Regulation of brain-specific kinases 1 and 2 (BRSK1/2) by Ca2+/calmodulin.

Calcium (Ca2+) is an essential mineral nutrient and a vital signaling molecule for plant growth and development. Ca2+/calmodulins (CaMs) can bind proteins, called CaM binding proteins (CBPs), to relay Ca2+ signals and modulate transcriptional activity. In this study, we demonstrate that AtCBP60b, a member of CBP60 family, is an essential factor in regulating plant growth and development. AtCBP60b loss of function plants showed impaired seedling growth, leaf development, and plant structural architecture under normal growth conditions. Transcriptomic profiles indicated a central role for AtCBP60b in the transcriptional regulation of Ca2+ signaling pathway, growth homeostasis, immune, and stress responses. Disruption of AtCBP60b leads to Ca2+ deficiency hypersensitivity, altered regulation of Ca2+ dynamics and Ca2+ signaling responses. AtCBP60b was found to bind calmodulin which was determined to be required for AtCBP60b function in transcriptional regulation of the genes encoding the pathways maintaining cytosolic Ca2+ homeostasis, regulating defense, and growth and development. In addition, we show that elevated temperatures can rescue the atcpb60b growth and development defects through reprogramming of the transcriptional profiles of the genes regulating these processes. Overall, our findings demonstrate that AtCBP60b plays an important function in regulating plant Ca2+ signaling response, growth and development, and responses to elevated temperature.

In addition to binding to and regulating over 400 different proteins, calmodulin (CaM) also binds to lipids. Binding occurs to the prenylated tails of various small GTPases, to specific lipids in biological membranes and to free lipids in the cytoplasm. Here, CaM binding to Rac1, RalA, and KRAS4b is covered, emphasizing its importance in protein translocation from the cell membrane to the cytosol and its resultant impact on cell signaling. Binding phosphatidylserine and phosphatidylethanolamine in membranes not only leads to the tethering of CaM, but also to the disruption of lipid bilayers. Binding to sphingolipids also occurs, an event that acts as a competitive inhibitor of CaM function. The mechanism through which CaM binds to lipids is also examined. In total, the current state of affairs regarding calcium-dependent CaM-lipid binding is reviewed, including potential therapeutic uses, setting the stage for future work on this important biological event.

The synaptic plasticity mechanisms that are thought to underlie learning and memory require Ca2+ influx mediated by N-methyl-D-aspartate receptors composed of glycine-binding GluN1 and glutamate-binding GluN2 subunits. Calmodulin (CaM) binding to the cytosolic regions in both GluN1 (residues 841-865, called GluN1-C0) and GluN2A (residues 1004-1023, called GluN2A-C0) may be important for Ca2+-dependent channel desensitization (CDD). Here, we report NMR, ITC and electrophysiological experiments to probe the structure and functional role of Ca2+-bound CaM (Ca2+-CaM) binding to both GluN1 and GluN2A subunits. Our ITC studies show that the GluN1-C0 peptide binds to both the N-lobe and C-lobe of Ca2+-CaM, whereas the GluN2A-C0 peptide binds to only the Ca2+-CaM C-lobe. Our NMR analysis reveals GluN2A residues (W1014 and V1018) interact with exposed hydrophobic residues in the Ca2+-CaM C-lobe. The NMR structure of Ca2+-CaM bound to the GluN1-C0 peptide indicates the two CaM lobes bind to opposite sides of the GluN1-C0 helix (C-lobe contacts M848, F852, A853 and N-lobe contacts A854, V855, W858). The GluN1 mutant F852E and the GluN2A mutant W1014E both perturbed CaM binding in ITC studies, and also diminished electrophysiologically-measured CDD, suggesting CaM interaction with these residues contributes to CDD. We propose a structural mechanism of CDD wherein channel desensitization is caused by the binding of four CaM per N-methyl-D-aspartate receptor subunit tetramer.

The HIV-1 Vpu membrane protein is crucial to the virus lifecycle. Our recent studies revealed soluble Vpu oligomers, prompting further investigation into their interactions with cellular proteins. Notably, Vpu may form a complex with calmodulin (CaM) due to its putative CaM-binding motif; however, experimental proof of this association is unavailable. Here, we present definitive experimental evidence that the soluble Vpu complex interacts in vitro with calcium-bound CaM (Ca2+-CaM), its active form. Using double electron-electron resonance (DEER) spectroscopy and protein spin labeling, we detected the formation of a soluble Vpu-Ca2+-CaM complex. Both the full-length (FL) and truncated C-terminal regions of Vpu bind Ca2+-CaM. DEER experiments on a spin-labeled CaM cysteine mutant S39C/A103C revealed that, upon association with Vpu, Ca2+-CaM undergoes a transition from an open to a more closed conformation, consistent with previous reports of Ca2+-CaM interactions with other proteins. Furthermore, we observed that the binding of Vpu to Ca2+-CaM leads to dissociation of soluble Vpu oligomers, as evidenced by a reduction in DEER modulation depth for FL Vpu spin-labeled at residue L42C. FRET analysis with a fluorescently labeled C-terminal cysteine mutant of Vpu confirmed this result. Like FL Vpu, the Vpu C-terminal region forms soluble homooligomers that dissociate upon binding to Ca2+-CaM. Collectively, our results suggest that soluble Vpu and Ca2+-CaM form an equimolar complex. DEER analysis of the Vpu C-terminal region spin-labeled at residues Q36C/I61C demonstrated that Vpu undergoes significant conformational changes to facilitate Ca2+-CaM binding. These findings could be relevant to Vpu-CaM interactions under physiological conditions.

Voltage-gated Na+ (Nav) channels, including Nav1.5, are responsible for the initiation of cardiac and neuronal action potentials. Regulation of Nav1.5 inactivation is linked to multiple accessory proteins that bind its C-terminal domain (CTD) including calmodulin (CaM) and intracellular fibroblast growth factors (iFGF). Previous results demonstrate that Ca2+-bound CaM preferentially binds to iFGF12A. The role of intracellular Ca2+ ([Ca2+]i) in regulating Nav1.5 gating, either directly or via auxiliary proteins like CaM, is controversial. We hypothesize that CaM binding to the Nav1.5 CTD and iFGF12A synergistically alters channel inactivation in a previously unobserved calcium-dependent manner. We performed Fluorescence Resonance Energy Transfer (FRET) imaging in live cells to observe the interaction between the Nav1.5 alpha subunit, CaM and iFGF12A. At resting [Ca2+]i, a 2-fold difference between acceptor and donor FRET efficiency was observed, implying that a single CaM acceptor is present on the Nav1.5 CTD even in the presence of FGF12A. After increasing [Ca2+]i, the donor and acceptor FRET efficiencies equalize, suggesting a 2:1:1 ratio between CaM, FGF12A, and the Nav1.5 CTD. We then compared the voltage-dependent gating kinetics of Nav1.5 with FGF12A in the presence/absence of calcium. With low [Ca2+]i, the steady-state inactivation of Nav1.5 with FGF12A was significantly shifted toward hyperpolarized potential compared to resting [Ca2+]i. Thus, the FGF12A:CaM complex confers a Ca2+-dependent mechanism enabling FGF12A modulates the Nav1.5 steady-state inactivation. Additionally, the ability of multiple subunits to bring CaM to the Nav1.5 CTD implies biological redundancy to prevent major alteration to Nav1.5 inactivation in the absence of CaM.

The matrix (MA) domain of the Mason-Pfizer monkey virus (M-PMV) Gag polyprotein plays a central role in retroviral assembly and trafficking, coordinating membrane association and proteolytic maturation. Unlike HIV-1, M-PMV assembles immature particles in the cytoplasm prior to plasma membrane targeting, but the molecular mechanisms governing this process remain poorly understood. Here, we identify calmodulin (CaM) as a calcium-dependent modulator of MA structural dynamics. Using a combination of biophysical and biochemical methods, we demonstrate that CaM directly interacts with myristoylated MA, promoting its oligomerization and enhancing its cleavage by the viral protease. In-depth characterization of MA-CaM complex by protein cross-linking mass spectrometry, hydrogen/deuterium exchange mass spectrometry and NMR spectroscopy reveals that the N-terminal parts of both proteins are in close proximity within the complex and that CaM binding induces increased conformational flexibility of key regions within MA, including the basic patch and C-terminal cleavage site. These dynamic changes suggest an allosteric mechanism by which CaM regulates MA function, potentially facilitating the temporal coordination of membrane targeting, the myristoyl switch and proteolytic processing. Our findings broaden the understanding of CaM as a regulatory factor in retroviral assembly and underscore the importance of conformational plasticity in viral maturation.

Eukaryotic elongation factor-2 kinase (eEF-2K), a member of the α-kinase family of atypical serine/threonine kinases, phosphorylates eEF-2 to slow ribosomal translocation and modulate translational elongation. eEF-2K activation requires Ca2+/calmodulin (CaM) and integrates upstream signals through specific sites within an intrinsically disordered regulatory loop (R-loop; ∼321–520) that links the α-kinase core to a C-terminal domain. Unlike canonical CaM-dependent kinases that are activated by displacement of an autoinhibitory segment that occludes the active site, eEF-2K is activated by CaM-driven stabilization of an active state; Ca2+/CaM engagement triggers rapid autophosphorylation at T348, which is essential for full activity. Phosphorylation on S500, by eEF-2K or PKA, lowers the CaM requirement (∼20-fold) without increasing maximal catalytic turnover. Here we show that the phosphomimetic S500D markedly enhances CaM binding in the T348-phosphorylated enzyme. S500D also elevates CaM-independent (“intrinsic”) activity even in the absence of phosphorylation at T348, although maximal activity requires modification at both sites. Hydrogen–deuterium exchange mass spectrometry reveals CaM-dependent conformational changes near S500, consistent with relief of inhibitory constraints. Deletion of residues near S500 mimics S500D, increasing intrinsic activity and CaM binding in vitro and enhancing eEF-2 phosphorylation in cells, supporting an inhibitory role for this segment. Prior studies have linked S500 phosphorylation to eEF-2K degradation, suggesting a dual regulatory role. We demonstrate that phosphorylation at T348 and S500 synergize to stabilize an active-like conformation and increase CaM responsiveness, effectively lowering the Ca2+/CaM threshold for eEF-2K activation and enabling the integration of Ca2+, cAMP/PKA, and metabolic cues.

The “wasabi receptor,” transient receptor potential ankyrin 1 (TRPA1), is a critical pain receptor that is activated by reactive environmental and endogenous irritants to initiate pain perception, local inflammation, and protective behaviors. TRPA1 is a calcium-permeable ion channel; calcium influx tightly controls channel function by first potentiating currents and then triggering rapid desensitization. Here, we provide evidence that the universal calcium sensor calmodulin (CaM) controls TRPA1 desensitization by engaging two distinct CaM-binding sites in the cytoplasmic C terminus: the distal C-terminal CaM binding element (DCTCaMBE) and the TRP CaMBD. Specifically, we found that CaM binds TRPA1 at the DCTCaMBE beginning at low calcium and the TRP CaMBD at high calcium concentrations. Pull-down experiments revealed that the DCTCaMBE and TRP CaMBD exhibit distinct CaM lobe binding specificities. Competition experiments showed that CaM can bind both sites simultaneously as isolated peptides. Molecular modeling identified residues predicted to contribute to the CaM–TRP CaMBD interaction. Mutation of these residues revealed that CaM binding to the TRP CaMBD controls a second kinetic step in TRPA1 desensitization. Finally, complete ablation of CaM binding at both the TRPA1 DCTCaMBE and TRP CaMBD showed that they contribute to a concerted desensitization mechanism. Together, these results support a model where CaM associates with TRPA1 at rest through the DCTCaMBE, which primes the channel for rapid desensitization. As intracellular calcium levels rise, CaM then binds the TRP CaMBD—through bridged or separate interactions—to promote a terminal desensitization step. Our work provides further mechanistic insight into how calcium and CaM tightly control TRPA1 channel function to promote nociceptive signaling.

Plasma membrane Ca2+-ATPases (PMCA) are activated by calmodulin (CaM) via a C-terminal calmodulin-binding domain, CaMBD. Although specific mutations in this domain have been linked to disease, the broader impact of alternative substitutions across the interface remains unexplored. We applied an integrative in silico workflow to test six substitutions within CaMBD positions 1-18, L5R, N6I, I8T, V14E/D, and F18S, across PMCA isoforms 1-4. CaMBD sequences were aligned across isoforms, and candidates for substitutions were selected by conservation and nucleotide feasibility, prioritizing conserved or co-evolutionarily relevant sites, with substitutions possible by single-nucleotide change. PolyPhen-2 screened the impact of the substitutions on the protein functionality, the DisGeNET database was used to contextualize ATP2B genes with clinical phenotypes, and structural models plus binding free energy changes were estimated with AlphaFold3, FoldX, and MutaBind2. Effects were isoform and subregion dependent, with the strongest weakening toward the CaMBD C-terminus. V14E/D and F18S showed the largest and consistent predicted destabilization, consistent with disruption of conserved hydrophobic anchors. I8T and L5R had mixed outcomes depending on isoform, while N6I presented various scenarios with no clear effect. PolyPhen-2 classified most tested substitutions as damaging. Gene-disease evidence linked ATP2B to neurological, endocrine, and oncologic phenotypes, consistent with roles in Ca2+ homeostasis. Overall, CaMBD appears highly sensitive to perturbation, with distal positions 14-18 particularly vulnerable to substitutions that can destabilize CaM binding and potentially impair PMCA-mediated Ca2+ clearance in susceptible tissues.

Calmodulin (CaM) serves an essential role in eukaryotic cells as a Ca2+ sensor. Ca2+ binding leads to conformation changes in CaM that enable engagement of a repertoire of enzymes and the regulation of their catalytic activities. Classically, Ca2+-CaM binds to an inhibitory pseudosubstrate sequence C-terminal to the kinase domain in members of the Ca2+-CaM-dependent protein kinase (CAMK) family and relieves inhibition to promote catalytic activity. Here, we report an unexpected mechanism by which CaM can bind CHK2 kinase to inhibit its kinase activity. Using biochemical, biophysical and structural mass spectrometry, we identify a direct interaction of Ca2+-CaM with the CHK2 kinase domain that suppresses CHK2 catalytic activity in vitro and identify K373 in CHK2 as crucial for cell proliferation in human cells following DNA damage. Our findings add direct suppression of kinase activity to the repertoire of CaM’s functions, complementing the paradigmatic mechanism of promoting kinase activity through autoinhibitory domain sequestration.

RsCAMTA11 positively regulates salt tolerance by repressing transcription of two RsRbohs in radish (Raphanus sativus L.).

Acute myeloid leukemia (AML) is a lethal clonal hematopoietic malignancy. Several reports have shown that serum metabolite alterations have been implicated in AML, but the causal relationship and underlying biological mechanisms remain unclear. We performed bidirectional Mendelian randomization (MR) to evaluate the association between 486 serum metabolites and AML. The analytical approaches used to minimize research bias included the inverse variance weighting (IVW), MR-Egger and weighted median (WM) methods. Sensitivity analyses were performed using Cochran’s Q Test, MR-Egger, MR pleiotropy residual sum and outlier (MR-PRESSO), and Leave-one-out (LOO) analysis. Metabolic pathway analysis was conducted using the MetaboAnalyst 6.0 platform. We utilized RNA-seq data to explore the potential genes and mechanisms underlying the regulation of AML occurrence by serum metabolites. We identified 23 serum metabolites (13 known and 10 unknown) significantly associated with AML. Sensitivity analyses further validated the robustness of these associations. No evidence of reverse causality was detected by reverse MR analysis. The core pathways were histidine metabolism and fructose/mannose metabolism. Transcriptomic integration revealed 39 overlapping genes (differentially expressed genes vs. metabolite-associated genes) as key mediators, enriched in neuroactive ligand signaling, synaptic vesicle cycle, and GABAergic synapse (KEGG), plus synapse assembly and calmodulin binding and neuron-to-neuron synapse (GO). This study establishes causal links between specific serum metabolites and AML, revealing neuro-related mechanistic pathways. These findings provide novel biomarkers and therapeutic targets for AML precision medicine.

In high-throughput screening (HTS) assays using fluorescence lifetime (FLT)-detected FRET, we have identified compounds that allosterically modulate the pathologically leaky ryanodine receptor (RyR) calcium release channels. These compounds may prevent or reduce the elevated Ca2+ that fuels arrhythmia, heart failure, and age-related neurodegeneration. RyRs are responsible for intracellular Ca2+ release from endoplasmic/sarcoplasmic reticulum (ER/SR). The resulting [Ca2+] pulse is a signal for many cellular processes, whereas sustained elevated [Ca2+] is pathologic. Our FRET-based HTS detects the pathology-linked RyR leaky state by monitoring binding of the accessory protein calmodulin and the DPc10 peptide (corresponding to RyR2 residues 2460–2495) known to perturb interdomain interactions within RyR2. Under conditions mimicking a pathological state, we have screened a 50,000-compound chemical library to identify small-molecule modulators of RyR2 in cardiac SR membranes. This screen yielded 603 compounds that reproducibly altered FRET. Based on FRET response profiles that align with therapeutic potential, 83 of those most promising compounds were purchased and validated by FRET dose response evaluation. Focusing on ten chemical scaffolds that desirably increase A-CaM binding, six representative compounds reduced RyR2 activity as measured by [3H]ryanodine binding. Ca2+ dynamics in HEK293 cells expressing human RyR2 or in cardiomyocytes highlighted the isoxazole group of hits as potentially therapeutic by targeting the pathological RyR2 leak state.

Abstract Background Ventricular arrhythmia, a major cause of sudden cardiac death, is driven by Ca2+ imbalance in cardiac myocytes, often linked to the overactivation of CaMKIIδ (Ca2+/calmodulin-dependent protein kinase II delta). As such, inhibiting CaMKIIδ represents a promising therapeutic strategy. Purpose Based on our previous finding that native secretoneurin (SN, 33 amino acids) is a weak CaMKIIδ inhibitor, we aimed to develop a more potent and shorter derivative of SN to effectively counter aberrant Ca2+ handling and arrhythmia risk. Methods Interaction of the novel SN derivative to CaMKIIδ was identified and analysed by structural homology modelling, pull-down, ELISA-based assays and surface plasmon resonance experiments. The inhibitory effect of the SN derivative on CaMKIIδ activity was investigated indirectly by measuring CaMKIIδ-dependent phosphorylation of Ser2814-RYR2 (ryanodine receptor) and Ser17-PLN (phospholamban). The internalisation into isolated rat cardiomyocytes was verified by confocal fluorescence microscopy of FITC-labelled peptides. Effects of the novel SN derivative on Ca2+ sparks and waves were examined by fluorescence in isolated rat cardiomyocytes treated with isoproterenol to mimic a β-adrenergic challenge. Results Various regions of SN were tested for CaMKIIδ binding, identifying the core region (amino acids 7-26), which is notably the most conserved across species, to have the strongest binding capacity. This region was subsequently optimized with two phenylalanine substitutions (L17F, V20F), resulting in the novel SN derivative; SN-db-short. Detailed mapping analyses show that SN-db-short bound both to the substrate-binding (S-site) region of CaMKIIδ, in addition to the ATP-binding region, with an 8-fold stronger binding compared to native SN. Consistent with only a partial calmodulin binding motif, SN-db-short exhibited no calmodulin binding, indicating a selective CaMKIIδ inhibition. In functional studies, SN-db-short inhibited CaMKIIδ-mediated phosphorylation of Ser2814-RYR2 and reduced the incidence of Ca2+ sparks and Ca2+ waves to a greater extent than native SN. SN-db-short also more markedly inhibited CaMKIIδ phosphorylation of Thr17-PLN, slowed Ca2+ reuptake, and reduced the magnitude of Ca2+ transients during isoproterenol stimulation. Conclusion SN-db-short effectively inhibits CaMKIIδ, and significantly counters aberrant Ca2+ handling in cardiomyocytes. Thus, this optimized peptide holds therapeutic potential for reducing the risk of ventricular arrhythmias.

Introduction: Heart failure with preserved ejection fraction (HFpEF) is a prevalent and morbid syndrome strongly linked with obesity. The Glucagon-like Peptide-1 (GLP-1) agonists have emerged as promising therapeutic tools, but their multifarious mechanisms remain under investigation. We investigated proteomic changes in the heart and liver in a pre-clinical model of cardiometabolic HFpEF and the effects of the GLP-1 agonist, semaglutide, with particular focus on lipid and fibrosis signaling. Methods: 10-week-old male ZSF1 obese rats (HFpEF) were treated with low-dose semaglutide (30 nmol/kg subcutaneously twice weekly, n=5) or vehicle (n=5) for 16 weeks. Myocardial and hepatic tissue were collected for mass spectrometry-based proteomics and histology evaluating lipid deposition (TEM and Oli red O) and fibrosis (Sirius Red/Fast Green collagen stain). Protein was labeled using tandem mass tags (TMT-16plex) for multiplexing and quantitation. Post-processing analysis included linear mixed modeling for differential abundance and pathway enrichment analysis (false discovery rate was 0.25). Results: The dose of semaglutide used in this study did not alter body weight. Myocardial enriched pathways for proteins upregulated by semaglutide were related to the ribosome and branch chain amino acid degradation (Fig. 1A). Myocardial enriched pathways for proteins downregulated by semaglutide were related to PPAR signaling, protein-lipid complex, and calmodulin binding. Hepatic enriched pathways for proteins upregulated by semaglutide were related to the ribosome and rRNA binding. Hepatic enriched pathways for proteins downregulated by semaglutide were related to the inflammatory response, oxidative phosphorylation, and SNARE proteins (Fig. 2A). Intracellular myocardial LD quantity and size were decreased as well as were numerous proteins related to lipid homeostasis with semaglutide treatment (Fig. 1B). Left ventricular fibrosis was also reduced with GLP-1 therapy (representative images in Fig. 1C). Similarly, hepatic lipid deposition and fatty acid/lipid protein signaling and fibroblast proliferation pathways were mitigated by semaglutide (Fig. 2B and 2C). Conclusion: These studies provide the first myocardial and hepatic proteomic atlas of the effects of GLP-1 agonism in a pre-clinical model of obese HFpEF. These results link the protective systemic actions of semaglutide on cardiac and hepatic lipid handling and fibrosis in a weight loss independent fashion.

The T1r family of receptors is essential for the detection of sweet and umami tastants, which are categorized as class C G protein-coupled receptors (GPCRs). Although these receptors share structural characteristics with other class C GPCRs, such as metabotropic glutamate receptors, they are uniquely characterized by a significantly shorter C-terminal intracellular domain, consisting of approximately 30-40 amino acid residues. Yoshida etal. recently demonstrated that the C-terminal region of mouse T1rs directly binds to calmodulin in a Ca2+-dependent manner. This interaction highlights a previously unrecognized aspect of the intracellular signalling mechanism of T1rs and indicates that the C-terminal region contributes to taste signal regulation, particularly through Ca2+-dependent feedback mechanisms.

Adenylyl Cyclase isoform 1 (AC1), responsible for synthesizing the signaling molecule cyclic adenosine monophosphate (cAMP), is key in synaptic plasticity, long-term chronic pain syndromes, osteosarcoma-associated pain, and drug abuse. The protein calmodulin (CaM) and the small molecule forskolin (Fsk) stimulate AC1 to form a catalytic site for ATP catalysis; however, molecular AC1 structural changes triggered by CaM and Fsk remain poorly understood. This study developed a computational model for AC1-cofactor complexes and used all-atom molecular dynamics (MD) simulations to determine how CaM and Fsk individually and jointly affect AC1 dynamics and assess whether they exhibit any synergistic effects. Four systems were investigated: AC1-No Partner, AC1-CaM, AC1-Fsk, and AC1-CaM-Fsk. Simulations revealed that individual and joint cofactor bindings induced unique structural changes within the regulatory C1b subdomain of AC1. CaM and Fsk binding result in a reduced cross section of the catalytic site, implying tighter binding for ATP. Notably, the results showed that the AC1-CaM-Fsk system exhibited unique features distinct from the AC1-CaM and AC1-Fsk systems, demonstrating synergistic effects of CaM and Fsk. Our understanding of AC1-cofactor interactions can guide future research toward modulating AC1 activity, potentially contributing to the development of novel treatments for AC1-associated diseases.

Mammalian cyclic nucleotide-gated (CNG) channels play crucial roles in visual and olfactory signal transduction. In olfactory sensory neurons, the native CNG channel functions as a heterotetramer consisting of CNGA2, CNGA4, and CNGB1b subunits and is activated by cAMP. Calmodulin (CaM) modulates the activity of the olfactory CNG channel, enabling rapid adaptation to odorants. Here we present cryo-EM structures of the native human olfactory CNGA2/A4/B1b channel in both CaM-bound closed and cAMP-bound open states, elucidating the molecular basis of the 2:1:1 subunit stoichiometry in channel assembly and the asymmetrical channel gating upon cAMP activation. Combining structural and functional analyses with AlphaFold prediction, we define two distinct CaM binding sites (CaM1 and CaM2) on the N- and C-terminal regions of CNGB1b, respectively, shedding light on the molecular mechanism of Ca2+/CaM-mediated rapid inhibition of the native olfactory CNG channel.

ABSTRACTVentricular arrhythmias, a major cause of sudden cardiac death, are driven by Ca2+ imbalance in cardiac myocytes, often linked to the overactivation of CaMKIIδ (Ca2+/calmodulin‐dependent protein kinase II delta). As such, inhibiting CaMKIIδ represents a promising therapeutic strategy. Based on our previous finding that native secretoneurin (SN) is a weak CaMKIIδ inhibitor, we aimed to develop a more potent derivative of SN to effectively counter aberrant Ca2+ handling and arrhythmia risk. Various regions of SN were tested for CaMKII binding, identifying the core region as the sequence with the strongest binding capacity. This region was subsequently optimised with two phenylalanine substitutions, resulting in the SN derivative SN‐db‐short. Structural homology modeling and ELISA‐based assays revealed that SN‐db‐short bound both the substrate‐binding (S‐site) region of CaMKIIδ, in addition to the ATP‐binding region, with 8‐fold stronger binding compared to SN. Surface plasmon resonance experiments confirmed that SN‐db‐short exhibited a higher association rate and affinity for CaMKIIδ compared to SN. Consistent with only a partial calmodulin binding motif, SN‐db‐short showed no calmodulin binding, indicating selective CaMKIIδ inhibition. In functional studies, SN‐db‐short inhibited CaMKIIδ‐mediated phosphorylation of ryanodine receptor 2 and appeared more effective than SN in reducing the incidence of Ca2+ sparks and Ca2+ waves. SN‐db‐short also more markedly inhibited CaMKIIδ phosphorylation of phospholamban, slowed Ca2+ reuptake, and reduced the magnitude of Ca2+ transients during isoproterenol stimulation. SN‐db‐short effectively inhibits CaMKIIδ and significantly counters aberrant Ca2+ handling in cardiomyocytes. Thus, this optimised peptide holds therapeutic potential for reducing the risk of ventricular arrhythmias.