Calcium-release channels: structure and function of IP3 receptors and ryanodine receptors.

Ca2+-release channels are giant membrane proteins that control the release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. The two members, ryanodine receptors (RyRs) and inositol-1,4,5-trisphosphate receptors (IP3Rs), are evolutionarily related and are both activated by cytosolic Ca2+. They share a common architecture, but RyRs have evolved additional modules in the cytosolic region. Their massive size allows for the regulation by tens of proteins and small molecules, which can affect the opening and closing of the channels. In addition to Ca2+, other major triggers include IP3 for the IP3Rs and depolarization of the plasma membrane for a particular RyR subtype expressed in skeletal muscle. Their size has made them popular targets for study via electron microscopic methods, with current structures culminating near 3 Å. The available structures have provided many new mechanistic insights into the binding of auxiliary proteins and small molecules, how these can regulate channel opening, and the mechanisms of disease-associated mutations. They also help scrutinize previously proposed binding sites, as some of these are now incompatible with the structures. Many questions remain around the structural effects of posttranslational modifications, additional binding partners, and the higher order complexes these channels can make in situ. This review summarizes our current knowledge about the structures of Ca2+-release channels and how this informs on their function.

The Cardiac Ryanodine Receptor Phosphorylation Hotspot Embraces PKA in a Phosphorylation-Dependent Manner.

Ryanodine receptors (RyRs) are intracellular Ca2+ release channels controlling essential cellular functions. RyRs are targeted by cyclic AMP (cAMP)-dependent protein kinase A (PKA), a controversial regulation implicated in disorders ranging from heart failure to Alzheimer's. Using crystal structures, we show that the phosphorylation hotspot domain of RyR2 embraces the PKA catalytic subunit, with an extensive interface not seen in PKA complexes with peptides. We trapped an intermediary open-form PKA bound to the RyR2 domain and an ATP analog, showing that PKA can engage substrates in an open form. Phosphomimetics or prior phosphorylation at nearby sites in RyR2 either enhance or reduce the activity of PKA. Finally, we show that a phosphomimetic at S2813, a well-known target site for calmodulin-dependent kinase II, induces the formation of an alpha helix in the phosphorylation domain, resulting in increased interactions and PKA activity. This shows that the different phosphorylation sites in RyR2 are not independent.

Calcium Channelopathies: Structural Insights into Disorders of the Muscle Excitation-Contraction Complex.

Ion channels are membrane proteins responsible for the passage of ions down their electrochemical gradients and across biological membranes. In this, they generate and shape action potentials and provide secondary messengers for various signaling pathways. They are often part of larger complexes containing auxiliary subunits and regulatory proteins. Channelopathies arise from mutations in the genes encoding ion channels or their associated proteins. Recent advances in cryo-electron microscopy have resulted in an explosion of ion channel structures in multiple states, generating a wealth of new information on channelopathies. Disease-associated mutations fall into different categories, interfering with ion permeation, protein folding, voltage sensing, ligand and protein binding, and allosteric modulation of channel gating. Prime examples of these are Ca2+-selective channels expressed in myocytes, for which multiple structures in distinct conformational states have recently been uncovered. We discuss the latest insights into these calcium channelopathies from a structural viewpoint.

Structures of the junctophilin/voltage-gated calcium channel interface reveal hot spot for cardiomyopathy mutations.

SignificanceIon channels have evolved the ability to communicate with one another, either through protein-protein interactions, or indirectly via intermediate diffusible messenger molecules. In special cases, the channels are part of different membranes. In muscle tissue, the T-tubule membrane is in proximity to the sarcoplasmic reticulum, allowing communication between L-type calcium channels and ryanodine receptors. This process is critical for excitation-contraction coupling and requires auxiliary proteins like junctophilin (JPH). JPHs are targets for disease-associated mutations, most notably hypertrophic cardiomyopathy mutations in the JPH2 isoform. Here we provide high-resolution snapshots of JPH, both alone and in complex with a calcium channel peptide, and show how this interaction is targeted by cardiomyopathy mutations.

Gap junctions mediate discrete regulatory steps during fly spermatogenesis.

Gametogenesis requires coordinated signaling between germ cells and somatic cells. We previously showed that Gap junction (GJ)-mediated soma-germline communication is essential for fly spermatogenesis. Specifically, the GJ protein Innexin4/Zero population growth (Zpg) is necessary for somatic and germline stem cell maintenance and differentiation. It remains unknown how GJ-mediated signals regulate spermatogenesis or whether the function of these signals is restricted to the earliest stages of spermatogenesis. Here we carried out comprehensive structure/function analysis of Zpg using insights obtained from the protein structure of innexins to design mutations aimed at selectively perturbing different regulatory regions as well as the channel pore of Zpg. We identify the roles of various regulatory sites in Zpg in the assembly and maintenance of GJs at the plasma membrane. Moreover, mutations designed to selectively disrupt, based on size and charge, the passage of cargos through the Zpg channel pore, blocked different stages of spermatogenesis. Mutations were identified that progressed through early germline and soma development, but exhibited defects in entry to meiosis or sperm individualisation, resulting in reduced fertility or sterility. Our work shows that specific signals that pass through GJs regulate the transition between different stages of gametogenesis.

Multiple regions within junctin drive its interaction with calsequestrin-1 and its localization to triads in skeletal muscle.

Junctin is a transmembrane protein of striated muscles, located at the junctional sarcoplasmic reticulum (SR). It is characterized by a luminal C-terminal tail, through which it functionally interacts with calsequestrin and the ryanodine receptor (RyR). Interaction with calsequestrin was ascribed to the presence of stretches of charged amino acids (aa). However, the regions able to bind calsequestrin have not been defined in detail. We report here that, in non-muscle cells, junctin and calsequestrin assemble in long linear regions within the endoplasmic reticulum, mirroring the formation of calsequestrin polymers. In differentiating myotubes, the two proteins colocalize at triads, where they assemble with other proteins of the junctional SR. By performing GST pull-down assays with distinct regions of the junctin tail, we identified two KEKE motifs that can bind calsequestrin. In addition, stretches of charged aa downstream these motifs were found to also bind calsequestrin and the RyR. Deletion of even one of these regions impaired the ability of junctin to localize at the junctional SR, suggesting that interaction with other proteins at this site represents a key element in junctin targeting.

Cardiac ryanodine receptor N-terminal region biosensors identify novel inhibitors via FRET-based high-throughput screening.

The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca2+ release during excitation-contraction (EC) coupling in muscle. The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators. We then screened a small validation library and identified several hits. Hits with saturable FRET dose-response profiles and previously unreported effects on RyR were further tested using [3H]ryanodine binding to isolated sarcoplasmic reticulum vesicles to determine effects on intact RyR opening in its natural membrane. We identified three novel inhibitors of both RyR1 and RyR2 and two RyR1-selective inhibitors effective at nanomolar Ca2+. Two of these hits activated RyR1 only at micromolar Ca2+, highlighting them as potential enhancers of excitation-contraction coupling. To determine whether such hits can inhibit RyR leak in muscle, we further focused on one, an FDA-approved natural antibiotic, fusidic acid (FA). In skinned skeletal myofibers and permeabilized cardiomyocytes, FA inhibited RyR leak with no detrimental effect on skeletal myofiber excitation-contraction coupling. However, in intact cardiomyocytes, FA induced arrhythmogenic Ca2+ transients, a cautionary observation for a compound with an otherwise solid safety record. These results indicate that HTS campaigns using the NTR biosensor can identify compounds with therapeutic potential.

Altered cyclic nucleotide binding and pore opening in a diseased human HCN4 channel.

A growing number of nonsynonymous mutations in the human HCN4 channel gene, the major component of the funny channel of the sinoatrial node, are associated with disease but how they impact channel structure and function, and, thus, how they result in disease, is not clear for any of them. Here, we study the S672R mutation, in the cyclic nucleotide-binding domain of the channel, which has been associated with an inherited bradycardia in an Italian family. This may be the best studied of all known mutations, yet the underlying molecular and atomistic mechanisms remain unclear and controversial. We combine measurements of binding by isothermal titration calorimetry to a naturally occurring tetramer of the HCN4 C-terminal region with a mathematical model to show that weaker binding of cAMP to the mutant channel contributes to a lower level of facilitation of channel opening at submicromolar ligand concentrations but that, in general, facilitation occurs over a range that is similar between the mutant and wild-type because of enhanced opening of the mutant channel when liganded. We also show that the binding affinity for cGMP, which produces the same maximum facilitation of HCN4 opening as cAMP, is weaker in the mutant HCN4 channel but that, for both wild-type and mutant, high-affinity binding of cGMP occurs in a range of concentrations below 1 µM. Thus, binding of cGMP to the HCN4 channel may be relevant normally in vivo and reduced binding of cGMP, as well as cAMP, to the mutant channel may contribute to the reduced resting heart rate observed in the affected family.

Structure and function of STAC proteins: Calcium channel modulators and critical components of muscle excitation-contraction coupling.

In skeletal muscle tissue, an intriguing mechanical coupling exists between two ion channels from different membranes: the L-type voltage-gated calcium channel (CaV1.1), located in the plasma membrane, and ryanodine receptor 1 (RyR1) located in the sarcoplasmic reticulum membrane. Excitable cells rely on Cavs to initiate Ca2+ entry in response to action potentials. RyRs can amplify this signal by releasing Ca2+ from internal stores. Although this process can be mediated through Ca2+ as a messenger, an overwhelming amount of evidence suggests that RyR1 has recruited CaV1.1 directly as its voltage sensor. The exact mechanisms that underlie this coupling have been enigmatic, but a recent wave of reports have illuminated the coupling protein STAC3 as a critical player. Without STAC3, the mechanical coupling between Cav1.1 and RyR1 is lost, and muscles fail to contract. Various sequence variants of this protein have been linked to congenital myopathy. Other STAC isoforms are expressed in the brain and may serve as regulators of L-type CaVs. Despite the short length of STACs, several points of contacts have been proposed between them and CaVs. However, it is currently unclear whether STAC3 also forms direct interactions with RyR1, and whether this modulates RyR1 function. In this review, we discuss the 3D architecture of STAC proteins, the biochemical evidence for their interactions, the relevance of these connections for functional modulation, and their involvement in myopathy.

Distinct protein architectures mediate species-specific beta-glucan binding and metabolism in the human gut microbiota.

Complex glycans that evade our digestive system are major nutrients that feed the human gut microbiota (HGM). The prevalence of Bacteroidetes in the HGM of populations worldwide is engendered by the evolution of polysaccharide utilization loci (PULs), which encode concerted protein systems to utilize the myriad complex glycans in our diets. Despite their crucial roles in glycan recognition and transport, cell-surface glycan-binding proteins (SGBPs) remained understudied cogs in the PUL machinery. Here, we report the structural and biochemical characterization of a suite of SGBP-A and SGBP-B structures from three syntenic ß(1,3)-glucan utilization loci (1,3GULs) from Bacteroides thetaiotaomicron (Bt), Bacteroides uniformis (Bu), and B. fluxus (Bf), which have varying specificities for distinct ß-glucans. Ligand complexes provide definitive insight into ß(1,3)-glucan selectivity in the HGM, including structural features enabling dual ß(1,3)-glucan/mixed-linkage ß(1,3)/ß(1,4)-glucan-binding capability in some orthologs. The tertiary structural conservation of SusD-like SGBPs-A is juxtaposed with the diverse architectures and binding modes of the SGBPs-B. Specifically, the structures of the trimodular BtSGBP-B and BuSGBP-B revealed a tandem repeat of carbohydrate-binding module-like domains connected by long linkers. In contrast, BfSGBP-B comprises a bimodular architecture with a distinct ß-barrel domain at the C terminus that bears a shallow binding canyon. The molecular insights obtained here contribute to our fundamental understanding of HGM function, which in turn may inform tailored microbial intervention therapies.

Binding of calcium and magnesium to human cardiac troponin C.

Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca2+ binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca2+-sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca2+ and Mg2+ and a regulatory site (II) that has been thought to bind only Ca2+. Binding of Ca2+ at this site initiates a series of conformational changes that culminate in force production. However, the mechanisms that underpin the regulation of binding at site II remain unclear. Here, we have quantified the interaction between site II and Ca2+/Mg2+ through isothermal titration calorimetry and thermodynamic integration simulations. Direct and competitive binding titrations with WT N-terminal cTnC and full-length cTnC indicate that physiologically relevant concentrations of both Ca2+/Mg2+ interacted with the same locus. Moreover, the D67A/D73A N-terminal cTnC construct in which two coordinating residues within site II were removed was found to have significantly reduced affinity for both cations. In addition, 1 mM Mg2+ caused a 1.4-fold lower affinity for Ca2+. These experiments strongly suggest that cytosolic-free Mg2+ occupies a significant population of the available site II. Interaction of Mg2+ with site II of cTnC likely has important functional consequences for the heart both at baseline as well as in diseased states that decrease or increase the availability of Mg2+, such as secondary hyperparathyroidism or ischemia, respectively.

Cell Surface Xyloglucan Recognition and Hydrolysis by the Human Gut Commensal Bacteroides uniformis.

Xyloglucan (XyG) is a ubiquitous plant cell wall hemicellulose that is targeted by a range of syntenic, microheterogeneous xyloglucan utilization loci (XyGUL) in Bacteroidetes species of the human gut microbiota (HGM), including Bacteroides ovatus and B. uniformis. Comprehensive biochemical and biophysical analyses have identified key differences in the protein complements of each locus that confer differential access to structurally diverse XyG side chain variants. A second, nonsyntenic XyGUL was previously identified in B. uniformis, although its function in XyG utilization compared to its syntenic counterpart was unclear. Here, complementary enzymatic product profiles and bacterial growth curves showcase the notable preference of BuXyGUL2 surface glycan-binding proteins (SGBPs) to bind full-length XyG, as well as a range of oligosaccharides produced by the glycoside hydrolase family 5 (GH5_4) endo-xyloglucanase from this locus. We use isothermal titration calorimetry (ITC) to characterize this binding capacity and pinpoint the specific contributions of each protein to nutrient capture. The high-resolution structure of BuXyGUL2 SGBP-B reveals remarkable putative binding site conservation with the canonical XyG-binding BoXyGUL SGBP-B, supporting similar roles for these proteins in glycan capture. Together, these data underpin the central role of complementary XyGUL function in B. uniformis and broaden our systems-based and mechanistic understanding of XyG utilization in the HGM. IMPORTANCE The omnipresence of xyloglucans in the human diet has led to the evolution of heterogeneous gene clusters in several Bacteroidetes species in the HGM, each specially tuned to respond to the structural variations of these complex plant cell wall polysaccharides. Our research illuminates the complementary roles of syntenic and nonsyntenic XyGUL in B. uniformis in conferring growth on a variety of XyG-derived substrates, providing evidence of glycan-binding protein microadaptation within a single species. These data serve as a comprehensive overview of the binding capacities of the SGBPs from a nonsyntenic B. uniformis XyGUL and will inform future studies on the roles of complementary loci in glycan targeting by key HGM species.

Functional characterization of a cellulose synthase, CtCESA1, from the marine red alga Calliarthron tuberculosum (Corallinales).

In land plants and algae, cellulose is important for strengthening cell walls and preventing breakage due to physical forces. Though our understanding of cellulose production by cellulose synthases (CESAs) has seen significant advances for several land plant and bacterial species, functional characterization of this fundamental protein is absent in red algae. Here we identify CESA gene candidates in the calcifying red alga Calliarthron tuberculosum using sequence similarity-based approaches, and elucidate their phylogenetic relationship with other CESAs from diverse taxa. One gene candidate, CtCESA1, was closely related to other putative red algal CESA genes. To test if CtCESA1 encoded a true cellulose synthase, CtCESA1 protein was expressed and purified from insect and yeast expression systems. CtCESA1 showed glucan synthase activity in glucose tracer assays. CtCESA1 activity was relatively low when compared with plant and bacterial CESA activity. In an in vitro assay, a predicted N-terminal starch-binding domain from CtCESA1 bound red algal floridean starch extracts, representing a unique domain in red algal CESAs not present in CESAs from other lineages. When the CtCESA1 gene was introduced into Arabidopsis thaliana cesa mutants, the red algal CtCESA1 partially rescued the growth defects of the primary cell wall cesa6 mutant, but not cesa3 or secondary cell wall cesa7 mutants. A fluorescently tagged CtCESA1 localized to the plasma membrane in the Arabidopsis cesa6 mutant background. This study presents functional evidence validating the sequence annotation of red algal CESAs. The relatively low activity of CtCESA1, partial complementation in Arabidopsis, and presence of unique protein domains suggest that there are probably functional differences between the algal and land plant CESAs.

Structural basis for diamide modulation of ryanodine receptor.

The diamide insecticide class is one of the top-selling insecticides globally. They are used to control a wide range of pests by targeting their ryanodine receptors (RyRs). Here, we report the highest-resolution cryo-electron microscopy (cryo-EM) structure of RyR1 in the open state, in complex with the anthranilic diamide chlorantraniliprole (CHL). The 3.2-Å local resolution map facilitates unambiguous assignment of the CHL binding site. The molecule induces a conformational change by affecting the S4-S5 linker, triggering channel opening. The binding site is further corroborated by mutagenesis data, which reveal how diamide insecticides are selective to the Lepidoptera group of insects over honeybee or mammalian RyRs. Our data reveal that several pests have developed resistance via two mechanisms, steric hindrance and loss of contact. Our results provide a foundation for the development of highly selective pesticides aimed at overcoming resistance and therapeutic molecules to treat human myopathies.

Crystal structures of Ca2+-calmodulin bound to NaV C-terminal regions suggest role for EF-hand domain in binding and inactivation.

Voltage-gated sodium (NaV) and calcium channels (CaV) form targets for calmodulin (CaM), which affects channel inactivation properties. A major interaction site for CaM resides in the C-terminal (CT) region, consisting of an IQ domain downstream of an EF-hand domain. We present a crystal structure of fully Ca2+-occupied CaM, bound to the CT of NaV1.5. The structure shows that the C-terminal lobe binds to a site ∼90° rotated relative to a previous site reported for an apoCaM complex with the NaV1.5 CT and for ternary complexes containing fibroblast growth factor homologous factors (FHF). We show that the binding of FHFs forces the EF-hand domain in a conformation that does not allow binding of the Ca2+-occupied C-lobe of CaM. These observations highlight the central role of the EF-hand domain in modulating the binding mode of CaM. The binding sites for Ca2+-free and Ca2+-occupied CaM contain targets for mutations linked to long-QT syndrome, a type of inherited arrhythmia. The related NaV1.4 channel has been shown to undergo Ca2+-dependent inactivation (CDI) akin to CaVs. We present a crystal structure of Ca2+/CaM bound to the NaV1.4 IQ domain, which shows a binding mode that would clash with the EF-hand domain. We postulate the relative reorientation of the EF-hand domain and the IQ domain as a possible conformational switch that underlies CDI.

In vitro analyses of suspected arrhythmogenic thin filament variants as a cause of sudden cardiac death in infants.

Sudden unexpected death of an infant (SUDI) is a devastating occurrence for families. To investigate the genetic pathogenesis of SUDI, we sequenced >70 genes from 191 autopsy-negative SUDI victims. Ten infants sharing a previously unknown variant in troponin I (TnI) were identified. The mutation (TNNI1 R37C+/-) is in the fetal/neonatal paralog of TnI, a gene thought to be expressed in the heart up to the first 24 months of life. Using phylogenetic analysis and molecular dynamics simulations, it was determined that arginine at residue 37 in TNNI1 may play a critical functional role, suggesting that the variant may be pathogenic. We investigated the biophysical properties of the TNNI1 R37C mutation in human reconstituted thin filaments (RTFs) using fluorometry. RTFs reconstituted with the mutant R37C TnI exhibited reduced Ca2+-binding sensitivity due to an increased Ca2+ off-rate constant. Furthermore, we generated TNNI1 R37C+/- mutants in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) using CRISPR-Cas9. In monolayers of hiPSC-CMs, we simultaneously monitored voltage and Ca2+ transients through optical mapping and compared them to their isogenic controls. We observed normal intrinsic beating patterns under control conditions in TNNI1 R37C+/- at stimulation frequencies of 55 beats/min (bpm), but these cells showed no restitution with increased stimulation frequency to 65 bpm and exhibited alternans at >75 bpm. The WT hiPSC-CMs did not exhibit any sign of arrhythmogenicity even at stimulation frequencies of 120 bpm. The approach used in this study provides critical physiological and mechanistic bases to investigate sarcomeric mutations in the pathogenesis of SUDI.

Molecular interactions between sex hormone-binding globulin and nonsteroidal ligands that enhance androgen activity.

Sex hormone-binding globulin (SHBG) determines the equilibrium between free and protein-bound androgens and estrogens in the blood and regulates their access to target tissues. Using crystallographic approaches and radiolabeled competitive binding-capacity assays, we report here how two nonsteroidal compounds bind to human SHBG, and how they influence androgen activity in cell culture. We found that one of these compounds, (-)3,4-divanillyltetrahydrofuran (DVT), present in stinging nettle root extracts and used as a nutraceutical, binds SHBG with relatively low affinity. By contrast, a synthetic compound, 3-(1H-imidazol-1-ylmethyl)-2phenyl-1H-indole (IPI), bound SHBG with an affinity similar to that of testosterone and estradiol. Crystal structures of SHBG in complex with DVT or IPI at 1.71-1.80 Šresolutions revealed their unique orientations in the SHBG ligand-binding pocket and suggested opportunities for the design of other nonsteroidal ligands of SHBG. As observed for estradiol but not testosterone, IPI binding to SHBG was reduced by ∼20-fold in the presence of zinc, whereas DVT binding was almost completely lost. Estradiol-dependent fibulin-2 interactions with SHBG similarly occurred for IPI-bound SHBG, but not with DVT-bound SHBG. Both DVT and IPI increased the activity of testosterone in a cell culture androgen reporter system by competitively displacing testosterone from SHBG. These findings indicate how nonsteroidal ligands of SHBG maybe designed to modulate the bioavailability of sex steroids.

The arrhythmogenic N53I variant subtly changes the structure and dynamics in the calmodulin N-terminal domain, altering its interaction with the cardiac ryanodine receptor.

Mutations in the genes encoding the highly conserved Ca2+-sensing protein calmodulin (CaM) cause severe cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia or long QT syndrome and sudden cardiac death. Most of the identified arrhythmogenic mutations reside in the C-terminal domain of CaM and mostly affect Ca2+-coordinating residues. One exception is the catecholaminergic polymorphic ventricular tachycardia-causing N53I substitution, which resides in the N-terminal domain (N-domain). It does not affect Ca2+ coordination and has only a minor impact on binding affinity toward Ca2+ and on other biophysical properties. Nevertheless, the N53I substitution dramatically affects CaM's ability to reduce the open probability of the cardiac ryanodine receptor (RyR2) while having no effect on the regulation of the plasmalemmal voltage-gated Ca2+ channel, Cav1.2. To gain more insight into the molecular disease mechanism of this mutant, we used NMR to investigate the structures and dynamics of both apo- and Ca2+-bound CaM-N53I in solution. We also solved the crystal structures of WT and N53I CaM in complex with the primary calmodulin-binding domain (CaMBD2) from RyR2 at 1.84-2.13 Å resolutions. We found that all structures of the arrhythmogenic CaM-N53I variant are highly similar to those of WT CaM. However, we noted that the N53I substitution exposes an additional hydrophobic surface and that the intramolecular dynamics of the protein are significantly altered such that they destabilize the CaM N-domain. We conclude that the N53I-induced changes alter the interaction of the CaM N-domain with RyR2 and thereby likely cause the arrhythmogenic phenotype of this mutation.

Homozygous SCN1B variants causing early infantile epileptic encephalopathy 52 affect voltage-gated sodium channel function.

We identified nine patients from four unrelated families harboring three biallelic variants in SCN1B (NM_001037.5: c.136C>T; p.[Arg46Cys], c.178C>T; p.[Arg60Cys], and c.472G>A; p.[Val158Met]). All subjects presented with early infantile epileptic encephalopathy 52 (EIEE52), a rare, severe developmental and epileptic encephalopathy featuring infantile onset refractory seizures followed by developmental stagnation or regression. Because SCN1B influences neuronal excitability through modulation of voltage-gated sodium (NaV ) channel function, we examined the effects of human SCN1BR46C (ß1R46C ), SCN1BR60C (ß1R60C ), and SCN1BV158M (ß1V158M ) on the three predominant brain NaV channel subtypes NaV 1.1 (SCN1A), NaV 1.2 (SCN2A), and NaV 1.6 (SCN8A). We observed a shift toward more depolarizing potentials of conductance-voltage relationships (NaV 1.2/ß1R46C , NaV 1.2/ß1R60C , NaV 1.6/ß1R46C , NaV 1.6/ß1R60C , and NaV 1.6/ß1V158M ) and channel availability (NaV 1.1/ß1R46C , NaV 1.1/ß1V158M , NaV 1.2/ß1R46C , NaV 1.2/ß1R60C , and NaV 1.6/ß1V158M ), and detected a slower recovery from fast inactivation for NaV 1.1/ß1V158M . Combined with modeling data indicating perturbation-induced structural changes in ß1, these results suggest that the SCN1B variants reported here can disrupt normal NaV channel function in the brain, which may contribute to EIEE52.

Arrhythmia mutations in calmodulin cause conformational changes that affect interactions with the cardiac voltage-gated calcium channel.

Calmodulin (CaM) represents one of the most conserved proteins among eukaryotes and is known to bind and modulate more than a 100 targets. Recently, several disease-associated mutations have been identified in the CALM genes that are causative of severe cardiac arrhythmia syndromes. Although several mutations have been shown to affect the function of various cardiac ion channels, direct structural insights into any CaM disease mutation have been lacking. Here we report a crystallographic and NMR investigation of several disease mutant CaMs, linked to long-QT syndrome, in complex with the IQ domain of the cardiac voltage-gated calcium channel (CaV1.2). Surprisingly, two mutants (D95V, N97I) cause a major distortion of the C-terminal lobe, resulting in a pathological conformation not reported before. These structural changes result in altered interactions with the CaV1.2 IQ domain. Another mutation (N97S) reduces the affinity for Ca2+ by introducing strain in EF hand 3. A fourth mutant (F141L) shows structural changes in the Ca2+-free state that increase the affinity for the IQ domain. These results thus show that different mechanisms underlie the ability of CaM disease mutations to affect Ca2+-dependent inactivation of the voltage-gated calcium channel.