Oluseye A. Ogunbayo

Use of Cells Expressing γ Subunit Variants to Identify Diverse Mechanisms of AMPK Activation

Summary A wide variety of agents activate AMPK, but in many cases the mechanisms remain unclear. We generated isogenic cell lines stably expressing AMPK complexes containing AMP-sensitive (wild-type, WT) or AMP-insensitive (R531G) γ2 variants. Mitochondrial poisons such as oligomycin and dinitrophenol only activated AMPK in WT cells, as did AICAR, 2-deoxyglucose, hydrogen peroxide, metformin, phenformin, galegine, troglitazone, phenobarbital, resveratrol, and berberine. Excluding AICAR, all of these also inhibited cellular energy metabolism, shown by increases in ADP:ATP ratio and/or by decreases in cellular oxygen uptake measured using an extracellular flux analyzer. By contrast, A769662, the Ca2+ ionophore, A23187, osmotic stress, and quercetin activated both variants to varying extents. A23187 and osmotic stress also increased cytoplasmic Ca2+, and their effects were inhibited by STO609, a CaMKK inhibitor. Our approaches distinguish at least six different mechanisms for AMPK activation and confirm that the widely used antidiabetic drug metformin activates AMPK by inhibiting mitochondrial respiration.

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 patients 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 Childrens Hospital Foundation and others.).

Cyclic Adenosine Diphosphate Ribose Activates Ryanodine Receptors, whereas NAADP Activates Two-pore Domain Channels

The mechanism by which cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mobilize intracellular Ca2+ stores remains controversial. It is open to question whether cADPR regulates ryanodine receptors (RyRs) directly, as originally proposed, or indirectly by promoting Ca2+ uptake into the sarco/endoplasmic reticulum by sarco/endoplasmic reticulum Ca2+-ATPases. Conversely, although we have proposed that NAADP mobilizes endolysosomal Ca2+ stores by activating two-pore domain channels (TPCs), others suggest that NAADP directly activates RyRs. We therefore assessed Ca2+ signals evoked by intracellular dialysis from a patch pipette of cADPR and NAADP into HEK293 cells that stably overexpress either TPC1, TPC2, RyR1, or RyR3. No change in intracellular Ca2+ concentration was triggered by cADPR in either wild-type HEK293 cells (which are devoid of RyRs) or in cells that stably overexpress TPC1 and TPC2, respectively. By contrast, a marked Ca2+ transient was triggered by cADPR in HEK293 cells that stably expressed RyR1 and RyR3. The Ca2+ transient was abolished following depletion of endoplasmic reticulum stores by thapsigargin and block of RyRs by dantrolene but not following depletion of acidic Ca2+ stores by bafilomycin. By contrast, NAADP failed to evoke a Ca2+ transient in HEK293 cells that expressed RyR1 or RyR3, but it induced robust Ca2+ transients in cells that stably overexpressed TPC1 or TPC2 and in a manner that was blocked following depletion of acidic stores by bafilomycin. We conclude that cADPR triggers Ca2+ release by activating RyRs but not TPCs, whereas NAADP activates TPCs but not RyRs.

Selective expression in carotid body type I cells of a single splice variant of the large conductance calcium- and voltage-activated potassium channel confers regulation by AMP-activated protein kinase

Inhibition of large conductance calcium-activated potassium (BKCa) channels mediates, in part, oxygen sensing by carotid body type I cells. However, BKCa channels remain active in cells that do not serve to monitor oxygen supply. Using a novel, bacterially derived AMP-activated protein kinase (AMPK), we show that AMPK phosphorylates and inhibits BKCa channels in a splice variant-specific manner. Inclusion of the stress-regulated exon within BKCa channel α subunits increased the stoichiometry of phosphorylation by AMPK when compared with channels lacking this exon. Surprisingly, however, the increased phosphorylation conferred by the stress-regulated exon abolished BKCa channel inhibition by AMPK. Point mutation of a single serine (Ser-657) within this exon reduced channel phosphorylation and restored channel inhibition by AMPK. Significantly, RT-PCR showed that rat carotid body type I cells express only the variant of BKCa that lacks the stress-regulated exon, and intracellular dialysis of bacterially expressed AMPK markedly attenuated BKCa currents in these cells. Conditional regulation of BKCa channel splice variants by AMPK may therefore determine the response of carotid body type I cells to hypoxia.

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.

From contraction to gene expression: nanojunctions of the sarco/endoplasmic reticulum deliver site- and function-specific calcium signals

Calcium signals determine, for example, smooth muscle contraction and changes in gene expression. How calcium signals select for these processes is enigmatic. We build on the “panjunctional sarcoplasmic reticulum” hypothesis, describing our view that different calcium pumps and release channels, with different kinetics and affinities for calcium, are strategically positioned within nanojunctions of the SR and help demarcate their respective cytoplasmic nanodomains. SERCA2b and RyR1 are preferentially targeted to the sarcoplasmic reticulum (SR) proximal to the plasma membrane (PM), i.e., to the superficial buffer barrier formed by PM-SR nanojunctions, and support vasodilation. In marked contrast, SERCA2a may be entirely restricted to the deep, perinuclear SR and may supply calcium to this sub-compartment in support of vasoconstriction. RyR3 is also preferentially targeted to the perinuclear SR, where its clusters associate with lysosome-SR nanojunctions. The distribution of RyR2 is more widespread and extends from this region to the wider cell. Therefore, perinuclear RyR3s most likely support the initiation of global calcium waves at L-SR junctions, which subsequently propagate by calcium-induced calcium release via RyR2 in order to elicit contraction. Data also suggest that unique SERCA and RyR are preferentially targeted to invaginations of the nuclear membrane. Site- and function-specific calcium signals may thus arise to modulate stimulus-response coupling and transcriptional cascades.

AMPK 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.

Organelle-specific subunit interactions of the vertebrate two-pore channel family.

Background: The role of two-pore channels (TPCs) in endolysosomal signaling remains controversial. Results: TPCs are targeted to different subpopulations of endolysosomes, and this determines subunit interaction and Ca2+ signaling by NAADP. Conclusion: All vertebrate TPC subtypes support Ca2+ signaling; lysosome-targeted, but not endosome-targeted, TPCs permit endoplasmic reticulum coupling. Significance: Organellar targeting and interaction of TPCs are likely critical to endolysosomal signaling in health and disease. The organellar targeting of two-pore channels (TPCs) and their capacity to associate as homo- and heterodimers may be critical to endolysosomal signaling. A more detailed understanding of the functional association of vertebrate TPC1–3 is therefore necessary. We report here that when stably expressed in HEK293 cells, human (h) TPC1 and chicken (c) TPC3 were specifically targeted to different subpopulations of endosomes, hTPC2 was specifically targeted to lysosomes, and rabbit (r) TPC3 was specifically targeted to both endosomes and lysosomes. Intracellular dialysis of NAADP evoked a Ca2+ transient in HEK293 cells that stably overexpressed hTPC1, hTPC2, and rTPC3, but not in cells that stably expressed cTPC3. The Ca2+ transients induced in cells that overexpressed endosome-targeted hTPC1 were abolished upon depletion of acidic Ca2+ stores by bafilomycin A1, but remained unaffected following depletion of endoplasmic reticulum stores by thapsigargin. In contrast, Ca2+ transients induced via lysosome-targeted hTPC2 and endolysosome-targeted rTPC3 were abolished by bafilomycin A1 and markedly attenuated by thapsigargin. NAADP induced marked Ca2+ transients in HEK293 cells that stably coexpressed hTPC2 with hTPC1 or cTPC3, but failed to evoke any such response in cells that coexpressed interacting hTPC2 and rTPC3 subunits. We therefore conclude that 1) all three TPC subtypes may support Ca2+ signaling from their designate acidic stores, and 2) lysosome-targeted (but not endosome-targeted) TPCs support coupling to the endoplasmic reticulum.

Modulation of the LKB1-AMPK Signalling Pathway Underpins Hypoxic Pulmonary Vasoconstriction and Pulmonary Hypertension

Perhaps the defining characteristic of pulmonary arteries is the process of hypoxic pulmonary vasoconstriction (HPV) which, under physiological conditions, supports ventilation-perfusion matching in the lung by diverting blood flow away from oxygen deprived areas of the lung to oxygen rich regions. However, when alveolar hypoxia is more widespread, either at altitude or with disease (e.g., cystic fibrosis), HPV may lead to hypoxic pulmonary hypertension. HPV is driven by the intrinsic response to hypoxia of pulmonary arterial smooth muscle and endothelial cells, which are acutely sensitive to relatively small changes in pO2 and have evolved to monitor oxygen supply and thus address ventilation-perfusion mismatch. There is now a consensus that the inhibition by hypoxia of mitochondrial oxidative phosphorylation represents a key step towards the induction of HPV, but the precise nature of the signalling pathway(s) engaged thereafter remains open to debate. We will consider the role of the AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1), an upstream kinase through which AMPK is intimately coupled to changes in oxygen supply via mitochondrial metabolism. A growing body of evidence, from our laboratory and others, suggests that modulation of the LKB1-AMPK signalling pathway underpins both hypoxic pulmonary vasoconstriction and the development of pulmonary hypertension.

Related flavonoids cause cooperative inhibition of the sarcoplasmic reticulum Ca2+ ATPase by multimode mechanisms

Flavonoids are group of plant‐derived hydroxylated polycyclic molecules found in fruit and vegetables. They are known to bio‐accumulate within humans and are considered to have beneficial health effects, including cancer chemoprotection. One mechanism proposed to explain this is that they are able to induce apoptosis in cancer cells by inhibiting a variety of kinases and also the Ca2+ ATPase. An investigation was undertaken with respect to the mechanism of inhibition for three flavonoids: quercetin, galangin and 3,6 dihydroxyflavone (3,6‐DHF). Each inhibited the Ca2+ ATPase with Ki values of 8.7, 10.3 and 5.4 μm, respectively, showing cooperative inhibition with n ~ 2. Given their similar structures, the flavonoids showed several differences in their mechanisms of inhibition. All three flavonoids stabilized the ATPase in the E1 conformation and reduced [32P]‐ATP binding. However, both galangin and 3,6‐DHF increased the affinity of Ca2+ for the ATPase by decreasing the Ca2+‐dissociation rate constant, whereas quercetin had little effect. Ca2+‐induced changes in tryptophan fluorescence levels were reduced in the presence of 3,6‐DHF and galangin (but not with quercetin), indicating that Ca2+‐associated changes within the transmembrane helices are altered. Both galangin and quercetin reduced the rates of ATP‐dependent phosphorylation and dephosphorylation, whereas 3,6‐DHF did not. Modelling studies suggest that flavonoids could potentially bind to two sites: one directly where nucleotides bind within ATP binding site and the other at a site close by. We hypothesize that interactions of these two neighbouring sites may account for both the cooperative inhibition and the multimode mechanisms of action seen with related flavonoids.