bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleolar Assembly of the Rrna Processing Machinery in Living Cells
Authors: Savino Tulia Maria , Gébrane-Younès Jeannine , De Mey Jan , Sibarita Jean-Baptiste , Hernandez-Verdun Danièle
Published: July 2002

To understand how nuclear machineries are targeted to accurate locations during nuclear assembly, we investigated the pathway of the ribosomal RNA (rRNA) processing machinery towards ribosomal genes (nucleolar organizer regions [NORs]) at exit of mitosis. To follow in living cells two permanently transfected green fluorescence protein–tagged nucleolar proteins, fibrillarin and Nop52, from metaphase to G1, 4-D time-lapse microscopy was used. In early telophase, fibrillarin is concentrated simultaneously in prenucleolar bodies (PNBs) and NORs, whereas PNB-containing Nop52 forms later. These distinct PNBs assemble at the chromosome surface. Analysis of PNB movement does not reveal the migration of PNBs towards the nucleolus, but rather a directional flow between PNBs and between PNBs and the nucleolus, ensuring progressive delivery of proteins into nucleoli. This delivery appeared organized in morphologically distinct structures visible by electron microscopy, suggesting transfer of large complexes. We propose that the temporal order of PNB assembly and disassembly controls nucleolar delivery of these proteins, and that accumulation of processing complexes in the nucleolus is driven by pre-rRNA concentration. Initial nucleolar formation around competent NORs appears to be followed by regroupment of the NORs into a single nucleolus 1 h later to complete the nucleolar assembly. This demonstrates the formation of one functional domain by cooperative interactions between different chromosome territories.
A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly
Authors: Sechler Jan L. , Rao Hongwei , Cumiskey Anne Marie , Vega-Colón Irbert , Smith Michael S. , Murata Takatoshi , Schwarzbauer Jean E.
Published: July 2002

Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1–7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNΔIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9–10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4–7, had no effect on assembly. In contrast, two deletions that included repeat III2, ΔIII1–2 and ΔIII2–5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN–FN interactions during fibril growth.
Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells
Authors: Haller Thomas , Dietl Paul , Pfaller Kristian , Frick Manfred , Mair Norbert , Paulmichl Markus , Hess Michael W. , Fürst Johannes , Maly Karl
Published: July 2002

In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579–1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Fick's law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation.
Kettin, a major source of myofibrillar stiffness in Drosophila indirect flight muscle
Authors: Kulke Michael , Neagoe Ciprian , Kolmerer Bernhard , Minajeva Ave , Hinssen Horst , Bullard Belinda , Linke Wolfgang A.
Published: July 2002

Kettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and α-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery. After extraction of the kettin-associated actin, the A-band edges were also stained. In contrast, the staining pattern of projectin, another IFM–I-band protein, was not altered by actin removal. Force measurements were performed on single IFM myofibrils to establish the passive length-tension relationship and record passive stiffness. Stiffness decreased within seconds during gelsolin incubation and to a similar degree upon kettin digestion with μ-calpain. Immunoblotting demonstrated the presence of kettin isoforms in normal Drosophila IFM myofibrils and in myofibrils from an actin-null mutant. Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin. We conclude that kettin is attached not only to actin but also to the end of the thick filament. Kettin along with projectin may constitute the elastic filament system of insect IFM and determine the muscle's high stiffness necessary for stretch activation. Possibly, the two proteins modulate myofibrillar stiffness by expressing different size isoforms.
A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast
Authors: Pavlov Evgeny V. , Priault Muriel , Pietkiewicz Dawn , Cheng Emily H.-Y. , Antonsson Bruno , Manon Stephen , Korsmeyer Stanley J. , Mannella Carmen A. , Kinnally Kathleen W.
Published: July 2002

During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is ∼4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis–induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.
Thin filaments elongate from their pointed ends during myofibril assembly in Drosophila indirect flight muscle
Authors: Mardahl-Dumesnil Michelle , Fowler Velia M.
Published: July 2002

Tropomodulin (Tmod) is an actin pointed-end capping protein that regulates actin dynamics at thin filament pointed ends in striated muscle. Although pointed-end capping by Tmod controls thin filament lengths in assembled myofibrils, its role in length specification during de novo myofibril assembly is not established. We used the Drosophila Tmod homologue, sanpodo (spdo), to investigate Tmod's function during muscle development in the indirect flight muscle. SPDO was associated with the pointed ends of elongating thin filaments throughout myofibril assembly. Transient overexpression of SPDO during myofibril assembly irreversibly arrested elongation of preexisting thin filaments. However, the lengths of thin filaments assembled after SPDO levels had declined were normal. Flies with a preponderance of abnormally short thin filaments were unable to fly. We conclude that: (a) thin filaments elongate from their pointed ends during myofibril assembly; (b) pointed ends are dynamically capped at endogenous levels of SPDO so as to allow elongation; (c) a transient increase in SPDO levels during myofibril assembly converts SPDO from a dynamic to a permanent cap; and (d) developmental regulation of pointed-end capping during myofibril assembly is crucial for specification of final thin filament lengths, myofibril structure, and muscle function.
A new role for BiP
Authors: Haigh Nora G. , Johnson Arthur E.
Published: July 2002

In mammalian cells, most membrane proteins are inserted cotranslationally into the ER membrane at sites termed translocons. Although each translocon forms an aqueous pore, the permeability barrier of the membrane is maintained during integration, even when the otherwise tight ribosome–translocon seal is opened to allow the cytoplasmic domain of a nascent protein to enter the cytosol. To identify the mechanism by which membrane integrity is preserved, nascent chain exposure to each side of the membrane was determined at different stages of integration by collisional quenching of a fluorescent probe in the nascent chain. Comparing integration intermediates prepared with intact, empty, or BiP-loaded microsomes revealed that the lumenal end of the translocon pore is closed by BiP in an ATP-dependent process before the opening of the cytoplasmic ribosome–translocon seal during integration. This BiP function is distinct from its previously identified role in closing ribosome-free, empty translocons because of the presence of the ribosome at the translocon and the nascent membrane protein that extends through the translocon pore and into the lumen during integration. Therefore, BiP is a key component in a sophisticated mechanism that selectively closes the lumenal end of some, but not all, translocons occupied by a nascent chain. By using collisional quenchers of different sizes, the large internal diameter of the ribosome-bound aqueous translocon pore was found to contract when BiP was required to seal the pore during integration. Therefore, closure of the pore involves substantial conformational changes in the translocon that are coupled to a complex sequence of structural rearrangements on both sides of the ER membrane involving the ribosome and BiP.
The utrophin actin-binding domain binds F-actin in two different modes
Authors: Galkin Vitold E. , Orlova Albina , VanLoock Margaret S. , Rybakova Inna N. , Ervasti James M. , Egelman Edward H.
Published: July 2002

Utrophin, like its homologue dystrophin, forms a link between the actin cytoskeleton and the extracellular matrix. We have used a new method of image analysis to reconstruct actin filaments decorated with the actin-binding domain of utrophin, which contains two calponin homology domains. We find two different modes of binding, with either one or two calponin-homology (CH) domains bound per actin subunit, and these modes are also distinguishable by their very different effects on F-actin rigidity. Both modes involve an extended conformation of the CH domains, as predicted by a previous crystal structure. The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments. When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin. The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.
Activated K-Ras and H-Ras display different interactions with saturable nonraft sites at the surface of live cells
Authors: Niv Hagit , Gutman Orit , Kloog Yoel , Henis Yoav I.
Published: July 2002

Ras–membrane interactions play important roles in signaling and oncogenesis. H-Ras and K-Ras have nonidentical membrane anchoring moieties that can direct them to different membrane compartments. Ras–lipid raft interactions were reported, but recent studies suggest that activated K-Ras and H-Ras are not raft resident. However, specific interactions of activated Ras proteins with nonraft sites, which may underlie functional differences and phenotypic variation between different Ras isoforms, are unexplored. Here we used lateral mobility studies by FRAP to investigate the membrane interactions of green fluorescent protein–tagged H- and K-Ras in live cells. All Ras isoforms displayed stable membrane association, moving by lateral diffusion and not by exchange with a cytoplasmic pool. The lateral diffusion rates of constitutively active K- and H-Ras increased with their expression levels in a saturable manner, suggesting dynamic association with saturable sites or domains. These sites are distinct from lipid rafts, as the activated Ras mutants are not raft resident. Moreover, they appear to be different for H- and K-Ras. However, wild-type H-Ras, the only isoform preferentially localized in rafts, displayed cholesterol-sensitive interactions with rafts that were independent of its expression level. Our findings provide a mechanism for selective signaling by different Ras isoforms.
Characterization of a mammalian Golgi-localized protein complex, COG, that is required for normal Golgi morphology and function
Authors: Ungar Daniel , Oka Toshihiko , Brittle Elizabeth E. , Vasile Eliza , Lupashin Vladimir V. , Chatterton Jon E. , Heuser John E. , Krieger Monty , Waters M. Gerard
Published: July 2002

Multiprotein complexes are key determinants of Golgi apparatus structure and its capacity for intracellular transport and glycoprotein modification. Three complexes that have previously been partially characterized include (a) the Golgi transport complex (GTC), identified in an in vitro membrane transport assay, (b) the ldlCp complex, identified in analyses of CHO cell mutants with defects in Golgi-associated glycosylation reactions, and (c) the mammalian Sec34 complex, identified by homology to yeast Sec34p, implicated in vesicular transport. We show that these three complexes are identical and rename them the conserved oligomeric Golgi (COG) complex. The COG complex comprises four previously characterized proteins (Cog1/ldlBp, Cog2/ldlCp, Cog3/Sec34, and Cog5/GTC-90), three homologues of yeast Sec34/35 complex subunits (Cog4, -6, and -8), and a previously unidentified Golgi-associated protein (Cog7). EM of ldlB and ldlC mutants established that COG is required for normal Golgi morphology. “Deep etch” EM of purified COG revealed an ∼37-nm-long structure comprised of two similarly sized globular domains connected by smaller extensions. Consideration of biochemical and genetic data for mammalian COG and its yeast homologue suggests a model for the subunit distribution within this complex, which plays critical roles in Golgi structure and function.
Tumor Necrosis Factor α and Lymphotoxin α Are Not Required for Induction of Acute Experimental Autoimmune Encephalomyelitis
Authors: Frei Karl , Eugster Hans-Pietro , Bopst Martin , Constantinescu Cris S. , Lavi Ehud , Fontana Adriano
Published: July 2002

Immunization of mice with myelin components results in experimental autoimmune encephalomyelitis (EAE), which is mediated by myelin-specific CD4+ T cells and anti-myelin antibodies. Tumor necrosis factor α (TNF-α) and lymphotoxin α (LT-α) are thought to be involved in the events leading to inflammatory demyelination in the central nervous system. To ascertain this hypothesis 129 × C57BL/6 mice with an inactivation of the tnf and lta genes (129 × C57BL/6−/−) and SJL/J mice derived from backcrosses of the above mentioned mutant mice (SJL−/−) were immunized with mouse spinal cord homogenate (MSCH) or proteolipid protein. Both 129 × C57BL/6−/− mice and SJL−/− mice developed EAE. In SJL−/− mice immunized with MSCH, a very severe form of EAE with weight loss, paralysis of all four limbs, and lethal outcome was observed. The histologic hallmark was an intense perivascular and parenchymal infiltration with predominantly CD4+ T cells and some CD8+ T cells associated with demyelination in both brain and spinal cord. These results indicate that TNF-α and LT-α are not essential for the development of EAE.
Identification of Amino Acid Residues Important for Ligand Binding to Fas
Authors: Starling Gary C. , Bajorath Jürgen , Emswiler John , Ledbetter Jeffrey A. , Aruffo Alejandro , Kiener Peter A.
Published: July 2002

The interaction of Fas (CD95), a member of the tumor necrosis factor receptor (TNFR) family, and its ligand (FasL) triggers programmed cell death (apoptosis) and is involved in the regulation of immune responses. Although the Fas–FasL interaction is conserved across species barriers, little is currently known about the molecular details of this interaction. Our aim was to identify residues in Fas that are important for ligand binding. With the aid of a Fas molecular model, candidate amino acid residues were selected in the Fas extracellular domain 2 (D2) and D3 and subjected to serine-scanning mutagenesis to produce mutant Fas molecules in the form of Ig fusion proteins. The effects of these mutations on FasL binding was examined by measuring the ability of these proteins to inhibit FasL-mediated apoptosis of Jurkat cells and bind FasL in ELISA and BIAcore™ assays. Mutation of two amino acids, R86 and R87 (D2), to serine totally abolished the ability of Fas to interact with its ligand, whereas mutants K84S, L90S, E93S (D2), or H126S (D3) showed reduced binding compared with wild-type Fas. Two mutants (K78S and H95S) bound FasL comparably to wild type. Therefore, the binding of FasL involves residues in two domains that correspond to positions critical for ligand binding in other family members (TNFR and CD40) but are conserved between murine and human Fas.
DNA as an Adjuvant: Capacity of Insect DNA and Synthetic Oligodeoxynucleotides to Augment T Cell Responses to Specific Antigen
Authors: Sun Siquan , Kishimoto Hidehiro , Sprent Jonathan
Published: July 2002

How strong adjuvants such as complete Freund's adjuvant (CFA) promote T cell priming to protein antigens in vivo is still unclear. Since the unmethylated CpG motifs in DNA of bacteria and other nonvertebrates are stimulatory for B cells and antigen-presenting cells, the strong adjuvanticity of CFA could be attributed, at least in part, to the presence of dead bacteria, i.e., a source of stimulatory DNA. In support of this possibility, evidence is presented that insect DNA in mineral oil has even stronger adjuvant activity than CFA by a number of parameters. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs mimic the effects of insect DNA and, even in soluble form, ODNs markedly potentiate clonal expansion of T cell receptor transgenic T cells responding to specific peptide.
Germinal Centers without T Cells
Authors: de Vinuesa Carola García , Cook Matthew C. , Ball Jennifer , Drew Marion , Sunners Yvonne , Cascalho Marilia , Wabl Matthias , Klaus Gerry G.B. , MacLennan Ian C.M.
Published: July 2002

Germinal centers are critical for affinity maturation of antibody (Ab) responses. This process allows the production of high-efficiency neutralizing Ab that protects against virus infection and bacterial exotoxins. In germinal centers, responding B cells selectively mutate the genes that encode their receptors for antigen. This process can change Ab affinity and specificity. The mutated cells that produce high-affinity Ab are selected to become Ab-forming or memory B cells, whereas cells that have lost affinity or acquired autoreactivity are eliminated. Normally, T cells are critical for germinal center formation and subsequent B cell selection. Both processes involve engagement of CD40 on B cells by T cells. This report describes how high-affinity B cells can be induced to form large germinal centers in response to (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll in the absence of T cells or signaling through CD40 or CD28. This requires extensive cross-linking of the B cell receptors, and a frequency of antigen-specific B cells of at least 1 in 1,000. These germinal centers abort dramatically at the time when mutated high-affinity B cells are normally selected by T cells. Thus, there is a fail-safe mechanism against autoreactivity, even in the event of thymus-independent germinal center formation.
Hematopoietic Stem Cells Need Two Signals to Prevent Apoptosis; Bcl-2 Can Provide One of These, Kitl/C-KIT Signaling the Other
Authors: Domen Jos , Weissman Irving L.
Published: July 2002

Growth factors can cause cells to proliferate, differentiate, survive, or die. Distinguishing between these responses is difficult in multicellular, multiparameter systems. Yet this is essential to understand the impact on cells like hematopoietic stem cells (HSCs), which have strict and still poorly understood growth factor requirements. Single cell plating in serum-free medium allows direct assessment of growth factor responses. The range of tested factors can be expanded if the cells are protected from growth factor deprivation–induced apoptosis. BCL-2 is overexpressed in HSCs of H2K-BCL-2 transgenic mice, protecting them from many apoptotic stimuli. The response of single wild-type and transgenic HSCs to stimulations with individual factors was tested. Surprisingly, we find that high level BCL-2 expression does not prevent rapid death under serum-free conditions, even though it does in the presence of serum. We also find that transgenic, but not wild-type cells, survive and proliferate rapidly in response to steel factor (Kit ligand). These studies show that two separate signals are necessary to prevent apoptosis in HSCs, and that Kit ligand by itself provides a strong proliferative stimulus to HSCs. However, the proliferative response does not result in self-renewal, but in differentiation to all known hematopoietic oligolineage progenitors.
Thymic Selection Generates a Large T Cell Pool Recognizing a Self-Peptide in Humans
Authors: Zippelius Alfred , Pittet Mikaël J. , Batard Pascal , Rufer Nathalie , de Smedt Magda , Guillaume Philippe , Ellefsen Kim , Valmori Danila , Liénard Danielle , Plum Jean , MacDonald H. Robson , Speiser Daniel E. , Cerottini Jean-Charles , Romero Pedro
Published: July 2002

The low frequency of self-peptide–specific T cells in the human preimmune repertoire has so far precluded their direct evaluation. Here, we report an unexpected high frequency of T cells specific for the self-antigen Melan-A/MART-1 in CD8 single–positive thymocytes from human histocompatibility leukocyte antigen-A2 healthy individuals, which is maintained in the peripheral blood of newborns and adults. Postthymic replicative history of Melan-A/MART-1–specific CD8 T cells was independently assessed by quantifying T cell receptor excision circles and telomere length ex vivo. We provide direct evidence that the large T cell pool specific for the self-antigen Melan-A/MART-1 is mostly generated by thymic output of a high number of precursors. This represents the only known naive self-peptide–specific T cell repertoire directly accessible in humans.
Single-Channel Kinetics, Inactivation, and Spatial Distribution of Inositol Trisphosphate (IP3) Receptors in Xenopus Oocyte Nucleus
Authors: Mak Don-On Daniel , Foskett J. Kevin
Published: July 2002

Single-channel properties of the Xenopus inositol trisphosphate receptor (IP3R) ion channel were examined by patch clamp electrophysiology of the outer nuclear membrane of isolated oocyte nuclei. With 140 mM K+ as the charge carrier (cytoplasmic [IP3] = 10 μM, free [Ca2+] = 200 nM), the IP3R exhibited four and possibly five conductance states. The conductance of the most-frequently observed state M was 113 pS around 0 mV and ∼300 pS at 60 mV. The channel was frequently observed with high open probability (mean Po = 0.4 at 20 mV). Dwell time distribution analysis revealed at least two kinetic states of M with time constants τ < 5 ms and ∼20 ms; and at least three closed states with τ ∼1 ms, ∼10 ms, and >1 s. Higher cytoplasmic potential increased the relative frequency and τ of the longest closed state. A novel “flicker” kinetic mode was observed, in which the channel alternated rapidly between two new conductance states: F1 and F2. The relative occupation probability of the flicker states exhibited voltage dependence described by a Boltzmann distribution corresponding to 1.33 electron charges moving across the entire electric field during F1 to F2 transitions. Channel run-down or inactivation (τ ∼ 30 s) was consistently observed in the continuous presence of IP3 and the absence of change in [Ca2+]. Some (∼10%) channel disappearances could be reversed by an increase in voltage before irreversible inactivation. A model for voltage-dependent channel gating is proposed in which one mechanism controls channel opening in both the normal and flicker modes, whereas a separate independent mechanism generates flicker activity and voltage- reversible inactivation. Mapping of functional channels indicates that the IP3R tends to aggregate into microscopic (<1 μm) as well as macroscopic (∼10 μm) clusters. Ca2+-independent inactivation of IP3R and channel clustering may contribute to complex [Ca2+] signals in cells.
Role of Transmembrane Segment S5 on Gating of Voltage-dependent K+ Channels
Authors: Shieh Char-Chang , Klemic Kathryn G. , Kirsch Glenn E.
Published: July 2002

The cytoplasmic half of S5 (5′S5) has been identified as part of the inner mouth of the pore based on evidence that mutations in this region greatly alter single channel conductance, 4-aminopyridine (4-AP) block and the rate of channel closing upon repolarization (deactivation). The latter effect, suggestive of a role for 5′S5 in channel gating was investigated in the present study. The biophysical properties of chimeric channels, in which the 5′S5 regions were exchanged between two host channels (Kv2.1 and Kv3.1) that differ in 4-AP sensitivity and deactivation rate, were examined in a Xenopus oocyte expression system. Exchange of 5′S5 between Kv2.1 and Kv3.1 confers steady-state voltage dependence of activation and rates of channel deactivation similar to those of the donor channel. The involvement of voltage-dependent gating was confirmed by the observation that exchanging the 5′S5 segment of Kv2.1 with that of Kv3.1 confers a change from slow to fast deactivation kinetics by accelerating the decay of off-gating charge movement. We suggest that a conformational change that extends from the voltage-sensor in S4 to the region of the pore lined by S5 regulates the stability of the open state. Therefore, the cytoplasmic end of S5, in addition to forming part of the conduction pathway near the inner mouth of the pore, also participates in the conformational rearrangements associated with late steps in channel activation and early steps in deactivation.
Selectivity Changes during Activation of Mutant Shaker Potassium Channels
Authors: Zheng Jie , Sigworth Fred J.
Published: July 2002

Mutations of the pore-region residue T442 in Shaker channels result in large effects on channel kinetics. We studied mutations at this position in the backgrounds of NH2-terminal–truncated Shaker H4 and a Shaker -NGK2 chimeric channel having high conductance (Lopez, G.A., Y.N. Jan, and L.Y. Jan. 1994. Nature (Lond.). 367: 179–182). While mutations of T442 to C, D, H, V, or Y resulted in undetectable expression in Xenopus oocytes, S and G mutants yielded functional channels having deactivation time constants and channel open times two to three orders of magnitude longer than those of the parental channel. Activation time courses at depolarized potentials were unaffected by the mutations, as were first-latency distributions in the T442S chimeric channel. The mutant channels show two subconductance levels, 37 and 70% of full conductance. From single-channel analysis, we concluded that channels always pass through the larger subconductance state on the way to and from the open state. The smaller subconductance state is traversed in ∼40% of activation time courses. These states apparently represent kinetic intermediates in channel gating having voltage-dependent transitions with apparent charge movements of ∼1.6 e0. The fully open T442S chimeric channel has the conductance sequence Rb+ > NH4+ > K+. The opposite conductance sequence, K+ > NH4+ > Rb+, is observed in each of the subconductance states, with the smaller subconductance state discriminating most strongly against Rb+.
Sodium Kinetics of Na,K-ATPase α Isoforms in Intact Transfected HeLa Cells
Authors: Zahler Raphael , Zhang Zhong-Ting , Manor Mira , Boron Walter F.
Published: July 2002

By participating in the regulation of ion and voltage gradients, the Na-K pump (i.e., Na,K-ATPase) influences many aspects of cellular physiology. Of the four α isoforms of the pump, α1 is ubiquitous, α2 is predominant in skeletal muscle, and α3 is found in neurons and the cardiac conduction system. To determine whether the isoforms have different intracellular Na+ affinities, we used the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI) to measure pump-mediated Na+ efflux as a function of [Na+]i in human HeLa cells stably transfected with rat Na-K pump isoforms. We Na+-loaded the cells, and then monitored the time course of the decrease in [Na+]i after removing external Na+. All transfected rat α subunits were highly ouabain resistant: the α1 isoform is naturally resistant, whereas the α2 and α3 isoforms had been mutagenized to render them resistant. Thus, the Na+ efflux mediated by endogenous and transfected pumps could be separated by studying the cells at low (1 μM) and high (4 mM) ouabain concentrations. We found that the apparent Km for Na+ efflux attributable to the native human α1 isoform was 12 mM, which was similar to the Km of rat α1. The α2 and α3 isoforms had apparent Km's of 22 and 33 mM, respectively. The cells expressing α3 had a high resting [Na+]i. The maximal activity of native α1 in the α3-transfected cells was only ∼56% of native α1 activity in untransfected HeLa cells, suggesting that transfection with α3 led to a compensatory decrease in endogenous α1 pumps. We conclude that the apparent Km(Na+) for rat Na-K pump isoforms increases in the sequence α1 < α2 < α3. The α3 isoform may be suited for handling large Na+ loads in electrically active cells.
Molecular Analysis of ATP-sensitive K Channel Gating and Implications for Channel Inhibition by ATP
Authors: Trapp Stefan , Proks Peter , Tucker Stephen J. , Ashcroft Frances M.
Published: July 2002

The β cell KATP channel is an octameric complex of four pore-forming subunits (Kir6.2) and four regulatory subunits (SUR1). A truncated isoform of Kir6.2 (Kir6.2ΔC26), which expresses independently of SUR1, shows intrinsic ATP sensitivity, suggesting that this subunit is primarily responsible for mediating ATP inhibition. We show here that mutation of C166, which lies at the cytosolic end of the second transmembrane domain, to serine (C166S) increases the open probability of Kir6.2ΔC26 approximately sevenfold by reducing the time the channel spends in a long closed state. Rundown of channel activity is also decreased. Kir6.2ΔC26 containing the C166S mutation shows a markedly reduced ATP sensitivity: the Ki is reduced from 175 μM to 2.8 mM. Substitution of threonine, alanine, methionine, or phenylalanine at position C166 also reduced the channel sensitivity to ATP and simultaneously increased the open probability. Thus, ATP does not act as an open channel blocker. The inhibitory effects of tolbutamide are reduced in channels composed of SUR1 and Kir6.2 carrying the C166S mutation. Our results are consistent with the idea that C166 plays a role in the intrinsic gating of the channel, possibly by influencing a gate located at the intracellular end of the pore. Kinetic analysis suggests that the apparent decrease in ATP sensitivity, and the changes in other properties, observed when C166 is mutated is largely a consequence of the impaired transition from the open to the long closed state.
Simultaneous Binding of Basic Peptides at Intracellular Sites on a Large Conductance Ca2+-activated K+ Channel
Authors: Favre Isabelle , Moczydlowski Edward
Published: July 2002

The homologous Kunitz inhibitor proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin I (DTX-I), interact with large conductance Ca2+-activated K+ channels (maxi-KCa) by binding to an intracellular site outside of the pore to produce discrete substate events. In contrast, certain homologues of the Shaker ball peptide produce discrete blocking events by binding within the ion conduction pathway. In this study, we investigated ligand interactions of these positively charged peptide molecules by analysis of single maxi-KCa channels in planar bilayers recorded in the presence of DTX-I and BPTI, or DTX-I and a high-affinity homologue of ball peptide. Both DTX-I (Kd, 16.5 nM) and BPTI (Kd, 1,490 nM) exhibit one-site binding kinetics when studied alone; however, records in the presence of DTX-I plus BPTI demonstrate simultaneous binding of these two molecules. The affinity of BPTI (net charge, +6) decreases by 11.7-fold (Kd, 17,500 nM) when DTX-I (net charge, +10) is bound and, conversely, the affinity of DTX-I decreases by 10.8-fold (Kd, 178 nM) when BPTI is bound. The ball peptide homologue (BP; net charge, +6) exhibits high blocking affinity (Kd, 7.2 nM) at a single site when studied alone, but has 8.0-fold lower affinity (Kd, 57 nM) for blocking the DTX-occupied channel. The affinity of DTX-I likewise decreases by 8.4-fold (Kd, 139 nM) when BP is bound. These results identify two types of negatively coupled ligand–ligand interactions at distinct sites on the intracellular surface of maxi-KCa channels. Such antagonistic ligand interactions explain how the binding of BPTI or DTX-I to four potentially available sites on a tetrameric channel protein can exhibit apparent one-site kinetics. We hypothesize that negatively coupled binding equilibria and asymmetric changes in transition state energies for the interaction between DTX-I and BP originate from repulsive electrostatic interactions between positively charged peptide ligands on the channel surface. In contrast, there is no detectable binding interaction between DTX-I on the inside and tetraethylammonium or charybdotoxin on the outside of the maxi-KCa channel.
Molecular Determinants of Electrical Rectification of Single Channel Conductance in Gap Junctions Formed by Connexins 26 and 32
Authors: Oh Seunghoon , Rubin Joshua B. , Bennett Michael V.L. , Verselis Vytas K. , Bargiello Thaddeus A.
Published: July 2002

The fully open state of heterotypic gap junction channels formed by pairing cells expressing connexin 32 (Cx32) with those expressing connexin 26 (Cx26) rectifies in a way that cannot be predicted from the current–voltage (I–V) relation of either homotypic channel. Using a molecular genetic analysis, we demonstrate that charged amino acids positioned in the amino terminus (M1 and D2) and first extracellular loop (E42) are major determinants of the current–voltage relation of the fully open state of homotypic and heterotypic channels formed by Cx26 and Cx32. The observed I–V relations of wild-type and mutant channels were closely approximated by those obtained with the electrodiffusive model of Chen and Eisenberg (Chen, D., and R. Eisenberg. 1993. Biophys. J. 64:1405–1421), which solves the Poisson-Nernst-Plank equations in one dimension using charge distribution models inferred from the molecular analyses. The rectification of the Cx32/Cx26 heterotypic channel results from the asymmetry in the number and position of charged residues. The model required the incorporation of a partial charge located near the channel surface to approximate the linear I–V relation observed for the Cx32*Cx26E1 homotypic channel. The best candidate amino acid providing this partial charge is the conserved tryptophan residue (W3). Incorporation of the partial charge of residue W3 and the negative charge of the Cx32E41 residue into the charge profile used in the Poisson-Nernst-Plank model of homotypic Cx32 and heterotypic Cx26/Cx32 channels resulted in I–V relations that closely resembled the observed I–V relations of these channels. We further demonstrate that some channel substates rectify. We suggest that the conformational changes associated with transjunctional voltage (Vj)-dependent gating to these substates involves a narrowing of the cytoplasmic entry of the channel that increases the electrostatic effect of charges in the amino terminus. The rectification that is observed in the Cx32/Cx26 heterotypic channel is similar although less steep than that reported for some rectifying electrical synapses. We propose that a similar electrostatic mechanism, which results in rectification through the open and substates of heterotypic channels, is sufficient to explain the properties of steeply rectifying electrical synapses.
The Lipid–Protein Interface of aShaker K+ Channel
Authors: Hong Kwang Hee , Miller Christopher
Published: July 2002

Tryptophan-substitution mutagenesis was applied to the first and third transmembrane segments (S1 and S3) of a Shaker-type K+ channel for the purpose of ascertaining whether these sequences are α-helical. Point mutants were examined for significant functional changes, indicated by the voltage-activation curves and gating kinetics. Helical periodicity of functional alteration was observed throughout the entire S1 segment. A similar result was obtained with the first 14 residues of S3, but this periodicity disappeared towards the extracellular side of this transmembrane sequence. In both helical stretches, tryptophan-tolerant positions are clustered on approximately half the α-helix surface, as if the sidechains are exposed to the hydrocarbon region of the lipid bilayer. These results, combined with an analogous study of S2 (Monks, S., D.J. Needleman, and C. Miller. 1999. J. Gen. Physiol. 113:415–423), locate S1, S2, and S3 on the lipid-facing periphery of Kv channels.
The Intrinsic Electrostatic Potential and the Intermediate Ring of Charge in the Acetylcholine Receptor Channel
Authors: Wilson Gary G. , Pascual Juan M. , Brooijmans Natasja , Murray Diana , Karlin Arthur
Published: July 2002

A ring of aligned glutamate residues named the intermediate ring of charge surrounds the intracellular end of the acetylcholine receptor channel and dominates cation conduction (Imoto et al. 1988). Four of the five subunits in mouse-muscle acetylcholine receptor contribute a glutamate to the ring. These glutamates were mutated to glutamine or lysine, and combinations of mutant and native subunits, yielding net ring charges of −1 to −4, were expressed in Xenopus laevis oocytes. In all complexes, the α subunit contained a Cys substituted for αThr244, three residues away from the ring glutamate αGlu241. The rate constants for the reactions of αThr244Cys with the neutral 2-hydroxyethyl-methanethiosulfonate, the positively charged 2-ammonioethyl-methanethiosulfonate, and the doubly positively charged 2-ammonioethyl-2′-ammonioethanethiosulfonate were determined from the rates of irreversible inhibition of the responses to acetylcholine. The reagents were added in the presence and absence of acetylcholine and at various transmembrane potentials, and the rate constants were extrapolated to zero transmembrane potential. The intrinsic electrostatic potential in the channel in the vicinity of the ring of charge was estimated from the ratios of the rate constants of differently charged reagents. In the acetylcholine-induced open state, this potential was −230 mV with four glutamates in the ring and increased linearly towards 0 mV by +57 mV for each negative charge removed from the ring. Thus, the intrinsic electrostatic potential in the narrow, intracellular end of the open channel is almost entirely due to the intermediate ring of charge and is strongly correlated with alkali-metal-ion conductance through the channel. The intrinsic electrostatic potential in the closed state of the channel was more positive than in the open state at all values of the ring charge. These electrostatic properties were simulated by theoretical calculations based on a simplified model of the channel.
Interaction between Quaternary Ammonium Ions in the Pore of Potassium Channels
Authors: Thompson Jill , Begenisich Ted
Published: July 2002

We have examined the interaction between internal and external ions in the pore of potassium channels. We found that external tetraethylammonium was able to antagonize block of Shaker channels by internal TEA when the external and internal solutions contained K+ ions. This antagonism was absent in solutions with Rb+ as the only permeant ion. An externally applied trivalent TEA analogue, gallamine, was less effective than the monovalent TEA in inhibiting block by internal TEA. In addition, block by external TEA was little affected by changes in the concentration of internal K+ ions, but was increased by the presence of internal Na+ ions in the pore. These results demonstrate that external and internal TEA ions, likely located at opposite ends of the pore selectivity filter, do not experience a mutual electrostatic repulsion. We found that these results can be simulated by a simple 4-barrier-3-site permeation model in which ions compete for available binding sites without long-range electrostatic interactions.
Voltage and Ca2+ Activation of Single Large-Conductance Ca2+-Activated K+ Channels Described by a Two-Tiered Allosteric Gating Mechanism
Authors: Rothberg Brad S. , Magleby Karl L.
Published: July 2002

The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (Po) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in Po; effective gating charge, qeff, of 2.3 ± 0.6 eo). Estimates of qeff were little changed for intracellular Ca2+ (Ca2+i) ranging from 0.0003 to 1,024 μM. Increasing Ca2+i from 0.03 to 1,024 μM shifted the voltage for half maximal activation (V1/2) 175 mV in the hyperpolarizing direction. V1/2 was independent of Ca2+i for Ca2+i ≤ 0.03 μM, indicating that the channel can be activated in the absence of Ca2+i. Open and closed dwell-time distributions for data obtained at different Ca2+i and voltage, but at the same Po, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of Po arose from a decrease in the mean closing rate with depolarization (qeff = −0.5 eo) and an increase in the mean opening rate (qeff = 1.8 eo), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+i (∼0 through 1,024 μM), voltage (+80 to −80 mV), and Po (10−4 to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.
The Eeee Locus Is the Sole High-Affinity Ca2+ Binding Structure in the Pore of a Voltage-Gated Ca2+ Channel
Authors: Cibulsky Susan M. , Sather William A.
Published: July 2002

Selective permeability in voltage-gated Ca2+ channels is dependent upon a quartet of pore-localized glutamate residues (EEEE locus). The EEEE locus is widely believed to comprise the sole high-affinity Ca2+ binding site in the pore, which represents an overturning of earlier models that had postulated two high-affinity Ca2+ binding sites. The current view is based on site-directed mutagenesis work in which Ca2+ binding affinity was attenuated by single and double substitutions in the EEEE locus, and eliminated by quadruple alanine (AAAA), glutamine (QQQQ), or aspartate (DDDD) substitutions. However, interpretation of the mutagenesis work can be criticized on the grounds that EEEE locus mutations may have additionally disrupted the integrity of a second, non-EEEE locus high-affinity site, and that such a second site may have remained undetected because the mutated pore was probed only from the extracellular pore entrance. Here, we describe the results of experiments designed to test the strength of these criticisms of the single high-affinity locus model of selective permeability in Ca2+ channels. First, substituted-cysteine accessibility experiments indicate that pore structure in the vicinity of the EEEE locus is not extensively disrupted as a consequence of the quadruple AAAA mutations, suggesting in turn that the quadruple mutations do not distort pore structure to such an extent that a second high affinity site would likely be destroyed. Second, the postulated second high-affinity site was not detected by probing from the intracellularly oriented pore entrance of AAAA and QQQQ mutants. Using inside-out patches, we found that, whereas micromolar Ca2+ produced substantial block of outward Li+ current in wild-type channels, internal Ca2+ concentrations up to 1 mM did not produce detectable block of outward Li+ current in the AAAA or QQQQ mutants. These results indicate that the EEEE locus is indeed the sole high-affinity Ca2+ binding locus in the pore of voltage-gated Ca2+ channels.