Can calmodulin bind to lipids of the cytosolic leaflet of plasma membranes?

Calmodulin (CaM) is a ubiquitous calcium-sensitive messenger in eukaryotic cells. It was previously shown that CaM possesses an affinity for diverse lipid moieties, including those found on CaM-binding proteins. These facts, together with our observation that CaM accumulates in membrane-rich protrusions of HeLa cells upon increased cytosolic calcium, motivated us to perform a systematic search for unmediated CaM interactions with model lipid membranes mimicking the cytosolic leaflet of plasma membranes. A range of experimental techniques and molecular dynamics simulations prove unambiguously that CaM interacts with lipid bilayers in the presence of calcium ions. The lipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) hold the key to CaM-membrane interactions. Calcium induces an essential conformational rearrangement of CaM, but calcium binding to the headgroup of PS also neutralizes the membrane negative surface charge. More intriguingly, PE plays a dual role-it not only forms hydrogen bonds with CaM, but also destabilizes the lipid bilayer increasing the exposure of hydrophobic acyl chains to the interacting proteins. Our findings suggest that upon increased intracellular calcium concentration, CaM and the cytosolic leaflet of cellular membranes can be functionally connected.

Unveiling the intricate role of S100A1 in regulating RyR1 activity: A commentary on "Structural insights into the regulation of RyR1 by S100A1".

S100A1, a calcium-binding protein, plays a crucial role in regulating Ca2+ signaling pathways in skeletal and cardiac myocytes via interactions with the ryanodine receptor (RyR) to affect Ca2+ release and contractile performance. Biophysical studies strongly suggest that S100A1 interacts with RyRs but have been inconclusive about both the nature of this interaction and its competition with another important calcium-binding protein, calmodulin (CaM). Thus, high-resolution cryo-EM studies of RyRs in the presence of S100A1, with or without additional CaM, were needed. The elegant work by Weninger et al. demonstrates the interaction between S100A1 and RyR1 through various experiments and confirms that S100A1 activates RyR1 at sub-micromolar Ca2+ concentrations, increasing the open probability of RyR1 channels.

Two new species of Fusarium in the F. incarnatum-equiseti species complex from Oryza sativa in Iran.

Identification of Fusarium species associated with diseases symptoms in plants is an important step toward understanding the ecology of plant-fungus associations. In this study, four Fusarium isolates were obtained from root rot of Oryza sativa L. in Izeh (southwest of Iran) and identified based on phylogenetic analyses combined with morphology. Phylogenetic analyses based on combined translation elongation factor 1-α, calmodulin, RNA polymerase II second largest subunit, and Beta-tubulin (tub2) sequence data delimited two new species, namely F. khuzestanicum and F. oryzicola spp. nov., from previously known species of Fusarium within F. incarnatum-equiseti species complex (FIESC). Morphologically, F. khuzestanicum produces the macroconidia with distinctly notched to foot-shaped basal cells, while basal cells in the macroconidia of F. oryzicola are more extended and distinctly elongated foot shape. Furthermore, these two new species are distinguished by the size of their sporodochial phialides and macroconidia. The results of the present show that the FIESC species complex represent more cryptic species.

Effects of calcium ions and cell wall deposition on the pollen viability of Paeonia lactiflora after cryopreservation.

MAIN CONCLUSION: Four cultivars of Paeonia lactiflora pollen have a different viability after cryopreservation, and that the difference of pollen viability is related to calcium ions and cell wall deposition. Cryopreservation is a vital technique for preserving germplasm resources, offering extensive application prospects. Understanding the factors influencing pollen viability after cryopreservation is crucial for the permanent preservation and exchange of pollen resources. This study investigated pollen from four Paeonia lactiflora cultivars with varying viability after cryopreservation, aiming to determine whether calcium ions (Ca2+) and cell wall deposition affect these viability changes. The results showed that Ca2+-ATPase activity and cytoplasmic Ca2+ of all four cultivars exhibited an increasing trend after cryopreservation; the calmodulin (CaM) content varied with cultivars. Correlation analysis showed that fresh pollen viability was significantly negatively correlated with cytoplasmic Ca2+ content and positively correlated with Ca2+-ATPase activity, while pollen viability after cryopreservation exhibited a significantly negative correlation with cytoplasmic Ca2+ content and a positive correlation with CaM content. The pollen cell wall of the cultivar 'Zi Feng Chao Yang' (ZFCY), which showed increased viability after cryopreservation, contained significantly higher levels of low-temperature tolerance-related phospholipids and proteins compared to other cultivars. Additionally, all cultivars maintained a clear Ca2+ gradient at the tips of pollen tubes after cryopreservation, without significant callose accumulation. These findings suggest that differences in Ca2+ signaling and cell wall components deposition influence changes in pollen viability after cryopreservation, and the Ca2+ gradient and callose at the tip of pollen tubes are not responsible for preventing pollen tube growth.

Atomistic mechanisms of the regulation of small-conductance Ca2+-activated K+ channel (SK2) by PIP2.

Small-conductance Ca2+-activated K+ channels (SK, KCa2) are gated solely by intracellular microdomain Ca2+. The channel has emerged as a therapeutic target for cardiac arrhythmias. Calmodulin (CaM) interacts with the CaM binding domain (CaMBD) of the SK channels, serving as the obligatory Ca2+ sensor to gate the channels. In heterologous expression systems, phosphatidylinositol 4,5-bisphosphate (PIP2) coordinates with CaM in regulating SK channels. However, the roles and mechanisms of PIP2 in regulating SK channels in cardiomyocytes remain unknown. Here, optogenetics, magnetic nanoparticles, combined with Rosetta structural modeling, and molecular dynamics (MD) simulations revealed the atomistic mechanisms of how PIP2 works in concert with Ca2+-CaM in the SK channel activation. Our computational study affords evidence for the critical role of the amino acid residue R395 in the S6 transmembrane segment, which is localized in propinquity to the intracellular hydrophobic gate. This residue forms a salt bridge with residue E398 in the S6 transmembrane segment from the adjacent subunit. Both R395 and E398 are conserved in all known isoforms of SK channels. Our findings suggest that the binding of PIP2 to R395 residue disrupts the R395:E398 salt bridge, increasing the flexibility of the transmembrane segment S6 and the activation of the channel. Importantly, our findings serve as a platform for testing of structural-based drug designs for therapeutic inhibitors and activators of the SK channel family. The study is timely since inhibitors of SK channels are currently in clinical trials to treat atrial arrhythmias.

Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites.

The autoinhibited plasma membrane calcium ATPase ACA8 from A. thaliana has an N-terminal autoinhibitory domain. Binding of calcium-loaded calmodulin at two sites located at residues 42-62 and 74-96 relieves autoinhibition of ACA8 activity. Through activity studies and a yeast complementation assay we investigated wild-type (WT) and N-terminally truncated ACA8 constructs (Δ20, Δ30, Δ35, Δ37, Δ40, Δ74 and Δ100) to explore the role of conserved motifs in the N-terminal segment preceding the calmodulin binding sites. Furthermore, we purified WT, Δ20- and Δ100-ACA8, tested activity in vitro and performed structural studies of purified Δ20-ACA8 stabilized in a lipid nanodisc to explore the mechanism of autoinhibition. We show that an N-terminal segment between residues 20 and 35 including conserved Phe32, upstream of the calmodulin binding sites, is important for autoinhibition and the activation by calmodulin. Cryo-EM structure determination at 3.3 Å resolution of a beryllium fluoride inhibited E2 form, and at low resolution for an E1 state combined with AlphaFold prediction provide a model for autoinhibition, consistent with the mutational studies.

Loop-Mediated Isothermal Amplification for the Diagnosis of Sporotrichosis by Sporothrix brasiliensis.

We aimed to develop and validate a Loop-mediated Isothermal Amplification (LAMP) assay to Sporothrix brasiliensis. LAMP reaction was developed using six primers designed based on calmodulin gene. In the LAMP reaction, we tested twenty isolates of S. brasiliensis from animals and humans, along with ten tissue samples extracted from the left footpad of mice that had been experimentally infected with S. brasiliensis. In addition, it included DNA samples from various other fungal species for specificity evaluation. All S. brasiliensis isolates yielded positive results in the LAMP, and the limit of DNA detection was 1 ng/µL. All murine samples were positive in the test while DNA from other fungal species were all negative, resulting in 100% of sensitivity and specificity of primers. LAMP diagnosis technique is a promising alternative to sporotrichosis diagnosis, in a simple and cost-effective way. Further studies are warranted to validate this technique using animal model samples obtained from both humans and animals.

Development and evaluation of a rapid diagnostic method for Sporothrix globosa in Asia using quantitative real-time PCR.

BACKGROUND: Sporotrichosis is a chronic granulomatous infection of the skin and subcutaneous tissue that can affect any organ through lymphatic spread. The prevalence of sporotrichosis infections is increasing and its treatment is challenging as there are no unified and standard diagnostic techniques or antifungal medications. Controlling further spread requires a rapid diagnosis. Assessment of clinical symptoms, histological analysis, serological testing, and pathogen culture are all necessary for the diagnosis of sporotrichosis. However, these procedures are unable to identify the species. The development of safe, reliable, and species-specific diagnostic techniques is essential. OBJECTIVE: To establish and evaluate a new quantitative real-time PCR assay for the rapid diagnosis of sporotrichosis and to identify relevant species. METHODS: Polymorphisms in calmodulin (CAL) gene sequences and the internal transcribed spacer (ITS) were used in a quantitative real-time PCR assay to identify S. globosa, S. schenckii, and non-target species. RESULTS: The quantitative real-time PCR assay had 100% sensitivity and specificity. The limit of detection was 6 fg/µl. Thirty-four clinical specimens were verified to be infected with S. globosa with a 100% positive detection rate. CONCLUSIONS: The quantitative PCR technique developed in this study is a quick, accurate, and targeted method of identifying S. globosa based on polymorphisms in CAL sequences and ITS. It can be used for a prompt clinical diagnosis to identify S. globosa in clinical specimens from patients with sporotrichosis.

Nanoparticle-mediated calmodulin dsRNA and cyantraniliprole co-delivery system: High-efficient control of two key pear pests while ensuring safety for natural enemy insects.

Currently, the predominant method for managing pests in orchards is chemical control. However, prolonged use of chemicals leads to resistance issues and raise ecological safety. A promising approach to tackle these challenges involves nanoparticles-mediated delivery system of dsRNA and pesticides. Despite its potential, this strategy has not been widely applied in controlling pests in pear orchards. In this study, we developed a nanoparticle-mediated ternary biopesticide to tackle resistance and safety concerns associated with calmodulin dsRNA and cyantraniliprole. Initially, we assessed the effectiveness of cyantraniliprole against two key pear pests, Grapholita molesta and Cacopsylla chinensis. Subsequently, we observed an upregualtion of genes CaM and CN following cyantraniliprole treatment. Furthermore, inhibiting or silencing GmCaM and CcGaM enhanced the sensitivity to cyantraniliprole more effectively. By introducing hairpin RNA into the pET30a-BL21 RNaseIII- system to silence GmCaM and CcCaM, we developed a nanoparticle-mediated co-delivery system that exhibited improved control over these two pests. Importantly, our research demonstrated that using reduced cyantraniliprole dosages through ternary biopesticides could help mitigate risks to natural enemies. Overall, our research emphasizes the enhanced effectiveness of ternary biopesticides in boosting the performance of dsRNA and pesticide against pear pests, while fostering environmental sustainability-a novel advancement in this field.

Calmodulin: a highly conserved and ubiquitous Ca2+ sensor.

Calcium ions (Ca2+) play critical roles in various biological phenomena. The free Ca2+ concentration in the cytoplasm of a resting cell is at the 10-7 M level, whereas that outside the cell is 10-3 M, creating a 10,000-fold gradient of Ca2+ concentrations across the cell membrane, separating the intracellular and extracellular solutions.1),2) When a cell is activated by external stimuli, the intracellular Ca2+ concentration increases to levels of 10-6-10-5 M through Ca2+ entry from the extracellular solution via plasma membrane Ca2+ channels and/or Ca2+ release from intracellular stores. This transient increase in Ca2+ functions as an important signal mediated by Ca2+ sensors. Thus, Ca2+ signals are transmitted to intracellular loci such as distinct, localized targets of Ca2+ sensors. Among numerous Ca2+ sensors present in cells, calmodulin is a highly conserved and ubiquitous Ca2+ sensor.3).

Characterization of Arabidopsis aldolases AtFBA4, AtFBA5, and their inhibition by morin and interaction with calmodulin.

Fructose bisphosphate aldolases (FBAs) catalyze the reversible cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. We analyzed two previously uncharacterized cytosolic Arabidopsis FBAs, AtFBA4 and AtFBA5. Based on a recent report, we examined the interaction of AtFBA4 with calmodulin (CaM)-like protein 11 (AtCML11). AtFBA4 did not bind AtCML11; however, we found that CaM bound AtFBA5 in a Ca2+-dependent manner with high specificity and affinity (KD ~ 190 nm) and enhanced its stability. AtFBA4 and AtFBA5 exhibited Michaelis-Menten kinetics with Km and Vmax values of 180 µm and 4.9 U·mg-1 for AtFBA4, and 6.0 µm and 0.30 U·mg-1 for AtFBA5, respectively. The flavonoid morin inhibited both isozymes. Our study suggests that Ca2+ signaling and flavanols may influence plant glycolysis/gluconeogenesis.

Genome-wide identification and expression analysis of calmodulin and calmodulin-like genes in passion fruit (Passiflora edulis) and their involvement in flower and fruit development.

BACKGROUND: The calmodulin (CaM) and calmodulin-like (CML) proteins play regulatory roles in plant growth and development, responses to biotic and abiotic stresses, and other biological processes. As a popular fruit and ornamental crop, it is important to explore the regulatory mechanism of flower and fruit development of passion fruit. RESULTS: In this study, 32 PeCaM/PeCML genes were identified from passion fruit genome and were divided into 9 groups based on phylogenetic analysis. The structural analysis, including conserved motifs, gene structure and homologous modeling, illustrates that the PeCaM/PeCML in the same subgroup have relative conserved structural features. Collinearity analysis suggested that the expansion of the CaM/CML gene family likely took place mainly by segmental duplication, and the whole genome replication events were closely related with the rapid expansion of the gene group. PeCaM/PeCMLs were potentially required for different floral tissues development. Significantly, PeCML26 had extremely high expression levels during ovule and fruit development compared with other PeCML genes, suggesting that PeCML26 had potential functions involved in the development of passion fruit flowers and fruits. The co-presence of various cis-elements associated with growth and development, hormone responsiveness, and stress responsiveness in the promoter regions of these PeCaM/PeCMLs might contribute to their diverse regulatory roles. Furthermore, PeCaM/PeCMLs were also induced by various abiotic stresses. This work provides a comprehensive understanding of the CaM/CML gene family and valuable clues for future studies on the function and evolution of CaM/CML genes in passion fruit. CONCLUSION: A total of 32 PeCaM/PeCML genes were divided into 9 groups. The PeCaM/PeCML genes showed differential expression patterns in floral tissues at different development stages. It is worth noting that PeCML26, which is highly homologous to AtCaM2, not only interacts with multiple BBR-BPC TFs, but also has high expression levels during ovule and fruit development, suggesting that PeCML26 had potential functions involved in the development of passion fruit flowers and fruits. This research lays the foundation for future investigations and validation of the potential function of PeCaM/PeCML genes in the growth and development of passion fruit.

Deficiency of IQCH causes male infertility in humans and mice.

IQ motif-containing proteins can be recognized by calmodulin (CaM) and are essential for many biological processes. However, the role of IQ motif-containing proteins in spermatogenesis is largely unknown. In this study, we identified a loss-of-function mutation in the novel gene IQ motif-containing H (IQCH) in a Chinese family with male infertility characterized by a cracked flagellar axoneme and abnormal mitochondrial structure. To verify the function of IQCH, Iqch knockout (KO) mice were generated via CRISPR-Cas9 technology. As expected, the Iqch KO male mice exhibited impaired fertility, which was related to deficient acrosome activity and abnormal structures of the axoneme and mitochondria, mirroring the patient phenotypes. Mechanistically, IQCH can bind to CaM and subsequently regulate the expression of RNA-binding proteins (especially HNRPAB), which are indispensable for spermatogenesis. Overall, this study revealed the function of IQCH, expanded the role of IQ motif-containing proteins in reproductive processes, and provided important guidance for genetic counseling and genetic diagnosis of male infertility.

Difference in Contractile Mechanisms between the Early and Sustained Components of Ionomycin-Induced Contraction in Rat Caudal Arterial Smooth Muscle.

We previously reported that the sustained component of contraction induced by depolarizing stimulation by high K+ concentration in rat caudal arterial smooth muscle involves a Ca2+-induced Ca2+ sensitization mechanism whereby Ca2+ entry through voltage-gated Ca2+ channels activates proline-rich tyrosine kinase 2 (Pyk2), leading to activation of RhoA/Rho-associated kinase (ROCK). In the present study, we investigated a potential role for Pyk2-mediated RhoA/ROCK activation in contraction mediated by elevation of cytosolic free Ca2+ concentration ([Ca2+]i) induced by a Ca2+ ionophore, ionomycin, rather than by depolarizing stimulation. Ionomycin (60 µM) induced slow and sustained contraction of rat caudal arterial smooth muscle due to influx of Ca2+. Pre-treatment with a myosin light chain kinase (MLCK) inhibitor, ML-9 (30 µM), inhibited both the early phase (4 min) and the sustained phase (30 min) of ionomycin-induced contraction. On the other hand, a ROCK inhibitor, HA-1077 (3 µM), and Pyk2 inhibitors, sodium salicylate (10 mM) and PF-431396 (3 µM), suppressed only the sustained phase of ionomycin-induced contraction. A calmodulin (CaM) inhibitor, W-7 (150 µM), but not W-5 (150 µM), suppressed the early phase of contraction. Early or sustained increase of ionomycin-induced 20 kDa light chain of myosin (LC20) phosphorylation was inhibited by each inhibitor in a manner similar to the attenuation of contraction. These results indicate that the early phase of ionomycin-induced contraction is mediated by MLCK activation by [Ca2+]i elevation, whereas the sustained phase of ionomycin-induced contraction involves RhoA/ROCK activation and inhibition of myosin light chain phosphatase (MLCP) through CaM-independent Pyk2 activation by [Ca2+]i elevation.

The Impact of Calcium Depletion on Proliferation of Chlorella sorokiniana Strain DSCG150.

This study analyzed the effects of Ca2+ metal ions among culture medium components on the Chlorella sorokiniana strain DSCG150 strain cell growth. The C. sorokiniana strain DSCG150 grew based on a multiple fission cell cycle and growth became stagnant in the absence of metal ions in the medium, particularly Ca2+. Flow cytometry and confocal microscopic image analysis results showed that in the absence of Ca2+, cell growth became stagnant as the cells accumulated into four autospores and could not transform into daughter cells. Genetic analysis showed that the absence of Ca2+ caused upregulation of calmodulin (calA) and cell division control protein 2 (CDC2_1) genes, and downregulation of origin of replication complex subunit 6 (ORC6) and dual specificity protein phosphatase CDC14A (CDC14A) genes. Analysis of gene expression patterns by qRT-PCR showed that the absence of Ca2+ did not affect cell cycle progression up to 4n autospore, but it inhibited Chlorella cell fission (liberation of autospores). The addition of Ca2+ to cells cultivated in the absence of Ca2+ resulted in an increase in n cell population, leading to the resumption of C. sorokiniana growth. These findings suggest that Ca2+ plays a crucial role in the fission process in Chlorella.

Reversing anxiety by targeting a stress-responsive signaling pathway.

Current treatments of anxiety and depressive disorders are plagued by considerable side effects and limited efficacies, underscoring the need for additional molecular targets that can be leveraged to improve medications. Here, we have identified a molecular cascade triggered by chronic stress that exacerbates anxiety- and depressive-like behaviors. Specifically, chronic stress enhances Src kinase activity and tyrosine phosphorylation of calmodulin, which diminishes MyosinVa (MyoVa) interaction with Neuroligin2 (NL2), resulting in decreased inhibitory transmission and heightened anxiety-like behaviors. Importantly, pharmacological inhibition of Src reinstates inhibitory synaptic deficits and effectively reverses heightened anxiety-like behaviors in chronically stressed mice, a process requiring the MyoVa-NL2 interaction. These data demonstrate the reversibility of anxiety- and depressive-like phenotypes at both molecular and behavioral levels and uncover a therapeutic target for anxiety and depressive disorders.

The CDPK-related protein kinase HvCRK2 and HvCRK4 interact with HvCML32 to negatively regulate drought tolerance in transgenic Arabidopsis thaliana.

Calcium-dependent protein kinase (CDPK) as one of calcium sensors were play important roles in stress responses. CDPK-related protein kinase (CRK) was identified as subgroup III of CDPK has been characterized in many plants, but the members and functions of CRK genes in hulless barley (Hordeum vulgare L.) has not been clarified. Here, we identified four HvCRK genes and named HvCRK1-4 according to chromosomes localization. Moreover, the physiological function of highly induced genes of HvCRK2 and HvCRK4 were investigated in drought stress tolerance by examining their overexpression transgenic lines functions generated in Arabidopsis thaliana. Under drought stress, both overexpression HvCRK2 and HvCRK4 displayed reduced drought resistance, and accompanied by higher accumulation levels of ROS. Notably, overexpression of HvCRK2 and HvCRK4 reduced sensitivity to exogenous ABA, meanwhile the expression of ABA-responsive genes in transgenic plants were down-regulated compared to the wild type in response to drought stress. Furthermore, the physically interaction of HvCRK2 and HvCRK4 with calmodulin (CaM) and calmodulin-like (CML) proteins were determined in vivo, the further results showed that HvCML32 binds to HvCRK2/4 S_TKC structural domains and negatively regulates drought tolerance. In summary, this study identified HvCRK members and indicated that HvCRK2 and HvCRK4 genes play negative roles in drought tolerance, and provide insight into potential molecular mechanism of HvCRK2 and HvCRK4 in response to drought stress.

Calmodulin Triggers Activity-Dependent rRNA Biogenesis via Interaction with DDX21.

Protein synthesis in response to neuronal activity, known as activity-dependent translation, is critical for synaptic plasticity and memory formation. However, the signaling cascades that couple neuronal activity to the translational events remain elusive. In this study, we identified the role of calmodulin (CaM), a conserved Ca2+-binding protein, in ribosomal RNA (rRNA) biogenesis in neurons. We found the CaM-regulated rRNA synthesis is Ca2+-dependent and necessary for nascent protein synthesis and axon growth in hippocampal neurons. Mechanistically, CaM interacts with nucleolar DEAD (Asp-Glu-Ala-Asp) box RNA helicase (DDX21) in a Ca2+-dependent manner to regulate nascent rRNA transcription within nucleoli. We further found CaM alters the conformation of DDX21 to liberate the DDX21-sequestered RPA194, the catalytic subunit of RNA polymerase I, to facilitate transcription of ribosomal DNA. Using high-throughput screening, we identified the small molecules batefenterol and indacaterol that attenuate the CaM-DDX21 interaction and suppress nascent rRNA synthesis and axon growth in hippocampal neurons. These results unveiled the previously unrecognized role of CaM as a messenger to link the activity-induced Ca2+ influx to the nucleolar events essential for protein synthesis. We thus identified the ability of CaM to transmit information to the nucleoli of neurons in response to stimulation.

CaM-dependent modulation of human CaV1.3 whole-cell and single-channel currents by C-terminal CaMKII phosphorylation site S1475.

Phosphorylation enables rapid modulation of voltage-gated calcium channels (VGCC) in physiological and pathophysiological conditions. How phosphorylation modulates human CaV1.3 VGCC, however, is largely unexplored. We characterized modulation of CaV1.3 gating via S1475, the human equivalent of a phosphorylation site identified in the rat. S1475 is highly conserved in CaV1.3 but absent from all other high-voltage activating calcium channel types co-expressed with CaV1.3 in similar tissues. Further, it is located in the C-terminal EF-hand motif, which binds calmodulin (CaM). This is involved in calcium-dependent channel inactivation (CDI). We used amino acid exchanges that mimic either sustained phosphorylation (S1475D) or phosphorylation resistance (S1475A). Whole-cell and single-channel recordings of phosphorylation state imitating CaV1.3 variants in transiently transfected HEK-293 cells revealed functional relevance of S1475 in human CaV1.3. We obtained three main findings: (1) CaV1.3_S1475D, imitating sustained phosphorylation, displayed decreased current density, reduced CDI and (in-) activation kinetics shifted to more depolarized voltages compared with both wildtype CaV1.3 and the phosphorylation-resistant CaV1.3_S1475A variant. Corresponding to the decreased current density, we find a reduced open probability of CaV1.3_S1475D at the single-channel level. (2) Using CaM overexpression or depletion, we find that CaM is necessary for modulating CaV1.3 through S1475. (3) CaMKII activation led to CaV1.3_WT-current properties similar to those of CaV1.3_S1475D, but did not affect CaV1.3_S1475A, confirming that CaMKII modulates human CaV1.3 via S1475. Given the physiological and pathophysiological importance of CaV1.3, our findings on the S1475-mediated interplay of phosphorylation, CaM interaction and CDI provide hints for approaches on specific CaV1.3 modulation under physiological and pathophysiological conditions. KEY POINTS: Phosphorylation modulates activity of voltage-gated L-type calcium channels for specific cellular needs but is largely unexplored for human CaV1.3 channels. Here we report that S1475, a CaMKII phosphorylation site identified in rats, is functionally relevant in human CaV1.3. Imitating phosphorylation states at S1475 alters current density and inactivation in a calmodulin-dependent manner. In wildtype CaV1.3 but not in the phosphorylation-resistant variant S1475A, CaMKII activation elicits effects similar to constitutively mimicking phosphorylation at S1475. Our findings provide novel insights on the interplay of modulatory mechanisms of human CaV1.3 channels, and present a possible target for CaV1.3-specific gating modulation in physiological and pathophysiological conditions.

Susceptibility Testing of Environmental and Clinical Aspergillus sydowii Demonstrates Potent Activity of Various Antifungals.

The genus Aspergillus consists of a vast number of medically and environmentally relevant species. Aspergillus species classified in series Versicolores are ubiquitous in the environment and include the opportunistic pathogen Aspergillus sydowii, which is associated with onychomycosis and superficial skin infections. Despite frequent clinical reports of A. sydowii and related series Versicolores species, antifungal susceptibility data are scarce, hampering optimal treatment choices and subsequent patient outcomes. Here, we employed antifungal susceptibility testing (AFST) based on microbroth dilution on a set of 155 series Versicolores strains using the common antifungals amphotericin B, itraconazole, voriconazole, posaconazole, isavuconazole and micafungin with the addition of luliconazole and olorofim. All strains were identified using partial calmodulin gene sequencing, with 145 being A. sydowii, seven A. creber and three A. versicolor, using the latest taxonomic insights. Overall, tested antifungals were potent against the entire strain collection. In comparison to A. fumigatus, azole and amphotericin B MICs were slightly elevated for some strains. AFST with luliconazole and olorofim, here reported for the first time, displayed the highest in vitro activity, making these antifungals interesting alternative drugs but clinical studies are warranted for future therapeutic use.