bluuebunny/crossref_metadata_2025 huggingface dataset:
A Mutational Analysis of the Binding of Staphylococcal Enterotoxins B and C3 to the T Cell Receptor β Chain and Major Histocompatibility Complex Class II
Authors: Leder Lukas , Llera Andrea , Lavoie Pascal M. , Lebedeva Marina I. , Li Hongmin , Sékaly Rafick-Pierre , Bohach Gregory A. , Gahr Pamala J. , Schlievert Patrick M. , Karjalainen Klaus , Mariuzza Roy A.
Published: July 2002

The three-dimensional structure of the complex between a T cell receptor (TCR) β chain (mouse Vβ8.2Jβ2.1Cβ1) and the superantigen (SAG) staphylococcal enterotoxin C3 (SEC3) has been recently determined to 3.5 Å resolution. To evaluate the actual contribution of individual SAG residues to stabilizing the β–SEC3 complex, as well as to investigate the relationship between the affinity of SAGs for TCR and MHC and their ability to activate T cells, we measured the binding of a set of SEC3 and staphylococcal enterotoxin B (SEB) mutants to soluble recombinant TCR β chain and to the human MHC class II molecule HLA-DR1. Affinities were determined by sedimentation equilibrium and/or surface plasmon detection, while mitogenic potency was assessed using T cells from rearrangement-deficient TCR transgenic mice. We show that there is a clear and simple relationship between the affinity of SAGs for the TCR and their biological activity: the tighter the binding of a particular mutant of SEC3 or SEB to the TCR β chain, the greater its ability to stimulate T cells. We also find that there is an interplay between TCR–SAG and SAG–MHC interactions in determining mitogenic potency, such that a small increase in the affinity of a SAG for MHC can overcome a large decrease in the SAG's affinity for the TCR. Finally, we observe that those SEC3 residues that make the greatest energetic contribution to stabilizing the β–SEC3 complex (“hot spot” residues) are strictly conserved among enterotoxins reactive with mouse Vβ8.2, thereby providing a basis for understanding why SAGs having other residues at these positions show different Vβ-binding specificities.
Distinct Methylation of the Interferon γ (IFN-γ) and Interleukin 3 (IL-3) Genes in Newly Activated Primary CD8+ T Lymphocytes: Regional IFN-γ Promoter Demethylation and mRNA Expression Are Heritable in CD44highCD8+ T Cells
Authors: Fitzpatrick David R. , Shirley Kym M. , McDonald Louise E. , Bielefeldt-Ohmann Helle , Kay Graham F. , Kelso Anne
Published: July 2002

Differential genomic DNA methylation has the potential to influence the development of T cell cytokine production profiles. Therefore, we have conducted a clonal analysis of interferon (IFN)-γ and interleukin (IL)-3 gene methylation and messenger (m)RNA expression in primary CD8+ T cells during the early stages of activation, growth, and cytokine expression. Despite similar distributions and densities of CpG methylation sites, the IFN-γ and IL-3 promoters exhibited differential demethylation in the same T cell clone, and heterogeneity between clones. Methylation patterns and mRNA levels were correlated for both genes, but demethylation of the IFN-γ promoter was widespread across >300 basepairs in clones expressing high levels of IFN-γ mRNA, whereas demethylation of the IL-3 promoter was confined to specific CpG sites in the same clones. Conversely, the majority of clones expressing low or undetectable levels of IFN-γ mRNA exhibited symmetrical methylation of four to six of the IFN-γ promoter CpG sites. Genomic DNA methylation also has the potential to influence the maintenance or stability of T cell cytokine production profiles. Therefore, we also tested the heritability of IFN-γ gene methylation and mRNA expression in families of clones derived from resting CD44lowCD8+ T cells or from previously activated CD44highCD8+ T cells. The patterns of IFN-γ gene demethylation and mRNA expression were faithfully inherited in all clones derived from CD44high cells, but variable in clones derived from CD44low cells. Overall, these findings suggest that differential genomic DNA methylation, including differences among cytokine genes, among individual T cells, and among T cells with different activation histories, is an important feature of cytokine gene expression in primary T cells.
The Functional Paradox of CD43 in Leukocyte Recruitment: A Study Using CD43-deficient Mice
Authors: Woodman Richard C. , Johnston Brent , Hickey Michael J. , Teoh Diane , Reinhardt Paul , Poon Betty Y. , Kubes Paul
Published: July 2002

Although there is considerable evidence implicating a role for CD43 (leukosialin) in leukocyte cell–cell interactions, its precise function remains uncertain. Using CD43-deficient mice (CD43−/−) and intravital microscopy to directly visualize leukocyte interactions in vivo, we investigated the role of CD43 in leukocyte–endothelial cell interactions within the cremasteric microcirculation under flow conditions. Our studies demonstrated significantly enhanced leukocyte rolling and adhesion after chemotactic stimuli in CD43−/− mice compared with wild type mice. Using an in vitro flow chamber, we established that the enhanced rolling interactions of CD43−/− leukocytes, primarily neutrophils, were also observed using immobilized E-selectin as a substrate, suggesting that passive processes related to steric hindrance or charge repulsion were likely mechanisms. Despite increased adhesion and rolling interactions by CD43−/− leukocytes, we uncovered a previously unrecognized impairment of CD43−/− leukocytes to infiltrate tissues. Oyster glycogen–induced neutrophil and monocyte infiltration into the peritoneum was significantly reduced in CD43−/− mice. In response to platelet activating factor, CD43−/− leukocytes were impaired in their ability to emigrate out of the vasculature. These results suggest that leukocyte CD43 has a dual function in leukocyte–endothelial interactions. In addition to its role as a passive nonspecific functional barrier, CD43 also facilitates emigration of leukocytes into tissues.
Receptor Editing Occurs Frequently during Normal B Cell Development
Authors: Retter Marc W. , Nemazee David
Published: July 2002

Allelic exclusion is established in development through a feedback mechanism in which the assembled immunoglobulin (Ig) suppresses further V(D)J rearrangement. But Ig expression sometimes fails to prevent further rearrangement. In autoantibody transgenic mice, reactivity of immature B cells with autoantigen can induce receptor editing, in which allelic exclusion is transiently prevented or reversed through nested light chain gene rearrangement, often resulting in altered B cell receptor specificity. To determine the extent of receptor editing in a normal, non-Ig transgenic immune system, we took advantage of the fact that λ light chain genes usually rearrange after κ genes. This allowed us to analyze κ loci in IgMλ+ cells to determine how frequently in-frame κ genes fail to suppress λ gene rearrangements. To do this, we analyzed recombined VκJκ genes inactivated by subsequent recombining sequence (RS) rearrangement. RS rearrangements delete portions of the κ locus by a V(D)J recombinase-dependent mechanism, suggesting that they play a role in receptor editing. We show that RS recombination is frequently induced by, and inactivates, functionally rearranged κ loci, as nearly half (47%) of the RS-inactivated VκJκ joins were in-frame. These findings suggest that receptor editing occurs at a surprisingly high frequency in normal B cells.
Hematopoietic Expression of Hoxb4 Is Regulated in Normal and Leukemic Stem Cells through Transcriptional Activation of the Hoxb4 Promoter by Upstream Stimulating Factor (Usf)-1 and Usf-2
Authors: Giannola Diane M. , Shlomchik Warren D. , Jegathesan Mithila , Liebowitz David , Abrams Charles S. , Kadesch Tom , Dancis Andrew , Emerson Stephen G.
Published: July 2002

The homeobox genes encode a family of transcription factors that regulate development and postnatal tissue homeostasis. Since HOXB4 plays a key role in regulating the balance between hematopoietic stem cell renewal and differentiation, we studied the molecular regulation of HOXB4 expression in human hematopoietic stem cells. HOXB4 expression in K562 cells is regulated at the level of transcription, and transient transfection defines primary HOXB4 regulatory sequences within a 99-bp 5′ promoter. Culture of highly purified human CD34+ bone marrow cells in thrombopoietin/Flt-3 ligand/stem cell factor induced HOXB4 3–10-fold, whereas culture in granulocyte/macrophage colony-stimulating factor, only increased HOXB4/luciferase expression 20–50%. Mutations within the HOXB4 promoter identified a potential E box binding site (HOX response element [HXRE]-2) as the most critical regulatory sequence, and yeast one hybrid assays evaluating bone marrow and K562 libraries for HXRE-2 interaction identified upstream stimulating factor (USF)-2 and micropthalmia transcription factor (MITF). Electrophoretic mobility shift assay with K562 extracts confirmed that these proteins, along with USF-1, bind to the HOXB4 promoter in vitro. Cotransfection assays in both K562 and CD34+ cells showed that USF-1 and USF-2, but not MITF, induce the HOXB4 promoter in response to signals stimulating stem cell self-renewal, through activation of the mitogen-activated protein kinase pathway. Thus hematopoietic expression of the human HOXB4 gene is regulated by the binding of USF-1 and USF-2, and this process may be favored by cytokines promoting stem cell self-renewal versus differentiation.
Migration and Differentiation of Autoreactive B-1 Cells Induced by Activated γ/δ T Cells in Antierythrocyte Immunoglobulin Transgenic Mice
Authors: Watanabe Norihiko , Ikuta Koichi , Fagarasan Sidonia , Yazumi Shujiro , Chiba Tsutomu , Honjo Tasuku
Published: July 2002

Using normal and transgenic (Tg) mice, we have shown that peritoneal B-1 cells are activated by administration of cytokines or lipopolysaccharide and migrate to other lymphoid organs where they differentiate into antibody-secreting cells. However, little is known about the process of B-1 cell migration and differentiation in vivo. We developed a mouse line by crossing the antierythrocyte antibody Tg mice (HL mice) with TCR-γ/δ Tg mice specific for a self-thymus leukemia (TL) antigen in the recombination activating gene (RAG)2−/− background. In the presence of the self-antigen, Tg γ/δ T cells increased in number and manifested activated phenotypes. Peritoneal B-1 cells in these mice migrated into mesenteric lymph nodes and differentiated into autoantibody-secreting cells, resulting in strong autoimmune hemolytic anemia. Furthermore, transfer of RAG2−/− × HL bone marrow or peritoneal cells into the peritoneal cavity of RAG2−/− × TCR-γ/δ Tg mice gave rise to donor-derived B-1 cells in mesenteric lymph nodes, and these cells produced the autoantibody. Thus, this study demonstrates that the migration of B-1 cells and differentiation into the antibody-secreting cells can be induced by noncognate T cell help and implies the possibility that γ/δ T cells may induce B-1 cell differentiation in vivo.
Transient Inhibition of Interleukin 4 Signaling by T Cell Receptor Ligation
Authors: Zhu Jinfang , Huang Hua , Guo Liying , Stonehouse Timothy , Watson Cynthia J. , Hu-Li Jane , Paul William E.
Published: July 2002

Interleukin (IL)-4 and IL-12 together with T cell receptor (TCR) engagement are crucial for the differentiation of CD4+ T cells into T helper (Th)2 or Th1 cells, respectively. Although IL-4 receptors (IL-4Rs) but not IL-12Rs are expressed on naive CD4+ T cells, IL-4 has no apparent advantage over IL-12 in driving naive T cell differentiation when the cells are primed with both IL-4 and IL-12 in vitro. It was found that IL-4–induced phosphorylation of Janus kinases 1 and 3, IL-4Rα, signal transducer and activator of transcription 6, and insulin receptor substrate 2 was strikingly but transiently inhibited by TCR ligation both in conventional and TCR transgenic T cells. TCR engagement also blocked the expression of an IL-4–inducible gene. Signals induced by other cytokines, including IL-2, IL-6, and interferon α, but not by insulin-like growth factor 1, were also blocked by TCR engagement. The capacity of various inhibitors to reverse TCR-mediated inhibition of IL-4 signaling suggested that activation of the Ras–mitogen-activated protein kinase pathway and of the calcineurin pathway contribute to desensitizing IL-4R. IL-4 responsiveness returned at about the time (∼12 h) that IL-12–mediated signaling was first observed. Thus, through different mechanisms, neither IL-4R nor IL-12R has any clear advantage in polarizing cells; rather, the availability of cytokine is probably the limiting factor in this process.
Antigen-Specific T Helper Cell Function
Authors: Panus Joanne Fanelli , McHeyzer-Williams Louise J. , McHeyzer-Williams Michael G.
Published: July 2002

Distinguishing between the development of functional potential in antigen-specific T helper (Th) cells and the delivery of these specialized functions in vivo has been difficult to resolve. Here, we quantify the frequency of cytokine-producing cells within the primary and memory B10.BR Th cell response to pigeon cytochrome c (PCC). In vitro analysis of acquired functional potential indicated no Th1/Th2 cytokine polarity at the peak of the primary response with surprisingly little evidence for the selective preservation of interleukin (IL)-2, tumor necrosis factor (TNF)-α, IL-4, and interferon (IFN)-γ potentials into the memory compartment. However, the expression of these functional potentials appears tightly regulated in vivo. The staggered appearance of primary response cytokines directly ex vivo contrasts markedly with their rapid coordinate expression in the memory response. Frequencies of IL-2–, TNF-α–, IFN-γ–, and IL-10–expressing memory responders increased over their primary response counterparts, but were still markedly lower than revealed in vitro. IL-4–, IFN-γ–, and IL-10–expressing Th cells remained at low but stable frequencies over the first 6 d of the memory response. Analysis of T cell receptor β chain sequences of IL-4– and TNF-α–expressing PCC-specific Th cells provides evidence for early functional commitment among clonal progeny. These data indicate that the development of functional potential is a consequence of initial antigen experience, but delivery of specialized functions is differentially regulated in primary and memory immune responses.
Importance of Primary Capture and L-Selectin–Dependent Secondary Capture in Leukocyte Accumulation in Inflammation and Atherosclerosis in Vivo
Authors: Eriksson Einar E. , Xie Xun , Werr Joachim , Thoren Peter , Lindbom Lennart
Published: July 2002

In the multistep process of leukocyte extravasation, the mechanisms by which leukocytes establish the initial contact with the endothelium are unclear. In parallel, there is a controversy regarding the role for L-selectin in leukocyte recruitment. Here, using intravital microscopy in the mouse, we investigated leukocyte capture from the free flow directly to the endothelium (primary capture), and capture mediated through interactions with rolling leukocytes (secondary capture) in venules, in cytokine-stimulated arterial vessels, and on atherosclerotic lesions in the aorta. Capture was more prominent in arterial vessels compared with venules. In venules, the incidence of capture increased with increasing vessel diameter and wall shear rate. Secondary capture required a minimum rolling leukocyte flux and contributed by ∼20–50% of total capture in all studied vessel types. In arteries, secondary capture induced formation of clusters and strings of rolling leukocytes. Function inhibition of L-selectin blocked secondary capture and thereby decreased the flux of rolling leukocytes in arterial vessels and in large (>45 μm in diameter), but not small (<45 μm), venules. These findings demonstrate the importance of leukocyte capture from the free flow in vivo. The different impact of blockage of secondary capture in venules of distinct diameter range, rolling flux, and wall shear rate provides explanations for the controversy regarding the role of L-selectin in various situations of leukocyte recruitment. What is more, secondary capture occurs on atherosclerotic lesions, a fact that provides the first evidence for roles of L-selectin in leukocyte accumulation in atherogenesis.
Major T Cell Progenitor Activity in Bone Marrow–derived Spleen Colonies
Authors: Lancrin Christophe , Schneider Elke , Lambolez Florence , Arcangeli Marie-Laure , Garcia-Cordier Corinne , Rocha Benedita , Ezine Sophie
Published: July 2002

Common lymphoid progenitors (CLP) are generated in adult bone marrow (BM), but the intermediate steps leading to T cell commitment are unknown, and so is the site at which this commitment occurs. Here, we show that colonies arising in the spleen 12 days after BM injection harbor T cell precursors that are undetectable in BM. These precursors did not generate myeloid cells in vivo but repopulated the thymus and the peripheral T cell compartment much faster than did CLP. Two lineage negative (Lin−) subpopulations were distinguished, namely CD44+ Thy1− cells still capable of natural killer generation and transient low-level B cell generation, and T cell–restricted CD44− Thy1+ cells. At a molecular level, frequency of CD3ε and preTα mRNA was very different in each subset. Furthermore, only the CD44− Thy1+ subset have initiated rearrangements in the T cell receptor β locus. Thus, this study identifies extramedullary T cell progenitors and will allow easy approach to T cell commitment studies.
Barium Influx Mediated by the Cardiac Sodium-Calcium Exchanger in Transfected Chinese Hamster Ovary Cells
Authors: Condrescu Madalina , Chernaya Galina , Kalaria Vijay , Reeves John P.
Published: July 2002

We examined Ba2+ influx using isotopic and fura-2 techniques in transfected Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger (CK1.4 cells). Ba2+ competitively inhibited exchange-me diated 45Ca2+ uptake with a Ki ∼ 3 mM. Ba2+ uptake was stimulated by pretreating the cells with ouabain and by removing extracellular Na+, as expected for Na+/Ba2+ exchange activity. The maximal velocity of Ba2+ accumulation was estimated to be 50% of that for Ca2+. When the monovalent cation ionophore gramicidin was used to equilibrate internal and external concentrations of Na+, Ba2+ influx was negligible in the absence of Na+ and increased to a maximum at 20–40 mM Na+. At higher Na+ concentrations, Ba2+ influx declined, presumably due to the competition between Na+ and Ba2+ for transport sites on the exchanger. Unlike Ca2+, Ba2+ did not appear to be taken up by intracellular organelles: Thus, 133Ba2+ uptake in ouabain-treated cells was not reduced by mitochondrial inhibitors such as Cl-CCP or oligomycin-rotenone. Moreover, intracellular Ca2+ stores that had been depleted of Ca2+ by pretreatment of the cells with ionomycin (a Ca2+ ionophore) remained empty during a subsequent period of Ba2+ influx. Ca2+ uptake or release by intracellular organelles secondarily regulated exchange activity through alterations in [Ca2+]i. Exchange-mediated Ba2+ influx was inhibited when cytosolic [Ca2+] was reduced to 20 nM or less and was accelerated at cytosolic Ca2+ concentrations of 25–50 nM. We conclude that (a) Ba2+ substitutes for Ca2+ as a transport substrate for the exchanger, (b) cytosolic Ba2+ does not appear to be sequestered by intracellular organelles, and (c) exchange-mediated Ba2+ influx is accelerated by low concentrations of cytosolic Ca2+.
Limited Bifurcation Asymmetry in Coronary Arterial Tree Models Generated by Constrained Constructive Optimization
Authors: Schreiner Wolfgang , Neumann Friederike , Neumann Martin , Karch Rudolf , End Adelheid , Roedler Susanne M.
Published: July 2002

Models of coronary arterial trees are generated by the algorithm of constrained constructive optimization (CCO). In a given perfusion area a binary branching network of straight cylindrical tubes is generated by successively adding terminal segments to the growing structure. In each step the site of connection is chosen according to an optimization target function (total intravascular volume), and in any stage of development the tree fulfills physiologic boundary conditions (constraints involving pressures, flows and bifurcation rules). CCO generates structures which in many aspects resemble real coronary arterial trees, except for very asymmetric bifurcations, occurring when a large branch gives off a tiny terminal segment. In the present work we evaluate an additional constraint within CCO, namely imposing a limit on the asymmetry of bifurcations during the construction process. Model trees are grown with different limits imposed, and the effects on structure are studied both phenomenologically and via statistical descriptors. As the limit to asymmetry is tightened, blood is conveyed to the perfusion sites via detours rather than directly and the comparison with measured data shows the structure to change from a conveying to a delivering type of function. Simultaneously total intravascular volume, surface and sum of segments' lengths increase. It is shown why and how local bifurcation asymmetry is able to determine the global structure of the optimized arterial tree model. Surprisingly, the pressure profile from inlet to terminals, being a functional characteristic, remains unaffected.
Calcium Current Activated by Depletion of Calcium Stores in Xenopus Oocytes
Authors: Yao Yong , Tsien Roger Y.
Published: July 2002

Ca2+ currents activated by depletion of Ca2+ stores in Xenopus oocytes were studied with a two-electrode voltage clamp. Buffering of cytosolic Ca2+ with EGTA and MeBAPTA abolished ICl(Ca) and unmasked a current in oocytes that was activated by InsP3 or ionomycin in minutes and by thapsigargin or the chelators themselves over hours. At −60 mV in 10 mM extracellular CaCl2, the current was typically around −90 or −160 nA in oocytes loaded with EGTA or MeBAPTA, respectively. This current was judged to be a Ca2+-selective current for the following reasons: (a) it was inwardly rectifying and reversed at membrane potentials usually more positive than +40 mV; (b) it was dependent on extracellular [CaCl2] with Km = 11.5 mM; (c) it was highly selective for Ca2+ against monovalent cations Na+ and K+, because replacing Na+ and K+ by N-methyl-d-glucammonium did not reduce the amplitude or voltage dependence of the current significantly; and (d) Ca2+, Sr2+, and Ba2+ currents had similar instantaneous conductances, but Sr2+ and Ba2+ currents appeared to inactivate more strongly than Ca2+. This Ca2+ current was blocked by metal ions with the following potency sequence: Mg2+ << Ni2+ ≈ Co2+ ≈ Mn2+ < Cd2+ << Zn2+ << La3+. It was also inhibited by niflumic acid, which is commonly used to block ICl(Ca). PMA partially inhibited the Ca2+ current, and this effect was mostly abolished by calphostin C, indicating that the Ca2+ current is sensitive to protein kinase C. These results are the first detailed electrophysiological characterization of depletion-activated Ca2+ current in nondialyzed cells. Because exogenous molecules and channels are easy to introduce into oocytes and the distortions in measuring ICl(Ca) can now be bypassed, oocytes are now a superior system in which to analyze the activation mechanisms of capacitative Ca2+ influx.
Swelling-activated Gd3+-sensitive Cation Current and Cell Volume Regulation in Rabbit Ventricular Myocytes
Authors: Clemo Henry F. , Baumgarten Clive M.
Published: July 2002

The role of swelling-activated currents in cell volume regulation is unclear. Currents elicited by swelling rabbit ventricular myocytes in solutions with 0.6–0.9× normal osmolarity were studied using amphotericin perforated patch clamp techniques, and cell volume was examined concurrently by digital video microscopy. Graded swelling caused graded activation of an inwardly rectifying, time-independent cation current (ICir,swell) that was reversibly blocked by Gd3+, but ICir,swell was not detected in isotonic or hypertonic media. This current was not related to IK1 because it was insensitive to Ba2+. The PK/PNa ratio for ICir,swell was 5.9 ± 0.3, implying that inward current is largely Na+ under physiological conditions. Increasing bath K+ increased gCir,swell but decreased rectification. Gd3+ block was fitted with a K0.5 of 1.7 ± 0.3 μM and Hill coefficient, n, of 1.7 ± 0.4. Exposure to Gd3+ also reduced hypotonic swelling by up to ∼30%, and block of current preceded the volume change by ∼1 min. Gd3+-induced cell shrinkage was proportional to ICir,swell when ICir,swell was varied by graded swelling or Gd3+ concentration and was voltage dependent, reflecting the voltage dependence of ICir,swell. Integrating the blocked ion flux and calculating the resulting change in osmolarity suggested that ICir,swell was sufficient to explain the majority of the volume change at –80 mV. In addition, swelling activated an outwardly rectifying Cl− current, ICl,swell. This current was absent after Cl− replacement, reversed at ECl, and was blocked by 1 mM 9-anthracene carboxylic acid. Block of ICl,swell provoked a 28% increase in swelling in hypotonic media. Thus, both cation and anion swelling-activated currents modulated the volume of ventricular myocytes. Besides its effects on cell volume, ICir,swell is expected to cause diastolic depolarization. Activation of ICir,swell also is likely to affect contraction and other physiological processes in myocytes.
Tetracaine Reports a Conformational Change in the Pore of Cyclic Nucleotide–gated Channels
Authors: Fodor Anthony A. , Black Kevin D. , Zagotta William N.
Published: July 2002

Local anesthetics are a diverse group of clinically useful compounds that act as pore blockers of both voltage- and cyclic nucleotide–gated (CNG) ion channels. We used the local anesthetic tetracaine to probe the nature of the conformational change that occurs in the pore of CNG channels during the opening allosteric transition. When applied to the intracellular side of wild-type rod CNG channels expressed in Xenopus oocytes from the α subunit, the local anesthetic tetracaine exhibits state-dependent block, binding with much higher affinity to closed states than to open states. Here we show that neutralization of a glutamic acid in the conserved P region (E363G) eliminated this state dependence of tetracaine block. Tetracaine blocked E363G channels with the same effectiveness at high concentrations of cGMP, when the channel spent more time open, and at low concentrations of cGMP, when the channel spent more time closed. In addition, Ni2+, which promotes the opening allosteric transition, decreased the effectiveness of tetracaine block of wild-type but not E363G channels. Similar results were obtained in a chimeric CNG channel that exhibits a more favorable opening allosteric transition. These results suggest that E363 is accessible to internal tetracaine in the closed but not the open configuration of the pore and that the conformational change that accompanies channel opening includes a change in the conformation or accessibility of E363.
Activation of Shaker Potassium Channels
Authors: Schoppa N.E. , Sigworth F.J.
Published: July 2002

This second of three papers, in which we functionally characterize activation gating in Shaker potassium channels, focuses on the properties of a mutant channel (called V2), in which the leucine at position 382 (in the Shaker B sequence) is mutated to valine. The general properties of V2's ionic and gating currents are consistent with changes in late gating transitions, in particular, with V2 disrupting the positively cooperative gating process of the normally activating wild type (WT) channel. An analysis of forward and backward rate constants, analogous to that used for WT in the previous paper, indicates that V2 causes little change in the rates for most of the transitions in the activation path, but causes large changes in the backward rates of the final two transitions. Single channel data indicate that the V2 mutation causes moderate changes in the rates of transitions to states that are not in the activation path, but little change in the rates from these states. V2's data also yield insights into the general properties of the activation gating process that could not be readily obtained from the WT channel, including evidence that intermediate transitions have rapid backward rates, and an estimate of a total charge 2 e0 for the final two transitions. Taken together, these data will help constrain an activation gating model in the third paper of this series, while also providing an explanation for V2's effects.
Regulatory Volume Decrease and Intracellular Ca2+ in Murine Neuroblastoma Cells Studied with Fluorescent Probes
Authors: Altamirano J. , Brodwick M.S. , Alvarez-Leefmans F.J.
Published: July 2002

The possible role of Ca2+ as a second messenger mediating regulatory volume decrease (RVD) in osmotically swollen cells was investigated in murine neural cell lines (N1E-115 and NG108-15) by means of novel microspectrofluorimetric techniques that allow simultaneous measurement of changes in cell water volume and [Ca2+]i in single cells loaded with fura-2. [Ca2+]i was measured ratiometrically, whereas the volume change was determined at the intracellular isosbestic wavelength (358 nm). Independent volume measurements were done using calcein, a fluorescent probe insensitive to intracellular ions. When challenged with ∼40% hyposmotic solutions, the cells expanded osmometrically and then underwent RVD. Concomitant with the volume response, there was a transient increase in [Ca2+]i, whose onset preceded RVD. For hyposmotic solutions (up to ∼−40%), [Ca2+]i increased steeply with the reciprocal of the external osmotic pressure and with the cell volume. Chelation of external and internal Ca2+, with EGTA and 1,2-bis-(o -aminophenoxy) ethane-N,N,N ′,N ′-tetraacetic acid (BAPTA), respectively, attenuated but did not prevent RVD. This Ca2+-independent RVD proceeded even when there was a concomitant decrease in [Ca2+]i below resting levels. Similar results were obtained in cells loaded with calcein. For cells not treated with BAPTA, restoration of external Ca2+ during the relaxation of RVD elicited by Ca2+-free hyposmotic solutions produced an increase in [Ca2+]i without affecting the rate or extent of the responses. RVD and the increase in [Ca2+]i were blocked or attenuated upon the second of two ∼40% hyposmotic challenges applied at an interval of 30–60 min. The inactivation persisted in Ca2+-free solutions. Hence, our simultaneous measurements of intracellular Ca2+ and volume in single neuroblastoma cells directly demonstrate that an increase in intracellular Ca2+ is not necessary for triggering RVD or its inactivation. The attenuation of RVD after Ca2+ chelation could occur through secondary effects or could indicate that Ca2+ is required for optimal RVD responses.
Potassium Currents in Freshly Dissociated Uterine Myocytes from Nonpregnant and Late-Pregnant Rats
Authors: Wang S.Y. , Yoshino M. , Sui J.L. , Wakui M. , Kao P.N. , Kao C.Y.
Published: July 2002

In freshly dissociated uterine myocytes, the outward current is carried by K+ through channels highly selective for K+. Typically, nonpregnant myocytes have rather noisy K+ currents; half of them also have a fast-inactivating transient outward current (ITO). In contrast, the current records are not noisy in late pregnant myocytes, and ITO densities are low. The whole-cell IK of nonpregnant myocytes respond strongly to changes in [Ca2+]o or changes in [Ca2+]i caused by photolysis of caged Ca2+ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca2+ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at −80, −40, or 0 mV and digital subtractions, the whole-cell IK of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately −61 and −22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca2+-activated K+ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca2+-sensitive K+ channels also have a half-open probability at 68 mV, and are less sensitive to Ca2+ than similar channels in taenia coli myocytes. Ca2+-activated K+ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30–35% of the total IK in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute ∼20% of total IK in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K+ currents that include a suppression of the ITO, a redistribution of IK expression from large-conductance Ca2+-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca2+ sensitivity, and a positive shift of the activation of some large-conductance Ca2+-activated channels.
Radial Localization of Inositol 1,4,5-Trisphosphate–sensitive Ca2+ Release Sites in Xenopus Oocytes Resolved by Axial Confocal Linescan Imaging
Authors: Callamaras Nick , Parker Ian
Published: July 2002

The radial localization and properties of elementary calcium release events (“puffs”) were studied in Xenopus oocytes using a confocal microscope equipped with a piezoelectric focussing unit to allow rapid (>100 Hz) imaging of calcium signals along a radial line into the cell with a spatial resolution of <0.7 μm. Weak photorelease of caged inositol 1,4,5-trisphosphate (InsP3) evoked puffs arising predominantly within a 6-μm thick band located within a few micrometers of the cell surface. Approximately 25% of puffs had a restricted radial spread, consistent with calcium release from a single site. Most puffs, however, exhibited a greater radial spread (3.25 μm), likely involving recruitment of radially neighboring release sites. Calcium waves evoked by just suprathreshold stimuli exhibited radial calcium distributions consistent with inward diffusion of calcium liberated at puff sites, whereas stronger flashes evoked strong, short-latency signals at depths inward from puff sites, indicating deep InsP3-sensitive stores activated at higher concentrations of InsP3. Immunolocalization of InsP3 receptors showed punctate staining throughout a region corresponding to the localization of puffs and subplasmalemmal endoplasmic reticulum. The radial organization of puff sites a few micrometers inward from the plasma membrane may have important consequences for activation of calcium-dependent ion channels and “capacitative” calcium influx. However, on the macroscopic (hundreds of micrometers) scale of global calcium waves, release can be considered to occur primarily within a thin, essentially two-dimensional subplasmalemmal shell.
ATP Dependence of the ICl, swell Channel Varies with Rate of Cell Swelling
Authors: Bond Tamara , Basavappa Srisaila , Christensen Michael , Strange Kevin
Published: July 2002

Swelling-induced activation of the outwardly rectifying anion current, ICl, swell, is modulated by intracellular ATP. The mechanisms by which ATP controls channel activation, however, are unknown. Whole cell patch clamp was employed to begin addressing this issue. Endogenous ATP production was inhibited by dialyzing N1E115 neuroblastoma cells for 4–5 min with solutions containing (μM): 40 oligomycin, 5 iodoacetate, and 20 rotenone. The effect of ATP on current activation was observed in the absence of intracellular Mg2+, in cells exposed to extracellular metabolic inhibitors for 25–35 min followed by intracellular dialysis with oligomycin, iodoacetate, and rotenone, after substitution of ATP with the nonhydrolyzable analogue AMP-PNP, and in the presence of AMP-PNP and alkaline phosphatase to dephosphorylate intracellular proteins. These results demonstrate that the ATP dependence of the channel requires ATP binding rather than hydrolysis and/or phosphorylation reactions. When cells were swollen at 15–55%/min in the absence of intracellular ATP, current activation was slow (0.3–0.8 pA/pF per min). ATP concentration increased the rate of current activation up to maximal values of 4–6 pA/pF per min, but had no effect on the sensitivity of the channel to cell swelling. Rate of current activation was a saturable, hyperbolic function of ATP concentration. The EC50 for ATP varied inversely with the rate of cell swelling. Activation of current was rapid (4–6 pA/pF per min) in the absence of ATP when cells were swollen at rates ≥65%/min. Intracellular ATP concentration had no effect on current activation induced by high rates of swelling. Current activation was transient when endogenous ATP was dialyzed out of the cytoplasm of cells swollen at 15%/min. Rundown of the current was reversed by increasing the rate of swelling to 65%/min. These results indicate that the channel and/or associated regulatory proteins are capable of sensing the rate of cell volume increase. We suggest that channel activation occurs via ATP-dependent and -independent mechanisms. Increasing the rate of cell swelling appears to increase the proportion of channels activating via the ATP-independent pathway. These findings have important physiological implications for understanding ICl, swell regulation, the mechanisms by which cells sense volume changes, and volume homeostasis under conditions where cell metabolism is compromised.
Potassium-dependent Changes in the Conformation of the Kv2.1 Potassium Channel Pore
Authors: Immke David , Wood Michael , Kiss Laszlo , Korn Stephen J.
Published: July 2002

The voltage-gated K+ channel, Kv2.1, conducts Na+ in the absence of K+. External tetraethylammonium (TEAo) blocks K+ currents through Kv2.1 with an IC50 of 5 mM, but is completely without effect in the absence of K+. TEAo block can be titrated back upon addition of low [K+]. This suggested that the Kv2.1 pore undergoes a cation-dependent conformational rearrangement in the external vestibule. Individual mutation of lysine (Lys) 356 and 382 in the outer vestibule, to a glycine and a valine, respectively, increased TEAo potency for block of K+ currents by a half log unit. Mutation of Lys 356, which is located at the outer edge of the external vestibule, significantly restored TEAo block in the absence of K+ (IC50 = 21 mM). In contrast, mutation of Lys 382, which is located in the outer vestibule near the TEA binding site, resulted in very weak (extrapolated IC50 = ∼265 mM) TEAo block in the absence of K+. These data suggest that the cation-dependent alteration in pore conformation that resulted in loss of TEA potency extended to the outer edge of the external vestibule, and primarily involved a repositioning of Lys 356 or a nearby amino acid in the conduction pathway. Block by internal TEA also completely disappeared in the absence of K+, and could be titrated back with low [K+]. Both internal and external TEA potencies were increased by the same low [K+] (30–100 μM) that blocked Na+ currents through the channel. In addition, experiments that combined block by internal and external TEA indicated that the site of K+ action was between the internal and external TEA binding sites. These data indicate that a K+-dependent conformational change also occurs internal to the selectivity filter, and that both internal and external conformational rearrangements resulted from differences in K+ occupancy of the selectivity filter. Kv2.1 inactivation rate was K+ dependent and correlated with TEAo potency; as [K+] was raised, TEAo became more potent and inactivation became faster. Both TEAo potency and inactivation rate saturated at the same [K+]. These results suggest that the rate of slow inactivation in Kv2.1 was influenced by the conformational rearrangements, either internal to the selectivity filter or near the outer edge of the external vestibule, that were associated with differences in TEA potency.
Modulation of N-Type Calcium Channel Activity by G-Proteins and Protein Kinase C
Authors: Barrett Curtis F. , Rittenhouse Ann R.
Published: July 2002

N-type voltage-gated calcium channel activity in rat superior cervical ganglion neurons is modulated by a variety of pathways. Activation of heterotrimeric G-proteins reduces whole-cell current amplitude, whereas phosphorylation by protein kinase C leads to an increase in current amplitude. It has been proposed that these two distinct pathways converge on the channel's pore-forming α1B subunit, such that the actions of one pathway can preclude those of the other. In this study, we have characterized further the actions of PKC on whole-cell barium currents in neonatal rat superior cervical ganglion neurons. We first examined whether the effects of G-protein–mediated inhibition and phosphorylation by PKC are mutually exclusive. G-proteins were activated by including 0.4 mM GTP or 0.1 mM GTP-γ-S in the pipette, and PKC was activated by bath application of 500 nM phorbol 12-myristate 13-acetate (PMA). We found that activated PKC was unable to reverse GTP-γ-S–induced inhibition unless prepulses were applied, indicating that reversal of inhibition by phosphorylation appears to occur only after dissociation of the G-protein from the channel. Once inhibition was relieved, activation of PKC was sufficient to prevent reinhibition of current by G-proteins, indicating that under phosphorylating conditions, channels are resistant to G-protein–mediated modulation. We then examined what effect, if any, phosphorylation by PKC has on N-type barium currents beyond antagonizing G-protein–mediated inhibition. We found that, although G-protein activation significantly affected peak current amplitude, fast inactivation, holding-potential–dependent inactivation, and voltage-dependent activation, when G-protein activation was minimized by dialysis of the cytoplasm with 0.1 mM GDP-β-S, these parameters were not affected by bath application of PMA. These results indicate that, under our recording conditions, phosphorylation by PKC has no effect on whole-cell N-type currents, other than preventing inhibition by G-proteins.
A Cl− Cotransporter Selective for Nh4+ over K+ in Glial Cells of Bee Retina
Authors: Marcaggi Païkan , Coles Jonathan A.
Published: July 2002

There appears to be a flux of ammonium (NH4+/NH3) from neurons to glial cells in most nervous tissues. In bee retinal glial cells, NH4+/NH3 uptake is at least partly by chloride-dependant transport of the ionic form NH4+. Transmembrane transport of NH4+ has been described previously on transporters on which NH4+ replaces K+, or, more rarely, Na+ or H+, but no transport system in animal cells has been shown to be selective for NH4+ over these other ions. To see if the NH4+-Cl− cotransporter on bee retinal glial cells is selective for NH4+ over K+ we measured ammonium-induced changes in intracellular pH (pHi) in isolated bundles of glial cells using a fluorescent indicator. These changes in pHi result from transmembrane fluxes not only of NH4+, but also of NH3. To estimate transmembrane fluxes of NH4+, it was necessary to measure several parameters. Intracellular pH buffering power was found to be 12 mM. Regulatory mechanisms tended to restore intracellular [H+] after its displacement with a time constant of 3 min. Membrane permeability to NH3 was 13 μm s−1. A numerical model was used to deduce the NH4+ flux through the transporter that would account for the pHi changes induced by a 30-s application of ammonium. This flux saturated with increasing [NH4+]o; the relation was fitted with a Michaelis-Menten equation with Km ≈ 7 mM. The inhibition of NH4+ flux by extracellular K+ appeared to be competitive, with an apparent Ki of ∼15 mM. A simple standard model of the transport process satisfactorily described the pHi changes caused by various experimental manipulations when the transporter bound NH4+ with greater affinity than K+. We conclude that this transporter is functionally selective for NH4+ over K+ and that the transporter molecule probably has a greater affinity for NH4+ than for K+.
A Point Mutation in the Pore Region Alters Gating, Ca2+Blockage, and Permeation of Olfactory Cyclic Nucleotide–Gated Channels
Authors: Gavazzo Paola , Picco Cristiana , Eismann Elisabeth , Kaupp U. Benjamin , Menini Anna
Published: July 2002

Upon stimulation by odorants, Ca2+ and Na+ enter the cilia of olfactory sensory neurons through channels directly gated by cAMP. Cyclic nucleotide–gated channels have been found in a variety of cells and extensively investigated in the past few years. Glutamate residues at position 363 of the α subunit of the bovine retinal rod channel have previously been shown to constitute a cation-binding site important for blockage by external divalent cations and to control single-channel properties. It has therefore been assumed, but not proven, that glutamate residues at the corresponding position of the other cyclic nucleotide–gated channels play a similar role. We studied the corresponding glutamate (E340) of the α subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca2+ and Mg2+. E340 was mutated into either an aspartate, glycine, glutamine, or asparagine residue and properties of mutant channels expressed in Xenopus laevis oocytes were measured in excised patches. By single-channel recordings, we demonstrated that the open probabilities in the presence of cGMP or cAMP were decreased by the mutations, with a larger decrease observed on gating by cAMP. Moreover, we observed that the mutant E340N presented two conductance levels. We found that both external Ca2+ and Mg2+ powerfully blocked the current in wild-type and E340D mutants, whereas their blockage efficacy was drastically reduced when the glutamate charge was neutralized. The inward current carried by external Ca2+ relative to Na+ was larger in the E340G mutant compared with wild-type channels. In conclusion, we have confirmed that the residue at position E340 of the bovine olfactory CNG channel is in the pore region, controls permeation and blockage by external Ca2+ and Mg2+, and affects channel gating by cAMP more than by cGMP.
Pore Block versus Intrinsic Gating in the Mechanism of Inward Rectification in Strongly Rectifying Irk1 Channels
Authors: Guo Donglin , Lu Zhe
Published: July 2002

The IRK1 channel is inhibited by intracellular cations such as Mg2+ and polyamines in a voltage-dependent manner, which renders its I-V curve strongly inwardly rectifying. However, even in excised patches exhaustively perfused with a commonly used artificial intracellular solution nominally free of Mg2+ and polyamines, the macroscopic I-V curve of the channels displays modest rectification. This observation forms the basis of a hypothesis, alternative to the pore-blocking hypothesis, that inward rectification reflects the enhancement of intrinsic channel gating by intracellular cations. We find, however, that residual rectification is caused primarily by the commonly used pH buffer HEPES and/or some accompanying impurity. Therefore, inward rectification in the strong rectifier IRK1, as in the weak rectifier ROMK1, can be accounted for by voltage-dependent block of its ion conduction pore by intracellular cations.