Adenosine triphosphate-sensitive potassium channel Kir subunits implicated in cardioprotection by diazoxide

BACKGROUND: ATP-sensitive potassium (K(ATP)) channel openers provide cardioprotection in multiple models. Ion flux at an unidentified mitochondrial K(ATP) channel has been proposed as the mechanism. The renal outer medullary kidney potassium channel subunit, potassium inward rectifying (Kir)1.1, has been implicated as a mitochondrial channel pore-forming subunit. We hypothesized that subunit Kir1.1 is involved in cardioprotection (maintenance of volume homeostasis and contractility) of the K(ATP) channel opener diazoxide (DZX) during stress (exposure to hyperkalemic cardioplegia [CPG]) at the myocyte and mitochondrial levels. METHODS AND RESULTS: Kir subunit inhibitor Tertiapin Q (TPN-Q) was utilized to evaluate response to stress. Mouse ventricular mitochondrial volume was measured in the following groups: isolation buffer; 200 μmol/L of ATP; 100 μmol/L of DZX+200 μmol/L of ATP; or 100 μmol/L of DZX+200 μmol/L of ATP+TPN-Q (500 or 100 nmol/L). Myocytes were exposed to Tyrode’s solution (5 minutes), test solution (Tyrode’s, cardioplegia [CPG], CPG+DZX, CPG+DZX+TPN-Q, Tyrode’s+TPN-Q, or CPG+TPN-Q), N=12 for all (10 minutes); followed by Tyrode’s (5 minutes). Volumes were compared. TPN-Q, with or without DZX, did not alter mitochondrial or myocyte volume. Stress (CPG) resulted in myocyte swelling and reduced contractility that was prevented by DZX. TPN-Q prevented the cardioprotection afforded by DZX (volume homeostasis and maintenance of contractility). CONCLUSIONS: TPN-Q inhibited myocyte cardioprotection provided by DZX during stress; however, it did not alter mitochondrial volume. Because TPN-Q inhibits Kir1.1, Kir3.1, and Kir3.4, these data support that any of these Kir subunits could be involved in the cardioprotection afforded by diazoxide. However, these data suggest that mitochondrial swelling by diazoxide does not involve Kir1.1, 3.1, or 3.4

Oncogenic Gq/11 signaling acutely drives and chronically sustains metabolic reprogramming in uveal melanoma

Metabolic reprogramming has been shown to occur in uveal melanoma (UM), the most common intraocular tumor in adults. Mechanisms driving metabolic reprogramming in UM are poorly understood. Elucidation of these mechanisms could inform development of new therapeutic strategies for metastatic UM, which has poor prognosis because existing therapies are ineffective. Here, we determined whether metabolic reprogramming is driven by constitutively active mutant α-subunits of the heterotrimeric G proteins Gq or G11 (Gq/11), the oncogenic drivers in ∼90% of UM patients. Using PET-computed tomography imaging, microphysiometry, and GC/MS, we found that inhibition of oncogenic Gq/11 with the small molecule FR900359 (FR) attenuated glucose uptake by UM cells in vivo and in vitro, blunted glycolysis and mitochondrial respiration in UM cell lines and tumor cells isolated from patients, and reduced levels of several glycolytic and tricarboxylic acid cycle intermediates. FR acutely inhibited glycolysis and respiration and chronically attenuated expression of genes in both metabolic processes. UM therefore differs from other melanomas that exhibit a classic Warburg effect. Metabolic reprogramming in UM cell lines and patient samples involved protein kinase C and extracellular signal-regulated protein kinase 1/2 signaling downstream of oncogenic Gq/11. Chronic administration of FR upregulated expression of genes involved in metabolite scavenging and redox homeostasis, potentially as an adaptive mechanism explaining why FR does not efficiently kill UM tumor cells or regress UM tumor xenografts. These results establish that oncogenic Gq/11 signaling is a crucial driver of metabolic reprogramming in UM and lay a foundation for studies aimed at targeting metabolic reprogramming for therapeutic development

Ligand-Induced Movements of Inner Transmembrane Helices of Glut1 Revealed by Chemical Cross-Linking of Di-Cysteine Mutants

The relative orientation and proximity of the pseudo-symmetrical inner transmembrane helical pairs 5/8 and 2/11 of Glut1 were analyzed by chemical cross-linking of di-cysteine mutants. Thirteen functional di-cysteine mutants were created from a C-less Glut1 reporter construct containing cysteine substitutions in helices 5 and 8 or helices 2 and 11. The mutants were expressed in Xenopus oocytes and the sensitivity of each mutant to intramolecular cross-linking by two homobifunctional thiol-specific reagents was ascertained by protease cleavage followed by immunoblot analysis. Five of 9 mutants with cysteine residues predicted to lie in close proximity to each other were susceptible to cross-linking by one or both reagents. None of 4 mutants with cysteine substitutions predicted to lie on opposite faces of their respective helices was susceptible to cross-linking. Additionally, the cross-linking of a di-cysteine pair (A70C/M420C, helices 2/11) predicted to lie near the exoplasmic face of the membrane was stimulated by ethylidene glucose, a non-transported glucose analog that preferentially binds to the exofacial substrate-binding site, suggesting that the binding of this ligand stimulates the closure of helices at the exoplasmic face of the membrane. In contrast, the cross-linking of a second di-cysteine pair (T158C/L325, helices 5/8), predicted to lie near the cytoplasmic face of the membrane, was stimulated by cytochalasin B, a glucose transport inhibitor that competitively inhibits substrate efflux, suggesting that this compound recruits the transporter to a conformational state in which closure of inner helices occurs at the cytoplasmic face of the membrane. This observation provides a structural explanation for the competitive inhibition of substrate efflux by cytochalasin B. These data indicate that the binding of competitive inhibitors of glucose efflux or influx induce occluded states in the transporter in which substrate is excluded from the exofacial or endofacial binding site

Expression of dicysteine mutants in Xenopus oocyte membranes.

<p>Stage 5 <i>Xenopus</i> oocytes were injected with water (Sham) or with 50 ng of mRNA encoding the parental reporter construct (C-Tev) or the indicated dicysteine mutant. Two days post injection total oocyte membranes were prepared and subjected to immunoblot analysis using a rabbit polyclonal ab raised against a peptide corresponding to the C-terminal 15 residues of human Glut1.</p

Cross-sectional model of the inner transmembrane helices of Glut1 in the exoplasmic conformation as viewed from the exoplasmic face of the membrane based on experimental results.

<p>Amino acid residues subjected to cysteine substitution in the dicysteine mutants are given by the single letter code. Red lines connect residues that were cross-linked by o-PDM or BMH within the helical pairs 2/11 and 5/8.</p

Construction of di-cysteine mutants in a C-Less GLUT 1 reporter molecule.

<p>An aglyco Glut1 reporter molecule was created by mutating the consensus site of N-linked glycosylation at N45 to a threonine residue and by inserting a Tev protease cleavage site (ENLYFQG) between residues 247 and 248 in the central cytoplasmic loop of Glut1. This reporter construct was then used to make the above 13 dicysteine mutants in helices 2/11 and helices 5/8.</p

Effect of non-transported ligands on chemical cross-linking.

<p>Oocyte membranes were incubated in presence of either vehicle alone (water or ethanol) or 50 µM cytochalasin B or 50 mM ethylidene glucose for 5 min prior to the addition of the indicated concentration of either o-PDM or BMH. Cross-linking efficiency was measured by protease cleavage followed by immunoblot analysis as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031412#s4" target="_blank">Materials and Methods</a>”. The water lanes represent the controls for the addition of ethylidene glucose and the ethanol lanes controlled for the addition of cytochalasin B. The DMSO lanes represent samples to which DMSO was added but no cross-linker. These lanes indicate the maximum cleavage observed for each mutant with TEV protease.</p

Effect of Ligands on the Cross-Linking of Glut1 Di-cysteine Mutants.

<p>Oocyte membranes expressing dicysteine mutants were subjected to chemical cross-linking as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031412#s4" target="_blank">Materials and Methods</a> in the presence or absence of cytochalasin B (CB) or ethylidene glucose (EG). Cross-linking efficiency is expressed as the intensity of the full-length transporter bands in the cross-linked lanes (o-PDM and BMH lanes, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031412#pone-0031412-g004" target="_blank">Figure 4</a>) divided by the intensity of the full-length transporter bands in the control lanes, after subtraction of background intensities. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031412#pone-0031412-g004" target="_blank">Figure 4</a> for a representative set of immunoblots. The results of 4–5 independent experiments are presented. Ethanol (ETOH) is the control for the addition of cytochalasin B (CB), and Water is the control for the addition of ethylidene glucose (EG).</p

Chemical Cross-linking of di-C Mutants.

<p>Stage 5 <i>Xenopus</i> oocytes were injected with 50 ng of mRNA encoding the parental reporter construct (C-Tev) or the indicated dicysteine mutants (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031412#pone-0031412-t001" target="_blank">Table 1</a>). After incubation of oocytes for 2 days, cross-linking analysis was conducted on purified oocyte membranes as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031412#s4" target="_blank">Materials and Methods</a>”. The reactions were quenched by the addition of 2 mM cysteine and oocyte membranes were digested with Tev protease then subjected to SDS-PAGE followed by immunoblotting with rabbit polylclonal ab raised against the C-terminal 15 residues of human Glut1 (red bands) and a mouse monoclonal ab that recognizes an epitope in the N-terminal half of the central cytoplasmic loop (green bands). Note that the full-length ∼54 kD bands were recognized by both antibodies and show up as yellow when the intensity of the detector was increased. “C-TEV” is the control cysteine-less parental construct. “Control” lanes were loaded with membranes that were not subjected to either chemical cross-linking or protease cleavage. “DMSO” lanes were loaded with membranes that were not subjected to chemical cross-linking but were digested with TEV protease. The “o-PDM” and “BMH” lanes were loaded with membranes that were subjected to cross-linking by the respective chemical and then were treated with TEV protease. The ratio of the intensities of the full-length bands in the DMSO lanes to those in the Control lanes thus provide the maximum level of protease cleavage for each mutant. The ratio of the intensities of the full-length bands in the “o-PDM” or ‘BMH” lanes to those in the Control lanes indicate the extent of cross-linking by either reagent. This ratio is termed the cross-linking efficiency or “fraction cross-linked” in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031412#pone-0031412-t002" target="_blank">Table 2</a>.</p