Acid-sensing ion channel 2 (ASIC2) modulates ASIC1 H+-activated currents in hippocampal neurons

Hippocampal neurons express subunits of the acid-sensing ion channel (ASIC1 and ASIC2) and exhibit large cation currents that are transiently activated by acidic extracellular solutions. Earlier work indicated that ASIC1 contributed to the current in these neurons and suggested its importance for normal behavior. However, the specific contribution of ASIC1 and ASIC2 subunits to acid-evoked currents in hippocampal neurons remained uncertain. To decipher the individual role of the ASIC subunits, we studied H(+)-gated currents in neurons from both ASIC1 and ASIC2 null mice. We found that much of the current was produced by ASIC1a/2a heteromultimeric channels, and individual subunits made distinct contributions. The ASIC1a subunit was key in establishing current amplitude. The ASIC2a subunit had little effect on amplitude but influenced desensitization, recovery from desensitization, pH sensitivity, and the response to modulatory agents. We also found heterogeneity in the contribution of ASIC2 throughout the neuronal population, with individual neurons expressing both ASIC1a homomultimeric and ASIC1a/2a heteromultimeric channels. Studies of neurons heterozygous for disrupted ASIC alleles indicated that the properties of H(+)-gated currents are dependent on the proportion of the individual subunits. These findings indicate that the absolute and relative amounts of ASIC subunits determine the amplitude and properties of hippocampal H(+)-gated currents and therefore may contribute to normal physiology and pathophysiology.     

PSD-95 and Lin-7b interact with acid-sensing ion channel-3 and have opposite effects on H+- gated current

The acid-sensing ion channel-3 (ASIC3) is a degenerin/epithelial sodium channel expressed in the peripheral nervous system. Previous studies indicate that it participates in the response to mechanical and painful stimuli, perhaps contributing to mechanoreceptor and/or H+ -gated nociceptor function. ASIC3 subunits contain intracellular N and C termini that may control channel localization and function. We found that a PDZ-binding motif at the ASIC3 C terminus interacts with four different proteins that contain PDZ domains: PSD-95, Lin-7b, MAGI-1b, and PIST. ASIC3 and these interacting proteins were expressed in dorsal root ganglia and spinal cord, and PSD-95 co-precipitated ASIC3 from spinal cord. When expressed in heterologous cells, PSD-95 reduced the amplitude of ASIC3 acid-evoked currents, whereas Lin-7b increased current amplitude. PSD-95 and Lin-7b altered current density by decreasing or increasing, respectively, the amount of ASIC3 on the cell surface. The finding that multiple PDZ-containing proteins bind ASIC3 and can influence its presence in the plasma membrane suggests that they may play an important role in the contribution of ASIC3 to nociception and mechanosensation.     

CLIC-5A functions as a chloride channel in vitro and associates with the cortical actin cytoskeleton in vitro and in vivo

CLIC-5A is a member of the chloride intracellular channel protein family, which is comprised of six related human genes encoding putative chloride channels. In this study, we found that reconstitution of purified recombinant CLIC-5A into artificial liposomes resulted in a dose-dependent chloride efflux that was sensitive to the chloride channel blocker IAA-94. CLIC-5A was originally isolated as a component of an ezrin-containing cytoskeletal complex from human placental microvilli. Here we show that similar protein complexes can be isolated using either immobilized CLIC-5A or the C-terminal F-actin-binding domain of ezrin and that actin polymerization is required for de novo assembly of these complexes. To investigate the behavior of CLIC-5A in vivo, JEG-3 placental choriocarcinoma cells were stably transfected with epitope-tagged CLIC-5A. In fixed cells, CLIC-5A displayed a polarized distribution and colocalized with ezrin in apical microvilli. Microvillar localization of CLIC-5A was retained after Triton X-100 extraction and was disrupted by treatment with latrunculin B. In transient transfections assays, we mapped a region between residues 20 and 54 of CLIC-5A that is required for targeting of CLIC-5A to microvilli in JEG-3 cells. Interestingly, expression of CLIC-5A in JEG-3 cells did not enhance the rate of iodide efflux in intact cells, suggesting that if CLIC-5A is a chloride channel, its channel activity may be restricted to intracellular membrane compartments in these cells. Regardless of its role in ion transport, CLIC-5A, like ezrin, may play an important role in the assembly or maintenance of F-actin-based structures at the cell cortex.     

The 1.3-Angstrom-resolution crystal structure of beta-ketoacyl-acyl carrier protein synthase II from Streptococcus pneumoniae

The beta-ketoacyl-acyl carrier protein synthases are members of the thiolase superfamily and are key regulators of bacterial fatty acid synthesis. As essential components of the bacterial lipid metabolic pathway, they are an attractive target for antibacterial drug discovery. We have determined the 1.3 A resolution crystal structure of the beta-ketoacyl-acyl carrier protein synthase II (FabF) from the pathogenic organism Streptococcus pneumoniae. The protein adopts a duplicated betaalphabetaalphabetaalphabetabeta fold, which is characteristic of the thiolase superfamily. The two-fold pseudosymmetry is broken by the presence of distinct insertions in the two halves of the protein. These insertions have evolved to bind the specific substrates of this particular member of the thiolase superfamily. Docking of the pantetheine moiety of the substrate identifies the loop regions involved in substrate binding and indicates roles for specific, conserved residues in the substrate binding tunnel. The active site triad of this superfamily is present in spFabF as His 303, His 337, and Cys 164. Near the active site is an ion pair, Glu 346 and Lys 332, that is conserved in the condensing enzymes but is unusual in our structure in being stabilized by an Mg(2+) ion which interacts with Glu 346. The active site histidines interact asymmetrically with Lys 332, whose positive charge is closer to His 303, and we propose a specific role for the lysine in polarizing the imidazole ring of this histidine. This asymmetry suggests that the two histidines have unequal roles in catalysis and provides new insights into the catalytic mechanisms of these enzymes.     

Field-induced reorganization of the neural membrane lipid bilayer: a proposed role in the regulation of ion-channel dynamics

We present a computational model demonstrating that an electric field propagating in the plane of the neural membrane during transmembrane ion movement creates lateral concentration gradients of the lipids. Due to this field-induced reorganization, ethenes of the lipid chains become aligned and polarized. This finding is interpreted within the context of molecular studies of protein folding in biological membranes. We propose that electrostatic interactions between membrane dipoles and charged amino acid residues of the unfolded ion-channel protein regulate protein-folding kinetics (channel closing). These electrostatic interactions thus regulate electrical signaling in neurons.     

Influence of coronary artery diameter on eNOS protein content

The purpose of this study was to test the hypothesis that the content of endothelial nitric oxide synthase (eNOS) protein (eNOS protein/g total artery protein) increases with decreasing artery diameter in the coronary arterial tree. Content of eNOS protein was determined in porcine coronary arteries with immunoblot analysis. Arteries were isolated in six size categories from each heart: large arteries [301- to 2,500-microm internal diameter (ID)], small arteries (201- to 300-microm ID), resistance arteries (151- to 200-microm ID), large arterioles (101- to 150-microm ID), intermediate arterioles (51- to 100-microm ID), and small arterioles(<50-microm ID). To obtain sufficient protein for analysis from small- and intermediate-sized arterioles, five to seven arterioles 1-2 mm in length were pooled into one sample for each animal. Results establish that the number of smooth muscle cells per endothelial cell decreases from a number of 10 to 15 in large coronary arteries to 1 in the smallest arterioles. Immunohistochemistry revealed that eNOS is located only in endothelial cells in all sizes of coronary artery and in coronary capillaries. Contrary to our hypothesis, eNOS protein content did not increase with decreasing size of coronary artery. Indeed, the smallest coronary arterioles had less eNOS protein per gram of total protein than the large coronary arteries. These results indicate that eNOS protein content is greater in the endothelial cells of conduit arteries, resistance arteries, and large arterioles than in small coronary arterioles.     

Ventricular-specific expression of angiotensin II type 2 receptors causes dilated cardiomyopathy and heart failure in transgenic mice

The angiotensin II type 2 (AT2) receptor is upregulated in the left ventricle in heart failure, but its pathophysiological roles in vivo are not understood. In the present study, AT2 receptors were expressed in transgenic (TG) mice using the ventricular-specific myosin light-chain (MLC-2v) promoter. In TG compared with nontransgenic (NTG) mice, in vivo left ventricular (LV) systolic pressure and peak +dP/dt were depressed while LV diastolic pressure was elevated (P < 0.05). Echocardiography showed severely depressed LV fractional shortening, increased systolic and diastolic dimensions, and wall thinning (P < 0.05). Confocal and electron microscopy studies revealed an increase in the size of myocytes and interstitial spaces as well as an increase in interstitial collagen, disruption of the Z-band, and changes in cytochrome c localization. The changes were most prominent in the highest-expressing TG line, which implies a dose-response relationship. AT2 overexpression was also directly associated with the increase of phosphorylated protein levels of PKC-alpha, PKC-beta, and p70S6 kinase. These data demonstrate that ventricular myocyte-specific expression of AT2 receptors promotes the development of dilated cardiomyopathy and heart failure in vivo.     

Integrin shedding as a mechanism of cellular adaptation during cardiac growth

Integrin-mediated cell-extracellular matrix (ECM) interactions are essential for multiple cellular processes; however, little is known regarding integrin turnover during these events. Recent studies have demonstrated shedding of cell surface molecules and suggested this as a potential mechanism for integrin turnover. Confocal microscopy of mouse hearts under different physiological conditions demonstrated the presence of beta(1)-integrin-immunoreactive material in the interstitium. Culture media from neonatal rat cardiac myocytes and fibroblasts contained a 55-kDa fragment of beta(1)-integrin. Attachment to ECM components, response to phorbol 12-myristate 13-acetate stimulation, and matrix metalloproteinase inhibition assays demonstrated that fibroblasts responded differently to the fragment compared with myocytes. The beta(1)-integrin fragment stimulated myocyte attachment to collagen and the fragment itself bound a variety of ECM proteins. These studies indicate that as myocytes and fibroblasts change size and shape, cellular contacts with the ECM are altered, resulting in the liberation of a beta(1)-integrin fragment from the cell surface. Integrin shedding may represent a novel mechanism of rapidly modifying cell-ECM contacts during various cellular processes.     

Distribution and oviposition preference of galling sawflies in arctic Canada

The distribution and ecology of insects in arctic regions are poorly known. The aim of this study was to determine the distribution of galling sawflies in the Canadian arctic and their oviposition preference. The Swedish Tundra Northwest 1999 expedition visited 17 sites in the Canadian arctic. We determined the occurrence of galling sawflies at all the sites and studied the oviposition preference of two leaf-galling sawflies, Eupontania arctica and Pontania nivalis, on Salix reticulata and S. glauca, respectively. Galling sawflies were abundant at only one site, the mainland site at Ivvavik National Park. Only a few galls in total were found at the remaining sites, suggesting that galling sawflies are rare in the higher arctic, and potential explanations for this pattern are discussed. Shoots with leaf galls were longer than shoots without galls on both S. reticulata and S. glauca. These differences could not be explained by a higher number of leaves on longer shoots. This suggests that long shoots are preferred by sawflies because of faster development and better survival of larvae on long shoots.