Modulation of potassium channel function by methionine oxidation and reduction

Oxidation of amino acid residues in proteins can be caused by a variety of oxidizing agents normally produced by cells. The oxidation of methionine in proteins to methionine sulfoxide is implicated in aging as well as in pathological conditions, and it is a reversible reaction mediated by a ubiquitous enzyme, peptide methionine sulfoxide reductase. The reversibility of methionine oxidation suggests that it could act as a cellular regulatory mechanism although no such in vivo activity has been demonstrated. We show here that oxidation of a methionine residue in a voltage-dependent potassium channel modulates its inactivation. When this methionine residue is oxidized to methionine sulfoxide, the inactivation is disrupted, and it is reversed by coexpression with peptide methionine sulfoxide reductase. The results suggest that oxidation and reduction of methionine could play a dynamic role in the cellular signal transduction process in a variety of systems.     

Structure and dynamics of hydronium in the ion channel gramicidin A

The effects of the hydronium ion, H(3)0+, on the structure of the ion channel gramicidin A and the hydrogen-bonded network of waters within the channel were studied to help elucidate a possible mechanism for proton transport through the channel. Several classical molecular dynamics studies were carried out with the hydronium in either the center of a gramicidin monomer or in the dimer junction. Structural reorganization of the channel backbone was observed for different hydronium positions, which were most apparent when the hydronium was within the monomer. In both cases the average O-O distance between the hydronium ion and its nearest neighbor water molecule was found to be approximately 2.55 A, indicating a rather strong hydrogen bond. Importantly, a subsequent break in the hydrogen-bonded network between the nearest neighbor and the next-nearest neighbor(approximately 2.7 -3.0 A) was repeatedly observed. Moreover, the carbonyl groups of gramicidin A were found to interact with the charge on the hydronium ion, helping in its stabilization. These facts may have significant implications for the proton hopping mechanism. The presence of the hydronium ion in the channel also inhibits to some degree the reorientational motions of the channel water molecules.     

Adoption of beta structure by the inactivating "ball" peptide of the Shaker B potassium channel

The conformation of the inactivating peptide of the Shaker B K+ channel (ShB peptide) and that of a noninactivating mutant (ShBL7E peptide) have been studied. Under all experimental conditions explored, the mutant peptide remains in a predominantly nonordered conformation. On the contrary, the inactivating ShB peptide has a great tendency to adopt a highly stable beta structure, particularly when challenged "in vitro" by anionic phospholipid vesicles. Because the putative peptide binding elements at the inner mouth of the channel comprise a ring of anionic residues and a hydrophobic pocket, we hypothesize that the conformational restrictions imposed on the ShB peptide by its interaction with the anionic lipid vesicles could partly imitate those imposed by the above ion channel elements. Thus, we propose that adoption of beta structure by the inactivating peptide may also occur during channel inactivation. Moreover, the difficulties encountered by the noninactivating ShBL7E peptide mutant to adopt beta structure and the observation that trypsin hydrolysis of the ShB peptide prevent both structure formation and channel inactivation lend further support to the hypothesis that adoption of beta structure by the inactivating peptide in a hydrophobic environment is important in determining channel blockade.     

The role of individual cysteine residues in the processing, structure, and function of human macrophage colony-stimulating factor

The ontogeny of the hematopoietic system in mammalian embryos occurs during the yolk sac (YS) and the fetal liver (FL) stages. Events leading to the establishment of hematopoiesis in the FL remain obscure. The appearance of colony-forming units-spleen (CFU-S) in the FL is preceded by a gradual increase of CFU-S in the YS and a more rapid increase in the AGM region (area comprising dorsal aorta, gonads, and mesonephros) during day 10 of development (Medvinsky et al, Nature 364:64, 1993). By this time, the AGM CFU-S attain a high frequency equivalent to that found in the adult bone marrow. The analogous area gives rise to adult hematopoiesis in amphibians and probably in birds. We present here a more complete picture of CFU-S development during transition from the pre-liver to liver stage of hematopoiesis. (1) Dissectional analysis of the mouse AGM region shows the presence of CFU-S both around the dorsal aorta and in the uro-genital ridges. (2) The embryonic gut also shows low but distinctive CFU-S activity. This initial intrabody pattern of CFU-S distribution in murine embryogenesis parallels that found for primordial germ cells. (3) The beginning of definitive liver hematopoiesis is accompanied by wide dissemination of CFU-S in the embryonic tissues. (4) Comparison of spleen colonies arising from the AGM and YS has shown morphologic differences. In contrast to simple erythroid constitution of the YS colonies, a broader variety of cells are found within the AGM-derived colonies that are similar to those derived from 11-day FL. These data suggest a lineage relationship for hematopoietic progenitors between the AGM region and the FL.     

Modulation of vascular function by diet and exercise

Clinical research is conducted in free living individuals who are always subject to the influences on vascular function and the major cardiovascular regulators of their lifestyle. The purpose of this paper is to review some lifestyle influences on cardiovascular function, particularly the sympathetic nervous system and endothelially mediated vasodilatation. There are highly differentiated sympathetic responses to feeding, and to acute exercise. Over a longer period obesity has a typical pattern of sympathetic activity. Reduced dietary salt intake elicits profound localised increases in sympathetic activity to the kidney. Marine oil supplementation attenuates the sympathetic responses to psychological stress and improves endothelially mediated vasodilatation in hypercholesterolaemics. Exercise training reduced total noradrenaline spillover, the major beds affected being the renal and skeletal muscle. These examples illustrate the dynamic nature of vascular dilatation and that, like the sympathetic nervous system, it is modulated by short, medium and long term influences. In both cases there is regulation both at a local and systemic level. Habitual, and recent, lifestyle can exert important cardiovascular effects which must be taken into account in clinical and epidemiological research.     

Modulation of the Kv1.3 potassium channel by receptor tyrosine kinases

The voltage-dependent potassium channel, Kv1.3, is modulated by the epidermal growth factor receptor (EGFr) and the insulin receptor tyrosine kinases. When the EGFr and Kv1.3 are coexpressed in HEK 293 cells, acute treatment of the cells with EGF during a patch recording can suppress the Kv1.3 current within tens of minutes. This effect appears to be due to tyrosine phosphorylation of the channel, as it is blocked by treatment with the tyrosine kinase inhibitor erbstatin, or by mutation of the tyrosine at channel amino acid position 479 to phenylalanine. Previous work has shown that there is a large increase in the tyrosine phosphorylation of Kv1.3 when it is coexpressed with the EGFr. Pretreatment of EGFr and Kv1.3 cotransfected cells with EGF before patch recording also results in a decrease in peak Kv1.3 current. Furthermore, pretreatment of cotransfected cells with an antibody to the EGFr ligand binding domain (alpha-EGFr), which blocks receptor dimerization and tyrosine kinase activation, blocks the EGFr-mediated suppression of Kv1.3 current. Insulin treatment during patch recording also causes an inhibition of Kv1.3 current after tens of minutes, while pretreatment for 18 h produces almost total suppression of current. In addition to depressing peak Kv1.3 current, EGF treatment produces a speeding of C-type inactivation, while pretreatment with the alpha-EGFr slows C-type inactivation. In contrast, insulin does not influence C-type inactivation kinetics. Mutational analysis indicates that the EGF-induced modulation of the inactivation rate occurs by a mechanism different from that of the EGF-induced decrease in peak current. Thus, receptor tyrosine kinases differentially modulate the current magnitude and kinetics of a voltage-dependent potassium channel.     

Modulation of C-type inactivation by K+ at the potassium channel selectivity filter

With prolonged or repetitive activation, voltage-gated K+ channels undergo a slow (C-type) inactivation mechanism, which decreases current flow through the channel. Previous observations suggest that C-type inactivation results from a localized constriction in the outer mouth of the channel pore and that the rate of inactivation is controlled by the-rate at which K+ leaves an unidentified binding site in the pore. We have functionally identified two K+ binding sites in the conduction pathway of a chimeric K+ channel that conducts Na+ in the absence of K+. One site has a high affinity for K+ and contributes to the selectivity filter mechanism for K+ over Na+. Another site, external to the high-affinity site, has a lower affinity for K+ and is not involved in channel selectivity. Binding of K+ to the high-affinity binding site slowed inactivation. Binding of cations to the external low-affinity site did not slow inactivation directly but could slow it indirectly, apparently by trapping K+ at the high-affinity site. These data support a model whereby C-type inactivation involves a constriction at the selectivity filter, and the constriction cannot proceed when the selectivity filter is occupied by K+.     

"Effect of mechanical deformation on structure and function of polymorphonuclear leukocytes"

The present studies were designed to test the hypothesis that mechanical deformation of polymorphonuclear leukocytes (PMN) leads to functional changes that might influence their transit in the pulmonary capillaries. Human leukocytes were passed through 5- or 3-micron-pore polycarbonate filters under controlled conditions. Morphometric analysis showed that the majority of PMN were deformed and that this deformation persisted longer after filtration through 3-micron filters than through 5-micron filters (P < 0.05) but did not result in the cytoskeletal polarization characteristic of migrating cells. Flow cytometric studies of the filtered PMN showed that there was a transient increase in the cytosolic free Ca2+ concentration after both 3- and 5-micron filtration (P < 0.01) with an increase in F-actin content after 3-micron filtration (P < 0.05). Although L-selectin expression on PMN was not changed by either 5- or 3-micron filtration, CD18 and CD11b were increased by 3-micron filtration (P < 0.05). Priming of the PMN with N-formyl-methionyl-leucyl-phenylalanine (0.5 nM) before filtration resulted in an increase of CD11b by both 5 (P < 0.05)- and 3-micron (P < 0.01) filtration. Neither 5- nor 3-micron filtration induced hydrogen peroxide production. We conclude that mechanical deformation of PMN, similar to what occurs in the pulmonary microvessels, induces both structural and functional changes in the cells, which might influence their passage through the pulmonary capillary bed.     

A comparison of the structure and function of the terminal ileum in Crohn's disease using radiology, the "Dicopac" Schilling test and [14C] G.C.A. breath test

Twenty patients with Crohn's disease were studied. Thirteen had radiological evidence of involvement of the terminal ileum. None had significant bacterial overgrowth of the small bowel contents and none had had resection of the terminal ileum. In all patients a [14C] glycocholic acid ([14C] G.C.A.) breath test and a "Dicopac" Schilling test were performed to assess terminal ileal function. The data showed poor correlation between the radiological appearance of the terminal ileum and the results of the functional tests. There was also poor correlation between the results of the [14C] G.C.A. breath test and the "Dicopac" Schilling test. Without terminal ileal histology, any assessment of the extent of Crohn's disease of the terminal ileum and of its effect on terminal ileal function, must include the [14C] G.C.A. breath test as well as radiology and the "Dicopac" Schilling test. The limitations of the [14C] G.C.A. breath test as a test of terminal ileal function in Crohn's disease are discussed.     

Modulation of RecA nucleoprotein function by the mutagenic UmuD'C protein complex

The RecA, UmuC, and UmuD' proteins are essential for error-prone, replicative bypass of DNA lesions. Normally, RecA protein mediates homologous pairing of DNA. We show that purified Umu(D')2C blocks this recombination function. Biosensor measurements establish that the mutagenic complex binds to the RecA nucleoprotein filament with a stoichiometry of one Umu(D')2C complex for every two RecA monomers. Furthermore, Umu(D')2C competitively inhibits LexA repressor cleavage but not ATPase activity, implying that Umu(D')2C binds in or proximal to the helical groove of the RecA nucleoprotein filament. This binding reduces joint molecule formation and even more severely impedes DNA heteroduplex formation by RecA protein, ultimately blocking all DNA pairing activity and thereby abridging participation in recombination function. Thus, Umu(D')2C restricts the activities of the RecA nucleoprotein filament and presumably, in this manner, recruits it for mutagenic repair function. This modulation by Umu(D')2C is envisioned as a key event in the transition from a normal mode of genomic maintenance by "error-free" recombinational repair, to one of "error-prone" DNA replication.     

Dialectic of the interrelationship between structure and function in biology and medicine

The paper deals with some aspects of the dialectics of structure and function relationships in biological objects normally and pathologically. Idealistic and metaphysical concepts of the structure-function relationships (morphological idealism, holism, physiological idealism, functionalism) are critisized, and historical premises of these concepts are characterized. The principle of indissoluble unity and interconnection of changes in structure and function is emphasized, while the thesis of the primacy of function in the shaping of the form and the concept of functional diseases are rejected. Much attention is paid to the methodological principles of the study of structure and function based on the systemic approach to the investigation of biological objects from the point of view of structural levels and integratism. The groundlessness of the principles of reductionism and organicism in the solution of this problem is indicated. The connection of the concepts of structure and function with categories and laws of materialistic dialectics is dwelt on.     

Relation between the structure and the pitting corrosion resistance of hypereutectoid U10 steel

Polarization pitting corrosion tests are used to investigate the effect of a structure on the corrosion resistance of hypereutectoid U10 steel. In the steel structure, coarse-lamellar and fine-lamellar pearlite forms as a result of isothermal decomposition at temperatures of 500 and 650°C and fine-lamellar pearlite forms during additional annealing at 650°C for 10 or 300 min. The nonequilibrium structure of fine-lamellar pearlite obtained in the process of isothermal decomposition at a temperature of 500°C is found to have the maximum pitting corrosion resistance among the structural states under study.     

Characterization of pKa values and titration shifts in the cytotoxic ribonuclease alpha-sarcin by NMR. Relationship between electrostatic interactions, structure, and catalytic function

The electrostatic behavior of titrating groups in alpha-sarcin was investigated using 1H NMR spectroscopy. A total of 209 chemical shift titration curves corresponding to different protons in the molecule were determined over the pH range of 3.0-8.5. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson-Hasselbalch equation led to the unambiguous determination of pKa values for all glutamic acid and histidine residues, as well as for the C-terminal carboxylate and most of the aspartic acids in the free enzyme. The ionization constants of catalytically relevant histidines, His50 and His137, and glutamic acid, Glu96, in the alpha-sarcin-2'-GMP complex were also determined. The pKa values of 15 ionizable groups (C-carboxylate, six aspartic acids, four glutamic acids, and four histidines) were found to be close to their normal values. On the other hand, a number of side chain groups, including those in the active center, showed pKa values far from their intrinsic values. Thus, the pKa values for active site residues His50, Glu96, and His137 were 7.7, 5.2, and 5.8 in the free enzyme and 7.6, approximately 4.8, and 6.8 in the alpha-sarcin-2'-GMP complex, respectively. The pKa values and the activity profile against ApA, as a function of pH, are in agreement with the proposed enzymatic mechanism (in common with RNase T1 and the family of the microbial ribonucleases), in which Glu96 and His137 act as a general base and general acid, respectively. In almost all microbial ribonucleases, a Phe-His interaction is present, which affects the pKa of one of the His residues at the active site (His137). The absence of this interaction in alpha-sarcin would explain the lower pKa value of this His residue, and provides an explanation for the decreased RNase activity of this protein as compared to those of other microbial ribonucleases.     

Modulation of Na+ channel inactivation by the beta 1 subunit: a deletion analysis

Na+ currents recorded from Xenopus oocytes expressing the Na+ channel alpha subunit alone inactivate with two exponential components. The slow component predominates in monomeric channels, while co-expression with the beta 1 subunit favors the fast component. Macropatch recordings show that the relative rates of these components are much greater than previously estimated from two-electrode measurements (approximately 30-fold vs approximately 5-fold). A re-assessment of steady-state inactivation, h infinity (V), shows that there is no depolarized shift of the slow component, provided a sufficiently long prepulse duration and repetition interval are used to achieve steady-state entry and recovery from inactivation, respectively. Deletion mutagenesis of the beta 1 subunit was used to define which regions of the subunit are required to modulate inactivation kinetics. The carboxy tail, comprising the entire predicted intracellular domain, can be deleted without a loss of activity; whereas small deletions in the extracellular amino domain or the signal peptide totally disrupt function.