Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable cation channel required for immune cell activation, insulin secretion, and body heat control. TRPM2 activated by cytosolic Ca2+, phosphatidyl-inositol-4,5-bisphosphate ADP ribose. Here, we present the ~3 Å resolution electron cryo-microscopic structure of from Nematostella vectensis, 63% similar in sequence to human TRPM2, Ca2+-bound closed state. Compared other TRPM channels, exhibits unique structural features that...

In normal beta-cells glucose induces insulin secretion by activating both a triggering pathway (closure of K(ATP) channels, depolarization, and rise in cytosolic [Ca(2+)](i)) an amplifying (augmentation Ca(2+) efficacy on exocytosis). It is unclear if how nutrients can regulate lacking channels (Sur1 knockout mice). We compared glucose- amino acid-induced [Ca(2+)](i) changes control Sur1KO islets. 1 mm (non-stimulatory for controls), the signal was high (loss regulation) stimulated This...

In adult beta-cells glucose-induced insulin secretion involves two mechanisms (a) a K(ATP) channel-dependent Ca(2+) influx and rise of cytosolic [Ca(2+)](c) (b) channel-independent amplification without further increase [Ca(2+)](c). Mice lacking the high affinity sulfonylurea receptor (Sur1KO), thus channels, have been developed as model congenital hyperinsulinism. Here, we compared in overnight cultured islets from 2-week-old normal Sur1KO mice. Control proved functionally mature: magnitude...

Protein kinase A (PKA) is a key regulator of cellular functions by selectively phosphorylating numerous substrates, including ion channels, enzymes, and transcription factors. It has long served as model system for understanding the eukaryotic kinases. Using cryoelectron microscopy, we present complex structures PKA catalytic subunit (PKA-C) bound to full-length protein substrate, cystic fibrosis transmembrane conductance (CFTR)—an channel vital human health. CFTR gating requires...

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) diverse functions. This involves formation of NBD dimers, with bound composite interfacial sites. In CFTR, intramolecular dimerization coupled opening. Channel closing triggered by molecule site 2. Site 1,...

Summary Protein kinase A (PKA) is a key regulator of cellular functions by selectively phosphorylating numerous substrates, including ion channels, enzymes, and transcription factors. It has long served as model system for understanding the eukaryotic kinases. Using cryo-electron microscopy, we present complex structures PKA catalytic subunit (PKA-C) bound to full-length protein substrate, cystic fibrosis transmembrane conductance (CFTR) – an channel vital human health. CFTR gating requires...

Background and purpose: ATP‐sensitive potassium channels (K ATP channels) in beta cells are a major target for insulinotropic drugs. Here, we studied the effects of selected stimulatory inhibitory pharmacological agents islets lacking K channels. Experimental approach: We compared insulin secretion (IS) cytosolic calcium ([Ca 2+ ] c ) changes isolated from control mice sulphonylurea receptor1 (SUR1), thus their ( Sur1 KO). Key results: While similarly increasing [Ca IS controls, binding to...

Islet antigen-2 (IA-2 or ICA 512) and IA-2β (or phogrin) are major autoantigens in type 1 diabetes. They located dense core secretory vesicles including insulin granules, but their role β-cell function is unclear. Targeted disruption of either IA-2 IA-2β, both, impaired glucose tolerance, an effect attributed to diminution secretion. In this study, we therefore characterized the dynamic changes cytosolic Ca 2+ ([Ca ] c ) secretion islets from IA-2/IA-2β double knockout (KO) mice. High (15...

Transient Receptor Potential Melastatin 2 (TRPM2) is a cation channel important for the immune response, insulin secretion, and body temperature regulation. It activated by cytosolic ADP ribose (ADPR) contains nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain homologous to ADPR phosphohydrolases (ADPRases). Human TRPM2 (hsTRPM2) catalytically inactive due mutations in conserved Nudix box sequence. Here, we show that motifs are canonical all invertebrates but vestigial vertebrates....

KtrAB belongs to the Trk/Ktr/HKT superfamily of monovalent cation (K+ and Na+) transport proteins that closely resemble K+ channels. These underlie a plethora cellular functions are crucial for environmental adaptation in plants, fungi, archaea, bacteria. The activation mechanism remains unknown. It has been shown ATP stimulates activity while ADP does not. Here, we present X-ray structural information on complex with bound ADP. A comparison KtrAB-ATP structure reveals conformational changes...

A current model ascribes glucose-induced insulin secretion to the interaction of a triggering pathway (K(ATP) channel-dependent Ca(2+) influx and rise in cytosolic [Ca(2+)](c)) an amplifying channel-independent augmentation without further increase [Ca(2+)](c)). However, several studies sulfonylurea receptor 1 null mice (Sur1KO) failed measure significant effects glucose their islets lacking K(ATP) channels. We addressed this issue that challenges model. Compared with controls, fresh Sur1KO...

A central step in the gating of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is association its two cytosolic nucleotide-binding domains (NBDs) into a head-to-tail dimer, with nucleotides bound at interface. Channel opening and closing, respectively, are coupled to formation disruption this tight NBD dimer. CFTR an asymmetric adenosine triphosphate (ATP)-binding cassette protein which interfacial-binding sites (composite 1 2) functionally different. During...

The chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) displays a typical adenosine trisphosphate (ATP)-binding cassette (ABC) protein architecture comprising two domains, intracellular nucleotide-binding domains (NBDs), and unique regulatory domain. Once phosphorylated in the domain, CFTR channels can open close when supplied with cytosolic ATP. Despite general agreement that formation of head-to-tail NBD dimer drives opening pore, little is known about how ATP...

Abstract CFTR, the anion channel mutated in cystic fibrosis (CF) patients, is activated by catalytic subunit of protein kinase A (PKA-C). PKA-C activates CFTR both reversibly, through binding, and irreversibly, phosphorylation multiple serines CFTR’s regulatory (R) domain. Here we identify key molecular determinants CFTR/PKA-C interaction essential for these processes. By comparing current activation presence ATP or an analog unsuitable phosphotransfer, as well pseudosubstrate peptides...

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the anion channel mutated in cystic fibrosis (CF) patients, is activated by catalytic subunit of protein kinase A (PKA-C). PKA-C activates CFTR both noncatalytically, through binding, and catalytically, phosphorylation multiple serines CFTR’s regulatory (R) domain. Here, we identify key molecular determinants CFTR/PKA-C interaction essential for these processes. By comparing current activation presence ATP or an analog unsuitable...

Transient Receptor Potential Melastatin 2 (TRPM2) cation channels contribute to immunocyte activation, insulin secretion, and central thermoregulation. TRPM2 opens upon binding cytosolic Ca 2+ ADP ribose (ADPR). We present here the 2.5 Å cryo-electronmicroscopy structure of from Nematostella vectensis (nvTRPM2) in a lipid nanodisc, complexed with ADPR-2′-phosphate. Comparison nvTRPM2 without nucleotide reveals that binding-induced movements protein’s three “core” layers deconvolve into set...

Transient receptor potential melastatin 2 (TRPM2) cation channel activity is required for insulin secretion, immune cell activation and body heat control. Channel upon oxidative stress involved in the pathology of stroke neurodegenerative disorders. Cytosolic Ca2+, ADP-ribose (ADPR) phosphatidylinositol-4,5-bisphosphate (PIP2) are obligate activators channel. Several TRPM2 cryo-EM structures have been resolved to date, yet functionality purified protein has not tested. Here we reconstituted...

CFTR, the anion channel mutated in cystic fibrosis patients, is a model ABC protein whose ATP-driven conformational cycle observable at single-molecule level patch-clamp recordings. Bursts of CFTR pore openings are coupled to tight dimerization its two nucleotide-binding domains (NBDs) and wild-type (WT) channels mostly terminated by ATP hydrolysis. The slow rate non-hydrolytic closure - which determines how tightly bursts hydrolysis unknown, as burst durations catalytic site mutants span...