A recurrent missense variant in ITPR3 causes demyelinating Charcot-Marie-Tooth with variable severity.

Charcot-Marie-Tooth (CMT) disease is a neuromuscular disorder affecting the peripheral nervous system. The diagnostic yield in demyelinating CMT (CMT1) is typically ∼80-95%, of which at least 60% is due to the PMP22 gene duplication. The remainder of CMT1 is more genetically heterogeneous.

Coupled transmembrane mechanisms control MCU-mediated mitochondrial Ca uptake.

Ca uptake by mitochondria regulates bioenergetics, apoptosis, and Ca signaling. The primary pathway for mitochondrial Ca uptake is the mitochondrial calcium uniporter (MCU), a Ca-selective ion channel in the inner mitochondrial membrane. MCU-mediated Ca uptake is driven by the sizable inner-membrane potential generated by the electron-transport chain.

ER-luminal [Ca] regulation of InsP receptor gating mediated by an ER-luminal peripheral Ca-binding protein.

Modulating cytoplasmic Ca concentration ([Ca]) by endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate receptor (InsPR) Ca-release channels is a universal signaling pathway that regulates numerous cell-physiological processes. Whereas much is known regarding regulation of InsPR activity by cytoplasmic ligands and processes, its regulation by ER-luminal Ca concentration ([Ca]) is poorly understood and controversial. We discovered that the InsPR is regulated by a peripheral membrane-associated ER-luminal protein that strongly inhibits the channel in the presence of high, physiological [Ca].

Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening.

The mitochondrial calcium uniporter complex is essential for calcium (Ca) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca signal transduction. Despite its involvement in several human diseases, we currently lack pharmacological agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specific small-molecule modulators in primary drug screens.

Biphasic regulation of InsP3 receptor gating by dual Ca2+ release channel BH3-like domains mediates Bcl-xL control of cell viability.

Antiapoptotic Bcl-2 family members interact with inositol trisphosphate receptor (InsP3R) Ca(2+)release channels in the endoplasmic reticulum to modulate Ca(2+)signals that affect cell viability. However, the molecular details and consequences of their interactions are unclear. Here, we found that Bcl-xL activates single InsP3R channels with a biphasic concentration dependence.

EMRE Is a Matrix Ca(2+) Sensor that Governs Gatekeeping of the Mitochondrial Ca(2+) Uniporter.

The mitochondrial uniporter (MCU) is an ion channel that mediates Ca(2+) uptake into the matrix to regulate metabolism, cell death, and cytoplasmic Ca(2+) signaling. Matrix Ca(2+) concentration is similar to that in cytoplasm, despite an enormous driving force for entry, but the mechanisms that prevent mitochondrial Ca(2+) overload are unclear. Here, we show that MCU channel activity is governed by matrix Ca(2+) concentration through EMRE.

General anesthetic isoflurane modulates inositol 1,4,5-trisphosphate receptor calcium channel opening.

Background: Pharmacological evidence suggests that inhalational general anesthetics induce neurodegeneration in vitro and in vivo through overactivation of inositol trisphosphate receptor (InsP3R) Ca-release channels, but it is not clear whether these effects are due to direct modulation of channel activity by the anesthetics.

Methods: Using single-channel patch clamp electrophysiology, the authors examined the gating of rat recombinant type 3 InsP3R (InsP3R-3) Ca-release channels in isolated nuclei (N = 3 to 15) from chicken lymphocytes modulated by isoflurane at clinically relevant concentrations in the absence and presence of physiological levels of the agonist inositol 1,4,5-trisphosphate (InsP3). The authors also examined the effects of isoflurane on InsP3R-mediated Ca release from the endoplasmic reticulum and changes in intracellular Ca concentration ([Ca]i).

Phosphorylated K-Ras limits cell survival by blocking Bcl-xL sensitization of inositol trisphosphate receptors.

K-Ras4B is targeted to the plasma membrane by a farnesyl modification that operates in conjunction with a polybasic domain. We characterized a farnesyl-electrostatic switch whereby protein kinase C phosphorylates K-Ras4B on serine 181 in the polybasic region and thereby induces translocation from the plasma membrane to internal membranes that include the endoplasmic reticulum (ER) and outer mitochondrial membrane. This translocation is associated with cell death.

Nuclear patch-clamp electrophysiology of Ca2+ channels.

Patch-clamping the outer or inner nuclear membrane of isolated nuclei is very similar to patch-clamping the plasma membrane of isolated cells. This protocol describes in detail all the steps required to successfully obtain nuclear membrane patches, in various configurations, from both the outer and inner nuclear membranes of isolated nuclei.

Isolating nuclei from cultured cells for patch-clamp electrophysiology of intracellular Ca(2+) channels.

Nuclear patch-clamp experiments can be performed with intact nuclei or with nuclei from which the outer nuclear membrane has been removed. This protocol presents procedures for harvesting different types of cultured cells, isolating nuclei, and exposing the inner nuclear membrane by agitating in the presence of sodium citrate. Particulars about obtaining and maintaining the cells of interest in culture are not described here.

Patch-clamp electrophysiology of intracellular Ca2+ channels.

The modulation of cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) is a universal intracellular signaling pathway that regulates numerous cellular physiological processes. Ubiquitous intracellular Ca(2+)-release channels localized to the endoplasmic/sarcoplasmic reticulum-inositol 1,4,5-trisphosphate receptor (InsP3R) and ryanodine receptor (RyR) channels-play a central role in [Ca(2+)]i signaling in all animal cells. Despite their intracellular localization, electrophysiological studies of the single-channel permeation and gating properties of these Ca(2+)-release channels using the powerful patch-clamp approach have been possible by application of this technique to isolated nuclei because the channels are present in membranes of the nuclear envelope.

Permeant calcium ion feed-through regulation of single inositol 1,4,5-trisphosphate receptor channel gating.

The ubiquitous inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) Ca(2+) release channel plays a central role in the generation and modulation of intracellular Ca(2+) signals, and is intricately regulated by multiple mechanisms including cytoplasmic ligand (InsP(3), free Ca(2+), free ATP(4-)) binding, posttranslational modifications, and interactions with cytoplasmic and endoplasmic reticulum (ER) luminal proteins. However, regulation of InsP(3)R channel activity by free Ca(2+) in the ER lumen ([Ca(2+)](ER)) remains poorly understood because of limitations of Ca(2+) flux measurements and imaging techniques. Here, we used nuclear patch-clamp experiments in excised luminal-side-out configuration with perfusion solution exchange to study the effects of [Ca(2+)](ER) on homotetrameric rat type 3 InsP(3)R channel activity.

Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) has InsP(3)-independent gating and disrupts intracellular Ca(2+) homeostasis.

The type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) is a ubiquitous intracellular Ca(2+) release channel that is vital to intracellular Ca(2+) signaling. InsP(3)R1 is a proteolytic target of calpain, which cleaves the channel to form a 95-kDa carboxyl-terminal fragment that includes the transmembrane domains, which contain the ion pore. However, the functional consequences of calpain proteolysis on channel behavior and Ca(2+) homeostasis are unknown.

Unitary Ca(2+) current through recombinant type 3 InsP(3) receptor channels under physiological ionic conditions.

The ubiquitous inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) channel, localized primarily in the endoplasmic reticulum (ER) membrane, releases Ca(2+) into the cytoplasm upon binding InsP(3), generating and modulating intracellular Ca(2+) signals that regulate numerous physiological processes. Together with the number of channels activated and the open probability of the active channels, the size of the unitary Ca(2+) current (i(Ca)) passing through an open InsP(3)R channel determines the amount of Ca(2+) released from the ER store, and thus the amplitude and the spatial and temporal nature of Ca(2+) signals generated in response to extracellular stimuli. Despite its significance, i(Ca) for InsP(3)R channels in physiological ionic conditions has not been directly measured.

Pseudomonas aeruginosa Homoserine lactone activates store-operated cAMP and cystic fibrosis transmembrane regulator-dependent Cl- secretion by human airway epithelia.

The ubiquitous bacterium Pseudomonas aeruginosa frequently causes hospital-acquired infections. P. aeruginosa also infects the lungs of cystic fibrosis (CF) patients and secretes N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) to regulate bacterial gene expression critical for P.

Essential regulation of cell bioenergetics by constitutive InsP3 receptor Ca2+ transfer to mitochondria.

Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of mitochondrial activity is incompletely understood.

Redox-regulated heterogeneous thresholds for ligand recruitment among InsP3R Ca2+-release channels.

To clarify the molecular mechanisms behind quantal Ca2+ release, the graded Ca2+ release from intracellular stores through inositol 1,4,5-trisphosphate receptor (InsP3R) channels responding to incremental ligand stimulation, single-channel patch-clamp electrophysiology was used to continuously monitor the number and open probability of InsP3R channels in the same excised cytoplasmic-side-out nuclear membrane patches exposed alternately to optimal and suboptimal cytoplasmic ligand conditions. Progressively more channels were activated by more favorable conditions in patches from insect cells with only one InsP3R gene or from cells solely expressing one recombinant InsP3R isoform, demonstrating that channels with identical primary sequence have different ligand recruitment thresholds. Such heterogeneity was largely abrogated, in a fully reversible manner, by treatment of the channels with sulfhydryl reducing agents, suggesting that it was mostly regulated by different levels of posttranslational redox modifications of the channels.

A polymorphism in CALHM1 influences Ca2+ homeostasis, Abeta levels, and Alzheimer's disease risk.

Alzheimer's disease (AD) is a genetically heterogeneous disorder characterized by early hippocampal atrophy and cerebral amyloid-beta (Abeta) peptide deposition. Using TissueInfo to screen for genes preferentially expressed in the hippocampus and located in AD linkage regions, we identified a gene on 10q24.33 that we call CALHM1.

Apoptosis regulation by Bcl-x(L) modulation of mammalian inositol 1,4,5-trisphosphate receptor channel isoform gating.

Members of the Bcl-2 family of proteins regulate apoptosis, with some of their physiological effects mediated by their modulation of endoplasmic reticulum (ER) Ca(2+) homeostasis. Antiapoptotic Bcl-x(L) binds to the inositol trisphosphate receptor (InsP(3)R) Ca(2+) release channel to enhance Ca(2+)- and InsP(3)-dependent regulation of channel gating, resulting in reduced ER [Ca(2+)], increased oscillations of cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), and apoptosis resistance. However, it is controversial which InsP(3)R isoforms mediate these effects and whether reduced ER [Ca(2+)] or enhanced [Ca(2+)](i) signaling is most relevant for apoptosis protection.

Graded recruitment and inactivation of single InsP3 receptor Ca2+-release channels: implications for quantal [corrected] Ca2+release.

Modulation of cytoplasmic free Ca2+ concentration ([Ca2+]i) by receptor-mediated generation of inositol 1,4,5-trisphosphate (InsP3) and activation of its receptor (InsP3R), a Ca2+-release channel in the endoplasmic reticulum, is a ubiquitous signalling mechanism. A fundamental aspect of InsP3-mediated signalling is the graded release of Ca2+ in response to incremental levels of stimuli. Ca2+ release has a transient fast phase, whose rate is proportional to [InsP3], followed by a much slower one even in constant [InsP3].

Novel adenoviral vectors coding for GFP-tagged wtCFTR and deltaF508-CFTR: characterization of expression and electrophysiological properties in A549 cells.

E1/E3-deleted adenoviral vectors expressing an N-terminal green fluorescent protein (GFP) reporter gene fused to either wtCFTR (H5.040CMVEGFP-wtCFTR) or deltaF508-CFTR (H5.040CMVEGFP-deltaF508CFTR) were generated.

Dibasic phosphorylation sites in the R domain of CFTR have stimulatory and inhibitory effects on channel activation.

To better understand the mechanisms by which PKA-dependent phosphorylation regulates CFTR channel activity, we have assayed open probabilities (P(o)), mean open time, and mean closed time for a series of CFTR constructs with mutations at PKA phosphorylation sites in the regulatory (R) domain. Forskolin-stimulated channel activity was recorded in cell-attached and inside-out excised patches from transiently transfected Chinese hamster ovary cells. Wild-type CFTR and constructs with a single Ser-to-Ala mutation as well as octa (Ser-to-Ala mutations at 8 sites) and constructs with one or two Ala-to-Ser mutations were studied.