Crystallographic, kinetic, and calorimetric investigation of PKA interactions with L-type calcium channels and Rad GTPase.

β-Adrenergic signaling activates cAMP-dependent PKA, which regulates the activity of L-type voltage-gated calcium channels such as Ca1.2. Several PKA target sites in the C-terminal tail of Ca1.

Elemental mapping in single-particle reconstructions by reconstructed electron energy-loss analysis.

For macromolecular structures determined by cryogenic electron microscopy, no technique currently exists for mapping elements to defined locations, leading to errors in the assignment of metals and other ions, cofactors, substrates, inhibitors and lipids that play essential roles in activity and regulation. Elemental mapping in the electron microscope is well established for dose-tolerant samples but is challenging for biological samples, especially in a cryo-preserved state. Here we combine electron energy-loss spectroscopy with single-particle image processing to allow elemental mapping in cryo-preserved macromolecular complexes.

Cryo-EM structures of ryanodine receptors and diamide insecticides reveal the mechanisms of selectivity and resistance.

The resistance of pests to common insecticides is a global issue that threatens food production worldwide. Diamide insecticides target insect ryanodine receptors (RyRs), causing uncontrolled calcium release from the sarcoplasmic and endoplasmic reticulum. Despite their high potency and species selectivity, several resistance mutations have emerged.

PirA- or PirB-binding nanobodies can protect whiteleg shrimp from the acute hepatopancreatic necrosis disease toxin.

Acute hepatopancreatic necrosis disease (AHPND) is a devastating shrimp disease caused by a binary toxin, PirAB, produced by Vibrio parahaemolyticus and other closely related bacteria. To address AHPND, over 300 unique single-domain antibodies (also known as nanobodies) derived from the VHH domains of Lama glama heavy-chain-only antibodies were raised against either PirA or PirB and characterized. Nanobodies were shortlisted based on their affinities for either PirA or PirB, their relative stability in intestinal fluids, and their ability to reduce PirAB-induced death in brine shrimp Artemia salina.

Cryo-EM investigation of ryanodine receptor type 3.

Ryanodine Receptor isoform 3 (RyR3) is a large ion channel found in the endoplasmic reticulum membrane of many different cell types. Within the hippocampal region of the brain, it is found in dendritic spines and regulates synaptic plasticity. It controls myogenic tone in arteries and is upregulated in skeletal muscle in early development.

Propofol binds and inhibits skeletal muscle ryanodine receptor 1.

Background: As the primary Ca release channel in skeletal muscle sarcoplasmic reticulum (SR), mutations in type 1 ryanodine receptor (RyR1) or its binding partners underlie a constellation of muscle disorders, including malignant hyperthermia (MH). In patients with MH mutations, triggering agents including halogenated volatile anaesthetics bias RyR1 to an open state resulting in uncontrolled Ca release, increased sarcomere tension, and heat production. Propofol does not trigger MH and is commonly used for patients at risk of MH.

Individuals and Families Affected by RYR1-Related Diseases: The Patient/Caregiver Perspective.

Background And Objective: Pathogenic variants of RYR1, the gene encoding the principal sarcoplasmic reticulum calcium release channel (RyR1) with a crucial role in excitation-contraction coupling, are among the most common genetic causes of non-dystrophic neuromuscular disorders. We recently conducted a questionnaire study focusing on functional impairments, fatigue, and quality of life (QoL) in patients with RYR1-related diseases (RYR1-RD) throughout the recognized disease spectrum. In this previous questionnaire study the medical perspective was taken, reflective of a study protocol designed by neurologists and psychologists.

Structural and functional characterization of the nucleotide-binding domains of ABCA4 and their role in Stargardt disease.

ABCA4 is an ATP-binding cassette (ABC) transporter that prevents the buildup of toxic retinoid compounds by facilitating the transport of N-retinylidene-phosphatidylethanolamine across membranes of rod and cone photoreceptor cells. Over 1500 missense mutations in ABCA4, many in the nucleotide-binding domains (NBDs), have been genetically linked to Stargardt disease. Here, we show by cryo-EM that ABCA4 is converted from an open outward conformation to a closed conformation upon the binding of adenylyl-imidodiphosphate.

The molecular basis of cereal mixed-linkage β-glucan utilization by the human gut bacterium Segatella copri.

Mixed-linkage β(1,3)/β(1,4)-glucan (MLG) is abundant in the human diet through the ingestion of cereal grains and is widely associated with healthful effects on metabolism and cholesterol levels. MLG is also a major source of fermentable glucose for the human gut microbiota (HGM). Bacteria from the family Prevotellaceae are highly represented in the HGM of individuals who eat plant-rich diets, including certain indigenous people and vegetarians in postindustrial societies.

ω-Grammotoxin-SIA inhibits voltage-gated Na+ channel currents.

ω-Grammotoxin-SIA (GrTX-SIA) was originally isolated from the venom of the Chilean rose tarantula and demonstrated to function as a gating modifier of voltage-gated Ca2+ (CaV) channels. Later experiments revealed that GrTX-SIA could also inhibit voltage-gated K+ (KV) channel currents via a similar mechanism of action that involved binding to a conserved S3-S4 region in the voltage-sensing domains (VSDs). Since voltage-gated Na+ (NaV) channels contain homologous structural motifs, we hypothesized that GrTX-SIA could inhibit members of this ion channel family as well.

Propofol directly binds and inhibits skeletal muscle ryanodine receptor 1 (RyR1).

As the primary Ca release channel in skeletal muscle sarcoplasmic reticulum (SR), mutations in the type 1 ryanodine receptor (RyR1) or its binding partners underlie a constellation of muscle disorders, including malignant hyperthermia (MH). In patients with MH mutations, exposure to triggering drugs such as the halogenated volatile anesthetics biases RyR1 to an open state, resulting in uncontrolled Ca release, sarcomere tension and heat production. Restoration of Ca into the SR also consumes ATP, generating a further untenable metabolic load.

RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET.

Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca-release channels contributes to heart failure and arrhythmias. Reducing the RyR2 activity, particularly during cardiac relaxation (diastole), is a desirable therapeutic goal. We previously reported that the unnatural enantiomer () of an insect-RyR activator, verticilide, inhibits porcine and mouse RyR2 at diastolic (nanomolar) Ca and has in vivo efficacy against atrial and ventricular arrhythmia.

Cryo-EM analysis of scorpion toxin binding to Ryanodine Receptors reveals subconductance that is abolished by PKA phosphorylation.

Calcins are peptides from scorpion venom with the unique ability to cross cell membranes, gaining access to intracellular targets. Ryanodine Receptors (RyR) are intracellular ion channels that control release of Ca from the endoplasmic and sarcoplasmic reticulum. Calcins target RyRs and induce long-lived subconductance states, whereby single-channel currents are decreased.

Allosteric modulation of ryanodine receptor RyR1 by nucleotide derivatives.

The coordinated release of Ca from the sarcoplasmic reticulum (SR) is critical for excitation-contraction coupling. This release is facilitated by ryanodine receptors (RyRs) that are embedded in the SR membrane. In skeletal muscle, activity of RyR1 is regulated by metabolites such as ATP, which upon binding increase channel open probability (P).

RNA Polymerase II transcription independent of TBP in murine embryonic stem cells.

Transcription by RNA Polymerase II (Pol II) is initiated by the hierarchical assembly of the pre-initiation complex onto promoter DNA. Decades of research have shown that the TATA-box binding protein (TBP) is essential for Pol II loading and initiation. Here, we report instead that acute depletion of TBP in mouse embryonic stem cells has no global effect on ongoing Pol II transcription.

Structural and functional conservation of the activating Ca binding site in inositol 1,4.5-trisphosphate and ryanodine receptors.

Inositol 1,4,5-trisphosphate receptors (IPRs) and Ryanodine Receptors (RyRs) dictate the release of Ca from the Endoplasmic (ER) and Sarcoplasmic Reticulum (SR). Arige et al [1] investigated the functional importance of specific Ca-coordinating residues, unambiguously confirming the activating Ca binding site in the IPR.

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.

The effect of Mg on Ca binding to cardiac troponin C in hypertrophic cardiomyopathy associated TNNC1 variants.

Cardiac troponin C (cTnC) is the critical Ca -sensing component of the troponin complex. Binding of Ca to cTnC triggers a cascade of conformational changes within the myofilament that culminate in force production. Hypertrophic cardiomyopathy (HCM)-associated TNNC1 variants generally induce a greater degree and duration of Ca binding, which may underly the hypertrophic phenotype.

It takes two to tango: Rycals and ATP snuggle up to bind ryanodine receptors.

In this issue of Structure, Melville and colleagues used cryo-EM to study the binding of ryanodine receptors to Rycals, compounds with the potential to treat skeletal and cardiac muscle disorders. Unexpectedly, they found that Rycal packs against an ATP in a peripheral pocket, which stabilizes the closed channel state.

Structural and electrophysiological basis for the modulation of KCNQ1 channel currents by ML277.

The KCNQ1 ion channel plays critical physiological roles in electrical excitability and K recycling in organs including the heart, brain, and gut. Loss of function is relatively common and can cause sudden arrhythmic death, sudden infant death, epilepsy and deafness. Here, we report cryogenic electron microscopic (cryo-EM) structures of Xenopus KCNQ1 bound to Ca/Calmodulin, with and without the KCNQ1 channel activator, ML277.

Structures of PKA-phospholamban complexes reveal a mechanism of familial dilated cardiomyopathy.

Several mutations identified in phospholamban (PLN) have been linked to familial dilated cardiomyopathy (DCM) and heart failure, yet the underlying molecular mechanism remains controversial. PLN interacts with sarco/endoplasmic reticulum Ca-ATPase (SERCA) and regulates calcium uptake, which is modulated by the protein kinase A (PKA)-dependent phosphorylation of PLN during the fight-or-flight response. Here, we present the crystal structures of the catalytic domain of mouse PKA in complex with wild-type and DCM-mutant PLNs.