mTORC1 controls lysosomal Ca²⁺ release through the two-pore channel TPC2

The ion channel TPC2 is required for Ca 2+ mobilization from lysosomes in response to mTORC1 inhibition and to NAADP. </jats:p

Exome Sequencing and the Management of Neurometabolic Disorders

BACKGROUND: Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. METHODS: To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient's clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. RESULTS: We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). CONCLUSIONS: Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children's Hospital Foundation and others.)

L-SR nanojunction study data

<p>Figure_1_ms_suppl.csv<br>Calcium fluorescence<br>Fura-2-AM calcium fluorescence imaging data set used to produce the graphs in Figure 1 of the manuscript as well as Figure 1 in the supplement file to the main manuscript. Data are in a csv file with columns headers for indicating the data in each column.</p> <p>Figure_2_B-D.csv<br>L-SR characterization<br>Data for image analysis statistics shown in Figure 2B-D and gathered from transmission electron micrographs. Data are in comma-separated columns in a simple text file. Column headers indicate the quantity and units listed in each column.</p> <p>Figure_6A.txt<br>TPC2 open probability vs lysosomal luminal [Ca2+]<br>Data used for plot in Figure 6A and obtained from analysis of experimental results in Pitt S et al 2010 (see manuscript references). Data are in space/tab-separated columns in a simple text file. Column headers indicate the quantity and units listed in each column.</p> <p>Figure_6B.txt<br>TPC2 Ca2+ release rate vs time<br>Data used for plot in Figure 6B and obtained as described in section “L-SR junctional Ca2+ signal”. Data are in space/tab-separated columns in a simple text file. Column headers indicate the quantity and units listed in each column.</p> <p>Figure_7A_20_nm.txt, Figure_7A_50_nm.txt, Figure_7A_100_nm.txt<br>Nanojunctional [Ca2+] vs time<br>Data for plot in Figure 7A and obtained as average over 100 simulations each started with a different random number generator seed (as described in section “L-SR junctional Ca2+ signal”). The files contain data from simulations with L-SR junctional separations of 20, 50 and 100 nm, respectively. Data are in space/tab-separated columns in a simple text file. Column headers indicate the quantity and units listed in each column.</p> <p>Figure_7B.txt<br>Nanojunctional [Ca2+] vs L-SR separation distance<br>Data for plot in Figure 7B, solid circles, obtained by temporal average of data in figure 7A over entire time interval. Data are in space/tab-separated columns in a simple text file. Column headers indicate the quantity and units listed in each column.</p> <p>Figure_7B_short_t_avg.txt<br>Nanojunctional [Ca2+] vs L-SR separation distance<br>Data for plot in Figure 7B, empty circles, obtained by temporal average of data in figure 7A over a shorter time interval, as described in section “Reconciling the temporal scales in simulation and experimental results”. Data are in space/tab-separated columns in a simple text file. Column headers indicate the quantity and units listed in each column.</p> <p> </p

Ion Channel Regulation by AMPK: The Route of Hypoxia-Response Coupling in the Carotid Body and Pulmonary Artery

Vital homeostatic mechanisms monitor O2 supply and adjust respiratory and circulatory function to meet demand. The pulmonary arteries and carotid bodies are key systems in this respect. Hypoxic pulmonary vasoconstriction (HPV) aids ventilation−perfusion matching in the lung by diverting blood flow from areas with an O2 deficit to those rich in O2, while a fall in arterial pO2 increases sensory afferent discharge from the carotid body to elicit corrective changes in breathing patterns. We discuss here the new concept that hypoxia, by inhibiting oxidative phosphorylation, activates AMP-activated protein kinase (AMPK) leading to consequent phosphorylation of target proteins, such as ion channels, which initiate pulmonary artery constriction and carotid body activation. Consistent with this view, AMPK knockout mice exhibit an impaired ventilatory response to hypoxia. Thus, AMPK may be sufficient and necessary for hypoxia-response coupling and may regulate O2 and thereby energy (ATP) supply at the whole body as well as the cellular level

The widely utilized brominated flame retardant tetrabromobisphenol A (TBBPA) is a potent inhibitor of the SERCA Ca2+ pump

TBBPA (tetrabromobisphenol A) is currently the most widely used type of BFR (brominated flame retardant) employed to reduce the combustibility of a large variety of electronic and other manufactured products. Recent studies have indicated that BFRs, including TBBPA, are bio-accumulating within animal and humans. BFRs including TBBPA have also been shown to be cytotoxic and potentially endocrine-disrupting to a variety of cells in culture. Furthermore, TBBPA has specifically been shown to cause disruption of Ca2+ homoeostasis within cells, which may be the underlying cause of its cytotoxicity. In this study, we have demonstrated that TBBPA is a potent non-isoform-specific inhibitor of the SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) (apparent Ki 0.46–2.3 μM), thus we propose that TBBPA inhibition of SERCA contributes in some degree to Ca2+ signalling disruption. TBBPA binds directly to the SERCA without the need to partition into the phospholipid bilayer. From activity results and Ca2+-induced conformational results, it appears that the major effect of TBBPA is to decrease the SERCA affinity for Ca2+ (increasing the Kd from approx. 1 μM to 30 μM in the presence of 10 μM TBBPA). Low concentrations of TBBPA can quench the tryptophan fluorescence of the SERCA and this quenching can be reversed by BHQ [2,5-di-(t-butyl)-1,4-hydroquinone] and 4-n-nonylphenol, but not thapsigargin, indicating that TBBPA and BHQ may be binding to similar regions in the SERCA

Ion Channel Regulation by AMPK: The Route of Hypoxia-Response Coupling in the Carotid Body and Pulmonary Artery

Vital homeostatic mechanisms monitor O2 supply and adjust respiratory and circulatory function to meet demand. The pulmonary arteries and carotid bodies are key systems in this respect. Hypoxic pulmonary vasoconstriction (HPV) aids ventilation−perfusion matching in the lung by diverting blood flow from areas with an O2 deficit to those rich in O2, while a fall in arterial pO2 increases sensory afferent discharge from the carotid body to elicit corrective changes in breathing patterns. We discuss here the new concept that hypoxia, by inhibiting oxidative phosphorylation, activates AMP-activated protein kinase (AMPK) leading to consequent phosphorylation of target proteins, such as ion channels, which initiate pulmonary artery constriction and carotid body activation. Consistent with this view, AMPK knockout mice exhibit an impaired ventilatory response to hypoxia. Thus, AMPK may be sufficient and necessary for hypoxia-response coupling and may regulate O2 and thereby energy (ATP) supply at the whole body as well as the cellular level

AMP-activated Protein Kinase Deficiency Blocks the Hypoxic Ventilatory Response and Thus Precipitates Hypoventilation and Apnea

Rationale: Modulation of breathing by hypoxia accommodates variations in oxygen demand and supply during, for example, sleep and ascent to altitude, but the precise molecular mechanisms of this phenomenon remain controversial. Among the genes influenced by natural selection in high-altitude populations is one for the adenosine monophosphate–activated protein kinase (AMPK) α1-catalytic subunit, which governs cell-autonomous adaptations during metabolic stress. Objectives: We investigated whether AMPK-α1 and/or AMPK-α2 are required for the hypoxic ventilatory response and the mechanism of ventilatory dysfunctions arising from AMPK deficiency. Methods: We used plethysmography, electrophysiology, functional magnetic resonance imaging, and immediate early gene (c-fos) expression to assess the hypoxic ventilatory response of mice with conditional deletion of the AMPK-α1 and/or AMPK-α2 genes in catecholaminergic cells, which compose the hypoxia-responsive respiratory network from carotid body to brainstem. Measurements and Main Results: AMPK-α1 and AMPK-α2 deletion virtually abolished the hypoxic ventilatory response, and ventilatory depression during hypoxia was exacerbated under anesthesia. Rather than hyperventilating, mice lacking AMPK-α1 and AMPK-α2 exhibited hypoventilation and apnea during hypoxia, with the primary precipitant being loss of AMPK-α1 expression. However, the carotid bodies of AMPK-knockout mice remained exquisitely sensitive to hypoxia, contrary to the view that the hypoxic ventilatory response is determined solely by increased carotid body afferent input to the brainstem. Regardless, functional magnetic resonance imaging and c-fos expression revealed reduced activation by hypoxia of well-defined dorsal and ventral brainstem nuclei. Conclusions: AMPK is required to coordinate the activation by hypoxia of brainstem respiratory networks, and deficiencies in AMPK expression precipitate hypoventilation and apnea, even when carotid body afferent input is normal