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Calmodulinopathies are caused by mutations in calmodulin (CaM), and result in debilitating cardiac arrythmias such as long-QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). In addition, many patients exhibit neurological comorbidities, including developmental delay and autism spectrum disorder. Until now, most work into these mutations has focused on cardiac effects, identifying impairment of Ca 2+ /CaM-dependent inactivation (CDI) of Ca V 1.2 channels as a major pathogenic mechanism. However, the impact of these mutations on neurological function has yet to be fully explored. CaM regulation of voltage-gated calcium channels (VGCCs) is a critical element of neuronal function, implicating multiple VGCC subtypes in the neurological pathogenesis of calmodulinopathies. Here, we explore the potential for pathological CaM variants to impair the Ca 2+ /CaM-dependent regulation of Ca V 1.3 and Ca V 2.1, both essential for neuronal function. We find that mutations in CaM can impair the CDI of Ca V 1.3 and reduce the Ca 2+ -dependent facilitation (CDF) of Ca V 2.1 channels. We find that mutations associated with significant neurological symptoms exhibit marked effects on Ca V 1.3 CDI, with overlapping but distinct impacts on Ca V 2.1 CDF. Moreover, while the majority of CaM variants demonstrated the ability to bind the IQ region of each channel, distinct differences were noted between Ca V 1.3 and Ca V 2.1, demonstrating distinct CaM interactions across the two channel subtypes. Further, C-domain CaM variants display a reduced ability to sense Ca 2+ when in complex with the Ca V IQ domains, explaining the Ca 2+ /CaM regulation deficits. Overall, these results support the possibility that disrupted Ca 2+ /CaM regulation of VGCCs may contribute to neurological pathogenesis of calmodulinopathies.

L-type Ca 2+ channels (Ca V 1.2/1.3) convey influx of calcium ions that orchestrate a bevy of biological responses including muscle contraction, neuronal function, and gene transcription. Deficits in Ca V 1 function play a vital role in cardiac and neurodevelopmental disorders. Here, we develop a genetically encoded enhancer of Ca V 1.2/1.3 channels (GeeC L ) to manipulate Ca 2+ entry in distinct physiological settings. We functionalized a nanobody that targets the Ca V complex by attaching a minimal effector domain from an endogenous Ca V modulator-leucine-rich repeat containing protein 10 (Lrrc10). In cardiomyocytes, GeeC L selectively increased L-type current amplitude. In neurons in vitro and in vivo, GeeC L augmented excitation-transcription (E-T) coupling. In all, GeeC L represents a powerful strategy to boost Ca V 1.2/1.3 function and lays the groundwork to illuminate insights on neuronal and cardiac physiology and disease.

NEDD4L is a HECT-type E3 ligase that catalyzes the addition of ubiquitin to intracellular substrates such as the cardiac voltage-gated sodium channel, Na V 1.5. The intramolecular interactions of NEDD4L regulate its enzymatic activity which is essential for proteostasis. For Na V 1.5, this process is critical as alterations in Na + current is involved in cardiac diseases including arrhythmias and heart failure. In this study, we perform extensive biochemical and functional analyses that implicate the C2 domain and the first WW-linker (1,2-linker) in the autoregulatory mechanism of NEDD4L. Through in vitro and electrophysiological experiments, the NEDD4L 1,2-linker was determined to be important in substrate ubiquitination of Na V 1.5. We establish the preferred sites of ubiquitination of NEDD4L to be in the second WW-linker (2,3-linker). Interestingly, NEDD4L ubiquitinates the cytoplasmic linker between the first and second transmembrane domains of the channel (DI-DII) of Na V 1.5. Moreover, we design a genetically encoded modulator of Nav1.5 that achieves Na + current reduction using the NEDD4L HECT domain as cargo of a Na V 1.5-binding nanobody. These investigations elucidate the mechanisms regulating the NEDD4 family and furnish a new molecular framework for understanding Na V 1.5 ubiquitination., Competing Interests: Conflict of interest The authors declare no conflicts of interest in regards to this manuscript. S. B. G. is a cofounder and equity holder in the company Advanced Molecular Sciences, LLC. S. B. G. has been or is a consultant for Scorpion Therapeutics and Xinthera. P. A. C. has been a consultant for Scorpion Therapeutics., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Voltage-dependent and Ca2+-dependent inactivation (VDI and CDI, respectively) of CaV channels are two biologically consequential feedback mechanisms that fine-tune Ca2+ entry into neurons and cardiomyocytes. Although known to be initiated by distinct molecular events, how these processes obstruct conduction through the channel pore remains poorly defined. Here, focusing on ultrahighly conserved tryptophan residues in the interdomain interfaces near the selectivity filter of CaV1.3, we demonstrate a critical role for asymmetric conformational changes in mediating VDI and CDI. Specifically, mutagenesis of the domain III-IV interface, but not others, enhanced VDI. Molecular dynamics simulations demonstrate that mutations in distinct selectivity filter interfaces differentially impact conformational flexibility. Furthermore, mutations in distinct domains preferentially disrupt CDI mediated by the N- versus C-lobes of CaM, thus uncovering a scheme of structural bifurcation of CaM signaling. These findings highlight the fundamental importance of the asymmetric arrangement of the pseudotetrameric CaV pore domain for feedback inhibition., (© 2024 del Rivero Morfin et al.)

The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac response depends on β-adrenergic-initiated reversal of the small RGK G protein Rad-mediated inhibition of voltage-gated calcium channels (CaV) acting through the Cavβ subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca2+ influx and increased cardiac contraction. We show that reversal required phosphorylation of Ser272 and Ser300 within Rad's polybasic, hydrophobic C-terminal domain (CTD). Phosphorylation of Ser25 and Ser38 in Rad's N-terminal domain (NTD) alone was ineffective. Phosphorylation of Ser272 and Ser300 or the addition of 4 Asp residues to the CTD reduced Rad's association with the negatively charged, cytoplasmic plasmalemmal surface and with CaVβ, even in the absence of CaVα, measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad's C-terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from CaVβ, is sufficient for sympathetic upregulation of Ca2+ currents.

L-type Ca 2+ channels (Ca V 1.2/1.3) convey influx of calcium ions (Ca 2+ ) that orchestrate a bevy of biological responses including muscle contraction and gene transcription. Deficits in Ca V 1 function play a vital role in cardiac and neurodevelopmental disorders. Yet conventional pharmacological approaches to upregulate Ca V 1 are limited, as excessive Ca 2+ influx leads to cytotoxicity. Here, we develop a genetically encoded enhancer of Ca V 1.2/1.3 channels (GeeC) to manipulate Ca 2+ entry in distinct physiological settings. Specifically, we functionalized a nanobody that targets the Ca V macromolecular complex by attaching a minimal effector domain from a Ca V enhancer-leucine rich repeat containing protein 10 (Lrrc10). In cardiomyocytes, GeeC evoked a 3-fold increase in L-type current amplitude. In neurons, GeeC augmented excitation-transcription (E-T) coupling. In all, GeeC represents a powerful strategy to boost Ca V 1.2/1.3 function in distinct physiological settings and, in so doing, lays the groundwork to illuminate new insights on neuronal and cardiac physiology and disease.

Two-hybrid Förster resonance energy transfer (FRET) provides proximity, affinity, and stoichiometry information in binding interactions. We present an image-based approach that surpasses traditional two-hybrid FRET assays in precision and robustness. We outline instrument setup and image acquisition and further describe steps for image preprocessing and two-hybrid FRET analysis using provided software to simplify the workflow. This protocol is compatible with confocal microscopes for high-precision and imaging plate readers for high-throughput applications. A plasmid-based reference system supports fast establishment of the protocol. For complete details on the use and execution of this protocol, please refer to Rivas et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Voltage-gated sodium and calcium channels are distinct, evolutionarily related ion channels that achieve remarkable ion selectivity despite sharing an overall similar structure. Classical studies have shown that ion selectivity is determined by specific binding of ions to the channel pore, enabled by signature amino acid sequences within the selectivity filter (SF). By studying ancestral channels in the pond snail (Lymnaea stagnalis), Guan et al. showed in a recent JBC article that this well-established mechanism can be tuned by alternative splicing, allowing a single Ca V 3 gene to encode both a Ca 2+ -permeable and an Na + -permeable channel depending on the cellular context. These findings shed light on mechanisms that tune ion selectivity in physiology and on the evolutionary basis of ion selectivity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Calcium influx through voltage-gated calcium channels, Ca v 1.2, in cardiomyocytes initiates excitation-contraction coupling in the heart. The force and rate of cardiac contraction are modulated by the sympathetic nervous system, mediated substantially by changes in intracellular calcium. Norepinephrine released from sympathetic neurons innervating the heart and epinephrine secreted by the adrenal chromaffin cells bind to β-adrenergic receptors on the sarcolemma of cardiomyocytes initiating a signaling cascade that generates cAMP and activates protein kinase A, the targets of which control calcium influx. For decades, the mechanisms by which PKA regulated calcium channels in the heart were not known. Recently, these mechanisms have been elucidated. In this chapter, we will review the history of the field and the studies that led to the identification of the evolutionarily conserved process., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Ca 2+ influx through high-voltage-activated calcium channels (HVACCs) controls diverse cellular functions. A critical feature enabling a singular signal, Ca 2+ influx, to mediate disparate functions is diversity of HVACC pore-forming α 1 and auxiliary Ca V β 1 -Ca V β 4 subunits. Selective Ca V α 1 blockers have enabled deciphering their unique physiological roles. By contrast, the capacity to post-translationally inhibit HVACCs based on Ca V β isoform is non-existent. Conventional gene knockout/shRNA approaches do not adequately address this deficit owing to subunit reshuffling and partially overlapping functions of Ca V β isoforms. Here, we identify a nanobody (nb.E8) that selectively binds Ca V β 1 SH3 domain and inhibits Ca V β 1 -associated HVACCs by reducing channel surface density, decreasing open probability, and speeding inactivation. Functionalizing nb.E8 with Nedd4L HECT domain yielded Chisel-1 which eliminated current through Ca V β 1 -reconstituted Ca V 1/Ca V 2 and native Ca V 1.1 channels in skeletal muscle, strongly suppressed depolarization-evoked Ca 2+ influx and excitation-transcription coupling in hippocampal neurons, but was inert against Ca V β 2 -associated Ca V 1.2 in cardiomyocytes. The results introduce an original method for probing distinctive functions of ion channel auxiliary subunit isoforms, reveal additional dimensions of Ca V β 1 signaling in neurons, and describe a genetically-encoded HVACC inhibitor with unique properties., (© 2022. The Author(s).)

The first pathogenic mutation in CaV1.2 was identified in 2004 and was shown to cause a severe multisystem disorder known as Timothy syndrome (TS). The mutation was localized to the distal S6 region of the channel, a region known to play a major role in channel activation. TS patients suffer from life-threatening cardiac symptoms as well as significant neurodevelopmental deficits, including autism spectrum disorder (ASD). Since this discovery, the number and variety of mutations identified in CaV1.2 have grown tremendously, and the distal S6 regions remain a frequent locus for many of these mutations. While the majority of patients harboring these mutations exhibit cardiac symptoms that can be well explained by known pathogenic mechanisms, the same cannot be said for the ASD or neurodevelopmental phenotypes seen in some patients, indicating a gap in our understanding of the pathogenesis of CaV1.2 channelopathies. Here, we use whole-cell patch clamp, quantitative Ca2+ imaging, and single channel recordings to expand the known mechanisms underlying the pathogenesis of CaV1.2 channelopathies. Specifically, we find that mutations within the S6 region can exert independent and separable effects on activation, voltage-dependent inactivation (VDI), and Ca2+-dependent inactivation (CDI). Moreover, the mechanisms underlying the CDI effects of these mutations are varied and include altered channel opening and possible disruption of CDI transduction. Overall, these results provide a structure-function framework to conceptualize the role of S6 mutations in pathophysiology and offer insight into the biophysical defects associated with distinct clinical manifestations., (© 2022 Bamgboye et al.)

Fight-or-flight responses involve β-adrenergic-induced increases in heart rate and contractile force. In the present study, we uncover the primary mechanism underlying the heart's innate contractile reserve. We show that four protein kinase A (PKA)-phosphorylated residues in Rad, a calcium channel inhibitor, are crucial for controlling basal calcium current and essential for β-adrenergic augmentation of calcium influx in cardiomyocytes. Even with intact PKA signaling to other proteins modulating calcium handling, preventing adrenergic activation of calcium channels in Rad-phosphosite-mutant mice (4SA-Rad) has profound physiological effects: reduced heart rate with increased pauses, reduced basal contractility, near-complete attenuation of β-adrenergic contractile response and diminished exercise capacity. Conversely, expression of mutant calcium-channel β-subunits that cannot bind 4SA-Rad is sufficient to enhance basal calcium influx and contractility to adrenergically augmented levels of wild-type mice, rescuing the failing heart phenotype of 4SA-Rad mice. Hence, disruption of interactions between Rad and calcium channels constitutes the foundation toward next-generation therapeutics specifically enhancing cardiac contractility., Competing Interests: Competing interests Columbia University, Harvard University and NY Presbyterian Hospital have filed a patent (WO/2021/003389), which is published and pending review, reporting a FRET-based method for screening small molecules that increase contractility for the treatment of heart failure. Inventors on this patent application are S.O.M., H.M.C., M.K., S.I.Z., A.N.K., M.B.J. and G.L. The FRET-based assay was utilized in this manuscript for assessing the effects of calyculin and whether 3DA-β2B and 2DA-β2B Ca2+ channel subunits bind to Rad.

Voltage-gated sodium (Nav1.5) channels support the genesis and brisk spatial propagation of action potentials in the heart. Disruption of Na V 1.5 inactivation results in a small persistent Na influx known as late Na current ( I Na,L ), which has emerged as a common pathogenic mechanism in both congenital and acquired cardiac arrhythmogenic syndromes. Here, using low-noise multi-channel recordings in heterologous systems, LQTS3 patient-derived iPSCs cardiomyocytes, and mouse ventricular myocytes, we demonstrate that the intracellular fibroblast growth factor homologous factors (FHF1-4) tune pathogenic I Na,L in an isoform-specific manner. This scheme suggests a complex orchestration of I Na,L in cardiomyocytes that may contribute to variable disease expressivity of Na V 1.5 channelopathies. We further leverage these observations to engineer a peptide-inhibitor of I Na,L with a higher efficacy as compared to a well-established small-molecule inhibitor. Overall, these findings lend insights into molecular mechanisms underlying FHF regulation of I Na,L in pathophysiology and outline potential therapeutic avenues., Competing Interests: Competing Interests N.C., S.O.M, and M.B.-J., (inventors) filed a provisional patent (attorney docket no. CoU1046P/CU22077; filed 14 February 2022) for application of FixR for inhibiting late Na current. The remaining authors declare no competing interests.

Voltage-gated sodium channels, Na V s, are responsible for the rapid rise of action potentials in excitable tissues. Na V channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-Na V nanobodies (Nbs), Nb17 and Nb82, that recognize the Na V 1.4 (skeletal muscle) and Na V 1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of Na V 1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human Na V isoform has been challenging because they usually show undesired crossreactivity for different Na V isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNa V 1.4 or CTNa V 1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNa V 1.4 and CTNa V 1.5 with high affinity (K D ∼ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect Na V 1.4 and Na V 1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo Na V 1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-Na V reagents to capture Na V s from cell lysates and as molecular visualization agents for Na V s., Competing Interests: Conflict of interest S. B. G. is a founder and holds equity in Advanced Molecular Sciences LLC. S. B. G. is consultant to Genesis Therapeutics and Xinthera, Inc. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Depolarizing sodium (Na + ) leak currents carried by the NALCN channel regulate the resting membrane potential of many neurons to modulate respiration, circadian rhythm, locomotion and pain sensitivity 1-8 . NALCN requires FAM155A, UNC79 and UNC80 to function, but the role of these auxiliary subunits is not understood 3,7,9-12 . NALCN, UNC79 and UNC80 are essential in rodents 2,9,13 , and mutations in human NALCN and UNC80 cause severe developmental and neurological disease 14,15 . Here we determined the structure of the NALCN channelosome, an approximately 1-MDa complex, as fundamental aspects about the composition, assembly and gating of this channelosome remain obscure. UNC79 and UNC80 are massive HEAT-repeat proteins that form an intertwined anti-parallel superhelical assembly, which docks intracellularly onto the NALCN-FAM155A pore-forming subcomplex. Calmodulin copurifies bound to the carboxy-terminal domain of NALCN, identifying this region as a putative modulatory hub. Single-channel analyses uncovered a low open probability for the wild-type complex, highlighting the tightly closed S6 gate in the structure, and providing a basis to interpret the altered gating properties of disease-causing variants. Key constraints between the UNC79-UNC80 subcomplex and the NALCN DI-DII and DII-DIII linkers were identified, leading to a model of channelosome gating. Our results provide a structural blueprint to understand the physiology of the NALCN channelosome and a template for drug discovery to modulate the resting membrane potential., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

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 .

Rationale: Changing activity of cardiac Ca V 1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac Ca V 1.2 channels are prominently upregulated via activation of PKA (protein kinase A), essential molecular details remained stubbornly enigmatic., Objective: The primary goal of this study was to determine how various factors converging at the Ca V 1.2 I-II loop interact to regulate channel activity under basal conditions, during β-adrenergic stimulation, and in heart failure., Methods and Results: We generated transgenic mice with expression of Ca V 1.2 α 1C subunits with (1) mutations ablating interaction between α 1C and β-subunits, (2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α 1C ), or (3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α 1C upregulated in the hypertrophied heart. Introducing 3 glycine residues that disrupt a rigid IS6-α-interaction domain helix markedly reduced basal open probability despite intact binding of Ca V β to α 1C I-II loop and eliminated β-adrenergic agonist stimulation of Ca V 1.2 current. In contrast, introduction of the exon 9* splice variant in the α 1C I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to β-adrenergic agonists when reconstituted heterologously with β 2B and Rad or transgenically expressed in cardiomyocytes., Conclusions: Ca 2+ channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α 1C I-II loop. Ca V β binding to α 1C stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the β-subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca 2+ channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β-binding to α 1C .

Voltage-gated sodium channels (NaVs) are membrane proteins responsible for the rapid upstroke of the action potential in excitable cells. There are nine human voltage-sensitive NaV1 isoforms that, in addition to their sequence differences, differ in tissue distribution and specific function. This review focuses on isoforms NaV1.4 and NaV1.5, which are primarily expressed in skeletal and cardiac muscle cells, respectively. The determination of the structures of several eukaryotic NaVs by single-particle cryo-electron microscopy (cryo-EM) has brought new perspective to the study of the channels. Alignment of the cryo-EM structure of the transmembrane channel pore with x-ray crystallographic structures of the cytoplasmic domains illustrates the complementary nature of the techniques and highlights the intricate cellular mechanisms that modulate these channels. Here, we review structural insights into the cytoplasmic C-terminal regulation of NaV1.4 and NaV1.5 with special attention to Ca2+ sensing by calmodulin, implications for disease, and putative channel dimerization., (© 2020 Nathan et al.)

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.)

Ion channels are macromolecular complexes whose functions are exquisitely tuned by interacting proteins. Fluorescence resonance energy transfer (FRET) is a powerful methodology that is adept at quantifying ion channel protein-protein interactions in living cells. For FRET experiments, the interacting partners are tagged with appropriate donor and acceptor fluorescent proteins. If the fluorescently-labeled molecules are in close proximity, then photoexcitation of the donor results in non-radiative energy transfer to the acceptor, and subsequent fluorescence emission of the acceptor. The stoichiometry of ion channel interactions and their relative binding affinities can be deduced by quantifying both the FRET efficiency and the total number of donors and acceptors in a given cell. In this chapter, we discuss general considerations for FRET analysis of biological interactions, various strategies for estimating FRET efficiencies, and detailed protocols for construction of binding curves and determination of stoichiometry. We focus on implementation of FRET assays using a flow cytometer given its amenability for high-throughput data acquisition, enhanced accessibility, and robust analysis. This versatile methodology permits mechanistic dissection of dynamic changes in ion channel interactions., (Copyright © 2021 Elsevier Inc. All rights reserved.)

S- Acylation, a reversible post-translational lipid modification of proteins, controls the properties and function of various proteins, including ion channels. Large conductance Ca 2+ -activated potassium (BK) channels are S- acylated at two sites that impart distinct functional effects. Whereas the enzymes that attach lipid groups are known, the enzymes mediating lipid removal ( i.e. deacylation) are largely unknown. Here, McClafferty et al. identify two enzymes, ABHD17a and ABHD17c, that excise BK channel lipid groups with remarkable precision. These findings lend insights into mechanisms that orchestrate the (de)acylation that fine-tunes ion channel function in physiology and disease., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 del Rivero Morfin and Ben-Johny.)

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.

To synthesize a coherent representation of the external world, the brain must integrate inputs across different types of stimuli. Yet the mechanistic basis of this computation at the level of neuronal populations remains obscure. Here, we investigate tactile-auditory integration using two-photon Ca 2+ imaging in the mouse primary (S1) and secondary (S2) somatosensory cortices. Pairing sound with whisker stimulation modulates tactile responses in both S1 and S2, with the most prominent modulation being robust inhibition in S2. The degree of inhibition depends on tactile stimulation frequency, with lower frequency responses the most severely attenuated. Alongside these neurons, we identify sound-selective neurons in S2 whose responses are inhibited by high tactile frequencies. These results are consistent with a hypothesized local mutually-inhibitory S2 circuit that spectrally selects tactile versus auditory inputs. Our findings enrich mechanistic understanding of multisensory integration and suggest a key role for S2 in combining auditory and tactile information.

Increased cardiac contractility during the fight-or-flight response is caused by β-adrenergic augmentation of Ca V 1.2 voltage-gated calcium channels 1-4 . However, this augmentation persists in transgenic murine hearts expressing mutant Ca V 1.2 α 1C and β subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of β-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mechanism by which β-adrenergic agonists stimulate voltage-gated calcium channels. We express α 1C or β 2B subunits conjugated to ascorbate peroxidase 5 in mouse hearts, and use multiplexed quantitative proteomics 6,7 to track hundreds of proteins in the proximity of Ca V 1.2. We observe that the calcium-channel inhibitor Rad 8,9 , a monomeric G protein, is enriched in the Ca V 1.2 microenvironment but is depleted during β-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for β subunits and relieves constitutive inhibition of Ca V 1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of Ca V 1.3 and Ca V 2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.

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.

Structural symmetry is a hallmark of homomeric ion channels. Nonobligatory regulatory proteins can also critically define the precise functional role of such channels. For instance, the pore-forming subunit of the large conductance voltage and calcium-activated potassium (BK, Slo1, or KCa 1.1 ) channels encoded by a single KCa1.1 gene assembles in a fourfold symmetric fashion. Functional diversity arises from two families of regulatory subunits, β and γ, which help define the range of voltages over which BK channels in a given cell are activated, thereby defining physiological roles. A BK channel can contain zero to four β subunits per channel, with each β subunit incrementally influencing channel gating behavior, consistent with symmetry expectations. In contrast, a γ1 subunit (or single type of γ1 subunit complex) produces a functionally all-or-none effect, but the underlying stoichiometry of γ1 assembly and function remains unknown. Here we utilize two distinct and independent methods, a Forster resonance energy transfer-based optical approach and a functional reporter in single-channel recordings, to reveal that a BK channel can contain up to four γ1 subunits, but a single γ1 subunit suffices to induce the full gating shift. This requires that the asymmetric association of a single regulatory protein can act in a highly concerted fashion to allosterically influence conformational equilibria in an otherwise symmetric K + channel., Competing Interests: The authors declare no conflict of interest.

Calmodulin (CaM) serves as a pervasive regulatory subunit of Ca V 1, Ca V 2, and Na V 1 channels, exploiting a functionally conserved carboxy-tail element to afford dynamic Ca 2+ -feedback of cellular excitability in neurons and cardiomyocytes. Yet this modularity counters functional adaptability, as global changes in ambient CaM indiscriminately alter its targets. Here, we demonstrate that two structurally unrelated proteins, SH3 and cysteine-rich domain (stac) and fibroblast growth factor homologous factors (fhf) selectively diminish Ca 2+ /CaM-regulation of Ca V 1 and Na V 1 families, respectively. The two proteins operate on allosteric sites within upstream portions of respective channel carboxy-tails, distinct from the CaM-binding interface. Generalizing this mechanism, insertion of a short RxxK binding motif into Ca V 1.3 carboxy-tail confers synthetic switching of CaM regulation by Mona SH3 domain. Overall, our findings identify a general class of auxiliary proteins that modify Ca 2+ /CaM signaling to individual targets allowing spatial and temporal orchestration of feedback, and outline strategies for engineering Ca 2+ /CaM signaling to individual targets., Competing Interests: JN, ID, WY, MB, DY, GT, TI, MB No competing interests declared, (© 2018, Niu et al.)

Ca V 1.1 is essential for skeletal muscle excitation-contraction coupling. Its functional expression is tuned by numerous regulatory proteins, yet underlying modulatory mechanisms remain ambiguous as Ca V 1.1 fails to function in heterologous systems. In this study, by dissecting channel trafficking versus gating, we evaluated the requirements for functional Ca V 1.1 in heterologous systems. Although coexpression of the auxiliary β subunit is sufficient for surface-membrane localization, this baseline trafficking is weak, and channels elicit a diminished open probability. The regulatory proteins calmodulin and stac3 independently enhance channel trafficking and gating via their interaction with the Ca V 1.1 carboxy terminus. Myopathic stac3 mutations weaken channel binding and diminish trafficking. Our findings demonstrate that multiple regulatory proteins orchestrate Ca V 1.1 function via duplex mechanisms. Our work also furnishes insights into the pathophysiology of stac3-associated congenital myopathy and reveals novel avenues for pharmacological intervention., (© 2018 Niu et al.)

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.

Two-pore channels (TPCs) are endolysosomal cation channels. Two members exist in humans, TPC1 and TPC2. Functional roles associated with the ubiquitously expressed TPCs include VEGF-induced neoangiogenesis, LDL-cholesterol trafficking and degradation, physical endurance under fasting conditions, autophagy regulation, the acrosome reaction in sperm, cancer cell migration, and intracellular trafficking of pathogens such as Ebola virus or bacterial toxins (e.g., cholera toxin). In a genome-wide association study for variants associated with human pigmentation characteristics two coding variants of TPC2, rs35264875 (encoding M484L) and rs3829241 (encoding G734E), have been found to be associated with a shift from brown to blond hair color. In two recent follow-up studies a role for TPC2 in pigmentation has been further confirmed. However, these human polymorphic variants have not been functionally characterized until now. The development of endolysosomal patch-clamp techniques has made it possible to investigate directly ion channel activities and characteristics in isolated endolysosomal organelles. We applied this technique here to scrutinize channel characteristics of the polymorphic TPC2 variants in direct comparison with WT. We found that both polymorphisms lead to a gain of channel function by independent mechanisms. We next conducted a clinical study with more than 100 blond- and brown/black-haired individuals. We performed a genotype/phenotype analysis and subsequently isolated fibroblasts from WT and polymorphic variant carriers for endolysosomal patch-clamp experimentation to confirm key in vitro findings., Competing Interests: The authors declare no conflict of interest.

The stoichiometry of macromolecular interactions is fundamental to cellular signalling yet challenging to detect from living cells. Fluorescence resonance energy transfer (FRET) is a powerful phenomenon for characterizing close-range interactions whereby a donor fluorophore transfers energy to a closely juxtaposed acceptor. Recognizing that FRET measured from the acceptor's perspective reports a related but distinct quantity versus the donor, we utilize the ratiometric comparison of the two to obtain the stoichiometry of a complex. Applying this principle to the long-standing controversy of calmodulin binding to ion channels, we find a surprising Ca 2+ -induced switch in calmodulin stoichiometry with Ca 2+ channels-one calmodulin binds at basal cytosolic Ca 2+ levels while two calmodulins interact following Ca 2+ elevation. This feature is curiously absent for the related Na channels, also potently regulated by calmodulin. Overall, our assay adds to a burgeoning toolkit to pursue quantitative biochemistry of dynamic signalling complexes in living 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.

Optogenetics describes the use of genetically encoded photosensitive proteins to direct intended biological processes with light in recombinant and native systems. While most of these light-responsive proteins were originally discovered in photosynthetic organisms, the past few decades have been punctuated by experiments that not only commandeer but also engineer and enhance these natural tools to explore a wide variety of physiological questions. In addition, the ability to tune dynamic range and kinetic rates of optogenetic actuators is a challenging question that is heavily explored with computational methods devised to facilitate optimization of these systems. Here, we explain the basic mechanisms of a few popular photodimerizing optogenetic systems, discuss applications, compare optogenetic tools against more traditional chemical methods, and propose a simple quantitative understanding of how actuators exert their influence on targeted processes., (Copyright © 2016. Published by Elsevier Inc.)

CaV1.3 channels are a major class of L-type Ca(2+) channels which contribute to the rhythmicity of the heart and brain. In the brain, these channels are vital for excitation-transcription coupling, synaptic plasticity, and neuronal firing. Moreover, disruption of CaV1.3 function has been associated with several neurological disorders. Here, we focus on the de novo missense mutation A760G which has been linked to autism spectrum disorder (ASD). To explore the role of this mutation in ASD pathogenesis, we examined the effects of A760G on CaV1.3 channel gating and regulation. Introduction of the mutation severely diminished the Ca(2+)-dependent inactivation (CDI) of CaV1.3 channels, an important feedback system required for Ca(2+) homeostasis. This reduction in CDI was observed in two major channel splice variants, though to different extents. Using an allosteric model of channel gating, we found that the underlying mechanism of CDI reduction is likely due to enhanced channel opening within the Ca(2+)-inactivated mode. Remarkably, the A760G mutation also caused an opposite increase in voltage-dependent inactivation (VDI), resulting in a multifaceted mechanism underlying ASD. When combined, these regulatory deficits appear to increase the intracellular Ca(2+) concentration, thus potentially disrupting neuronal development and synapse formation, ultimately leading to ASD.

David T. Yue was a renowned biophysicist who dedicated his life to the study of Ca(2+) signaling in cells. In the wake of his passing, we are left not only with a feeling of great loss, but with a tremendous and impactful body of work contributed by a remarkable man. David's research spanned the spectrum from atomic structure to organ systems, with a quantitative rigor aimed at understanding the fundamental mechanisms underlying biological function. Along the way he developed new tools and approaches, enabling not only his own research but that of his contemporaries and those who will come after him. While we cannot hope to replicate the eloquence and style we are accustomed to in David's writing, we nonetheless undertake a review of David's chosen field of study with a focus on many of his contributions to the calcium channel field.

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:

Calcineurin (CN) is a highly conserved Ca(2+)-calmodulin (CaM)-dependent phosphatase that senses Ca(2+) concentrations and transduces that information into cellular responses. Ca(2+) homeostasis is disrupted by α-synuclein (α-syn), a small lipid binding protein whose misfolding and accumulation is a pathological hallmark of several neurodegenerative diseases. We report that α-syn, from yeast to neurons, leads to sustained highly elevated levels of cytoplasmic Ca(2+), thereby activating a CaM-CN cascade that engages substrates that result in toxicity. Surprisingly, complete inhibition of CN also results in toxicity. Limiting the availability of CaM shifts CN's spectrum of substrates toward protective pathways. Modulating CN or CN's substrates with highly selective genetic and pharmacological tools (FK506) does the same. FK506 crosses the blood brain barrier, is well tolerated in humans, and is active in neurons and glia. Thus, a tunable response to CN, which has been conserved for a billion years, can be targeted to rebalance the phosphatase's activities from toxic toward beneficial substrates. These findings have immediate therapeutic implications for synucleinopathies.

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.)

CaV1.3 ion channels are dominant Ca(2+) portals into pacemaking neurons, residing at the epicenter of brain rhythmicity and neurodegeneration. Negative Ca(2+) feedback regulation of CaV1.3 channels (CDI) is therefore critical for Ca(2+) homeostasis. Intriguingly, nearly half the CaV1.3 transcripts in the brain are RNA edited to reduce CDI and influence oscillatory activity. It is then mechanistically remarkable that this editing occurs precisely within an IQ domain, whose interaction with Ca(2+)-bound calmodulin (Ca(2+)/CaM) is believed to induce CDI. Here, we sought the mechanism underlying the altered CDI of edited channels. Unexpectedly, editing failed to attenuate Ca(2+)/CaM binding. Instead, editing weakened the prebinding of Ca(2+)-free CaM (apoCaM) to channels, which proves essential for CDI. Thus, editing might render CDI continuously tunable by fluctuations in ambient CaM, a prominent effect we substantiate in substantia nigral neurons. This adjustability of Ca(2+) regulation by CaM now looms as a key element of CNS Ca(2+) homeostasis., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

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.