmodulin (apoCaM) often appears inert, modulating target molecules only upon conversion to its Ca(2+)-bound form. This schema has appeared to govern voltage-gated Ca(2+) channels, where apoCaM has been considered a dormant Ca(2+) sensor, associated with channels but awaiting the binding of Ca(2+) ions before inhibiting channel opening to provide vital feedback inhibition. Using single-molecule measurements of channels and chemical dimerization to elevate apoCaM, we find that apoCaM binding on its own markedly upregulates opening, rivaling the strongest forms of modulation. Upon Ca(2+) binding to this CaM, inhibition may simply reverse the initial upregulation. As RNA-edited and -spliced channel variants show different affinities for apoCaM, the apoCaM-dependent control mechanisms may underlie the functional diversity of these variants and explain an elongation of neuronal action potentials by apoCaM. More broadly, voltage-gated Na channels adopt this same modulatory principle. ApoCaM thus imparts potent and pervasive ion-channel regulation. PAPERCLIP:

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 interactions 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+ photorelease onto 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. Channelopathic 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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Calmodulin regulation (calmodulation) of the family of voltage-gated CaV1-2 channels comprises a prominent prototype for ion channel regulation, remarkable for its powerful Ca(2+) sensing capabilities, deep in elegant mechanistic lessons, and rich in biological and therapeutic implications. This field thereby resides squarely at the epicenter of Ca(2+) signaling biology, ion channel biophysics, and therapeutic advance. This review summarizes the historical development of ideas in this field, the scope and richly patterned organization of Ca(2+) feedback behaviors encompassed by this system, and the long-standing challenges and recent developments in discerning a molecular basis for calmodulation. We conclude by highlighting the considerable synergy between mechanism, biological insight, and promising therapeutics., (© 2014 Ben-Johny and Yue.)

Calmodulin regulation of CaV channels is a prominent Ca(2+) feedback mechanism orchestrating vital adjustments of Ca(2+) entry. The long-held structural correlation of this regulation has been Ca(2+)-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 (individually transformed Langmuir analysis). Accordingly, we uncover frank exchange of Ca(2+)-calmodulin to interfaces beyond the IQ domain, initiating substantial rearrangements of the calmodulin/channel complex. The N-lobe of Ca(2+)-calmodulin binds an N-terminal spatial Ca(2+) transforming element module on the channel amino terminus, whereas 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.

Ca(2+)/calmodulin- and voltage-dependent inactivation (CDI and VDI) comprise vital prototypes of Ca(2+) channel modulation, rich with biological consequences. Although the events initiating CDI and VDI are known, their downstream mechanisms have eluded consensus. Competing proposals include hinged-lid occlusion of channels, selectivity filter collapse, and allosteric inhibition of the activation gate. Here, novel theory predicts that perturbations of channel activation should alter inactivation in distinctive ways, depending on which hypothesis holds true. Thus, we systematically mutate the activation gate, formed by all S6 segments within Ca(V)1.3. These channels feature robust baseline CDI, and the resulting mutant library exhibits significant diversity of activation, CDI, and VDI. For CDI, a clear and previously unreported pattern emerges: activation-enhancing mutations proportionately weaken inactivation. This outcome substantiates an allosteric CDI mechanism. For VDI, the data implicate a "hinged lid-shield" mechanism, similar to a hinged-lid process, with a previously unrecognized feature. Namely, we detect a "shield" in Ca(V)1.3 channels that is specialized to repel lid closure. These findings reveal long-sought downstream mechanisms of inactivation and may furnish a framework for the understanding of Ca(2+) channelopathies involving S6 mutations.