Cyclic Nucleotide-Gated Channels Contribute to Thromboxane A2-Induced Contraction of Rat Small Mesenteric Arteries

Background: Thromboxane A 2 (TxA 2)-induced smooth muscle contraction has been implicated in cardiovascular, renal and respiratory diseases. This contraction can be partly attributed to TxA2-induced Ca 2+ influx, which resulted in vascular contraction via Ca 2+-calmodulin-MLCK pathway. This study aims to identify the channels that mediate TxA2-induced Ca 2+ influx in vascular smooth muscle cells. Methodology/Principal Findings: Application of U-46619, a thromboxane A2 mimic, resulted in a constriction in endothelium-denuded small mesenteric artery segments. The constriction relies on the presence of extracellular Ca 2+, because removal of extracellular Ca 2+ abolished the constriction. This constriction was partially inhibited by an L-type Ca 2+ channel inhibitor nifedipine (0.5–1 mM). The remaining component was inhibited by L-cis-diltiazem, a selective inhibitor for CNG channels, in a dose-dependent manner. Another CNG channel blocker LY83583 [6-(phenylamino)-5,8-quinolinedione] had similar effect. In the primary cultured smooth muscle cells derived from rat aorta, application of U46619 (100 nM) induced a rise in cytosolic Ca 2+ ([Ca 2+]i), which was inhibited by L-cis-diltiazem. Immunoblot experiments confirmed the presence of CNGA2 protein in vascular smooth muscle cells. Conclusions/Significance: These data suggest a functional role of CNG channels in U-46619-induced Ca 2+ influx and contraction of smooth muscle cells

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Klonierung und Expression von cGMP-gesteuerten Kanälen aus Sehstäbchen und Zapfen der Hühnernetzhaut

Die Außensegmente von Zapfensehzellen und Sehstäbchen enthalten einen Kationenkanal, der direkt durch cGMP gesteuert wird. Mit einer eDNA-Sonde aus dem Rindersehstäbchenkanal wurden aus einer Hühnerretina-cDNA-Bibliothek Klone isoliert, die für CNG-Kanäle aus Sehstäbchen und Zapfen kodieren. Die aus den Nukleinsäuresequenzen abgeleiteten Primarstrukturen der beiden Kanäle weisen sowohl untereinander, als auch zu anderen CNG-Kanälen eine hohe Ähnlichkeit auf. Diese ist besonders in der Zentralregion der Proteine ausgeprägt, die die hydrophoben, putativ transmembranalen Bereiche und die C-terminal gelegene cGMP-Bindestelle umfaßt. Im N-terminalen Bereich beider Kanäle befindet sich ein hydrophiles Segment, das charakteristisch für die CNG-Kanäle aus Photorezeptorzellen ist. Die Kanäle weisen keine Sequenzähnlichkeiten zu ligandenaktivierten Ionenkanälen auf. Sie besitzen jedoch Strukturelemente, die in spannungsabhängigen Kaliumkanälen vorkommen. Hinweise auf den Ursprung der wässrigen Pore von CNG-Kanalen und den Mechanismus der Öffnung der Kanäle können aus denSequenzähnlichkeiten zu Proteinkinasen abgeleitet werden. Durch immunhistochemische Untersuchungen konnte ein Kanal den Zapfensehzellen zugeordnet werden. Weiterhin wurde gezeigt, daß der Zapfenkanal in der Hühnerretina ausschließlich in den äußeren Segmenten von Zapfen lokalisiert ist und in allen Zapfentypen vorkommt. Der zweite Kanal konnte durch Sequenzvergleiche und Western Blot-Analysen als Sehstäbchenkanal identifiziert werden. Die reifen Untereinheiten beider Kanäle aus der Hühnerretina sind durch eine co- oder posttranslationale, proteolytische Modifikation im N-terminalen Bereich verkürzt. Verschiedene Spaltstellen beim Sehstäbchen- und Zapfenkanal weisen auf unterschiedliche Mechanismen dieser Prozessierung hin. Die elektrophysiologischen Eigenschaften der Kanäle wurden, nach Veränderung der nichtkodierenden Bereiche der ursprünglichen Klone, durch heterologe Expression in Xenopus Oozyten bestimmt. Die Kanäle werden kooperativ durch cGMP geöffnet, wobei der Sehstäbchenkanal eine etwa zweifach höhere Ligandenempfindlichkeit als der Zapfenkanal aufweist. Durch cAMP werdenbeide Kanäle nur sehr schlecht aktiviert. Die makroskopischen Ströme, die ein nichtlineares, spannungsabhangiges Verhalten zeigen und die relativen Ionenpermeabilitäten des exprimierten Sehstäbchen- und Zapfenkanals aus dem Huhn sind ähnlich

Assembly of retinal rod or cone Na+/Ca2+/K+-exchanger oligomers with cGMP-gated channel subunits as probed with heterologously expressed cDNAs

Proper control of intracellular free Ca(2+) is thought to involve subsets of proteins that co-localize to mediate coordinated Ca(2+) entry and Ca(2+) extrusion. The outer segments of vertebrate rod and cone photoreceptors present one example: Ca(2+) influx is exclusively mediated via cGMP-gated channels (CNG), whereas the Na(+)/Ca(2+)-K(+) exchanger (NCKX) is the only Ca(2+) extrusion protein present. In situ, a rod NCKX homodimer and a CNG heterotetramer are thought to be part of a single protein complex. However, NCKX-NCKX and NCKX-CNG interactions have been described so far only in bovine rod outer segment membranes. We have used thiol-specific cross-linking and co-immunoprecipitation to examine NCKX self-assembly and CNG-NCKX co-assembly after heterologous expression of either the rod or cone NCKX/CNG isoforms. Co-immunoprecipitation clearly demonstrated both NCKX homooligomerization and interactions between NCKX and CNG. The NCKX-NCKX and NCKX-CNG interactions were observed for both the rod and the cone isoforms. Thiol-specific cross-linking led to rod NCKX1 dimers and to cone NCKX2 adducts of an apparent molecular weight higher than that predicted for a NCKX2 dimer. The mass of the cross-link product critically depended on the location of the particular cysteine residue used by the cross-linker, and we cannot exclude that NCKX forms a higher oligomer. The NCKX-NCKX and NCKX-CNG interactions were not isoform-specific (i.e., rod NCKX could interact with cone NCKX, rod NCKX could interact with cone CNGA, and vice versa). Deletion of the two large hydrophilic loops from the NCKX protein did not abolish the NCKX oligomerization, suggesting that it is mediated by the highly conserved transmembrane spanning segments

Exploring fluorescence antibunching in solution to determine the stoichiometry of molecular complexes

Fluorescence antibunching is a well-known technique for determining the number of independent emitters per molecule or molecular complex. It was rarely applied to autofluorescent proteins due to the necessity of collecting large numbers of fluorescence photons from a single molecule, which is usually impossible to achieve with rather photolabile autofluorescent proteins. Here, we measure fluorescence antibunching on molecules in solution, allowing us to accumulate data over a large number of molecules. We use that method for determining an average stoichiometry of molecular complexes. The proposed method is absolute in the sense that it does not need any calibration or referencing. We develop the necessary theoretical background and check the method on pure dye solutions and on molecular complexes with known stoichiometry

The solute carrier SLC9C1 is a Na+/H+-exchanger gated by an S4-type voltage-sensor and cyclic-nucleotide binding

Voltage-sensing (VSD) and cyclic nucleotide-binding domains (CNBD) gate ion channels for rapid electrical signaling. By contrast, solute carriers (SLCs) that passively redistribute substrates are gated by their substrates themselves. Here, we study the orphan sperm-specific solute carriers SLC9C1 that feature a unique tripartite structure: an exchanger domain, a VSD, and a CNBD. Voltage-clamp fluorimetry shows that SLC9C1 is a genuine Na+/H+ exchanger gated by voltage. The cellular messenger cAMP shifts the voltage range of activation. Mutations in the transport domain, the VSD, or the CNBD strongly affect Na+/H+ exchange, voltage gating, or cAMP sensitivity, respectively. Our results establish SLC9C1 as a phylogenetic chimaera that combines the ion-exchange mechanism of solute carriers with the gating mechanism of ion channels. Classic SLCs slowly readjust changes in the intra-and extracellular milieu, whereas voltage gating endows the Na+/H+ exchanger with the ability to produce a rapid pH response that enables downstream signaling events