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1.
Neuregulins stimulate the functional expression of Ca2+-activated K+ channels in developing chicken parasympathetic neurons
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Priya Subramony Stuart E. Dryer 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(11):5934-5938
The developmental expression of macroscopic Ca2+-activated K+ currents (IK[Ca]) in chicken ciliary ganglion (CG) neurons is dependent in part on trophic factors released from preganglionic nerve terminals. Neuregulins are expressed in the preganglionic neurons that innervate the chicken CG and are therefore plausible candidates for this activity. Application of 1 nM β1-neuregulin peptide for 12 hr evokes a large (7- to 10-fold) increase in IK[Ca] in embryonic day 9 CG neurons, even in the presence of a translational inhibitor. A similar posttranslational effect is produced by high concentrations (10 nM) of epidermal growth factor and type α transforming growth factor but not by 10 nM α2-neuregulin peptide or by neurotrophins at 40 ngml−1. β1-neuregulin treatment for 12 hr also confers Ca2+ sensitivity onto large-conductance (285 pS) K+ channels observed in inside–out patches. β-Neuregulins have no effect on voltage-activated Ca2+ currents of CG neurons. These data support the hypothesis that β-neuregulins mediate the trophic effects of preganglionic nerve terminals on the electrophysiological differentiation of developing CG neurons. 相似文献
2.
Ca2+-dependent and -independent interactions of the isoforms of the α1A subunit of brain Ca2+ channels with presynaptic SNARE proteins
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D. Kyle Kim William A. Catterall 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(26):14782-14786
Fast neurotransmission requires that docked synaptic vesicles be located near the presynaptic N-type or P/Q-type calcium channels. Specific protein–protein interactions between a synaptic protein interaction (synprint) site on N-type and P/Q-type channels and the presynaptic SNARE proteins syntaxin, SNAP-25, and synaptotagmin are required for efficient, synchronous neurotransmitter release. Interaction of the synprint site of N-type calcium channels with syntaxin and SNAP-25 has a biphasic calcium dependence with maximal binding at 10–20 μM. We report here that the synprint sites of the BI and rbA isoforms of the α1A subunit of P/Q-type Ca2+ channels have different patterns of interactions with synaptic proteins. The BI isoform of α1A specifically interacts with syntaxin, SNAP-25, and synaptotagmin independent of Ca2+ concentration and binds with high affinity to the C2B domain of synaptotagmin but not the C2A domain. The rbA isoform of α1A interacts specifically with synaptotagmin and SNAP-25 but not with syntaxin. Binding of synaptotagmin to the rbA isoform of α1A is Ca2+-dependent, with maximum affinity at 10–20 μM Ca2+. Although the rbA isoform of α1A binds well to both the C2A and C2B domains of synaptotagmin, only the interaction with the C2A domain is Ca2+-dependent. These differential, Ca2+-dependent interactions of Ca2+ channel synprint sites with SNARE proteins may modulate the efficiency of transmitter release triggered by Ca2+ influx through these channels. 相似文献
3.
A Xenopus oocyte β subunit: Evidence for a role in the assembly/expression of voltage-gated calcium channels that is separate from its role as a regulatory subunit
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Erwin Tareilus Michel Roux Ning Qin Riccardo Olcese Jianming Zhou Enrico Stefani Lutz Birnbaumer 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(5):1703-1708
Two closely related β subunit mRNAs (xo28 and xo32) were identified in Xenopus oocytes by molecular cloning. One or both appear to be expressed as active proteins, because: (i) injection of Xenopus β antisense oligonucleotides, but not of sense or unrelated oligonucleotides, significantly reduced endogenous oocyte voltage-gated Ca2+ channel (VGCC) currents and obliterated VGCC currents that arise after injection of mammalian α1 cRNAs (α1C and α1E); (ii) coinjection of a Xenopus β antisense oligonucleotide and excess rat β cRNA rescued expression of α1 Ca2+ channel currents; and (iii) coinjection of mammalian α1 cRNA with cRNA encoding either of the two Xenopus β subunits facilitated both activation and inactivation of Ca2+ channel currents by voltage, as happens with most mammalian β subunits. The Xenopus β subunit cDNAs (β3xo cDNAs) predict proteins of 484 aa that differ in only 22 aa and resemble most closely the sequence of the mammalian type 3 β subunit. We propose that “α1 alone” channels are in fact tightly associated α1β3xo channels, and that effects of exogenous β subunits are due to formation of higher-order [α1β]βn complexes with an unknown contribution of β3xo. It is thus possible that functional mammalian VGCCs, rather than having subunit composition α1β, are [α1β]βn complexes that associate with α2δ and, as appropriate, other tissue-specific accessory proteins. In support of this hypothesis, we discovered that the last 277-aa of α1E have a β subunit binding domain. This β binding domain is distinct from the previously known interaction domain located between repeats I and II of calcium channel α1 subunits. 相似文献
4.
Sho-Ya Wang Ging Kuo Wang 《Proceedings of the National Academy of Sciences of the United States of America》1998,95(5):2653-2658
Batrachotoxin (BTX) is a steroidal alkaloid that causes Na+ channels to open persistently. This toxin has been used widely as a tool for studying Na+ channel gating processes and for estimating Na+ channel density. In this report we used point mutations to identify critical residues involved in BTX binding and to examine if such mutations affect channel gating. We show that a single asparagine→lysine substitution of the rat muscle Na+ channel α-subunit, μ1-N434K, renders the channel completely insensitive to 5 μM BTX when expressed in mammalian cells. This mutant channel nonetheless displays normal current kinetics with minimal changes in gating properties. Another substitution, μ1-N434A, yields a partial BTX-sensitive mutant. Unlike wild-type currents, the BTX-modified μ1-N434A currents continue to undergo fast and slow inactivation as if the inactivation processes remain functional. This finding implies that the μ1-N434 residue upon binding with BTX is critical for subsequent changes on gating; alanine at the μ1–434 position apparently diminishes the efficacy of BTX on eliminating Na+ channel inactivation. Mutants of two adjacent residues, μ1-I433K and μ1-L437K, also were found to exhibit the identical BTX-resistant phenotype. We propose that the μ1-I433, μ1-N434, and μ1-L437 residues in transmembrane segment I-S6 probably form a part of the BTX receptor. 相似文献
5.
Molecular mechanism of use-dependent calcium channel block by phenylalkylamines: Role of inactivation
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Steffen Hering Stefan Aczl Richard L. Kraus Stanislav Berjukow Jrg Striessnig Eugen N. Timin 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(24):13323-13328
The role of channel inactivation in the molecular mechanism of calcium (Ca2+) channel block by phenylalkylamines (PAA) was analyzed by designing mutant Ca2+ channels that carry the high affinity determinants of the PAA receptor site [Hockerman, G. H., Johnson, B. D., Scheuer, T., and Catterall, W. A. (1995) J. Biol. Chem. 270, 22119–22122] but inactivate at different rates. Use-dependent block by PAAs was studied after expressing the mutant Ca2+ channels in Xenopus oocytes. Substitution of single putative pore-orientated amino acids in segment IIIS6 by alanine (F-1499-A, F-1500-A, F-1510-A, I-1514-A, and F-1515-A) gradually slowed channel inactivation and simultaneously reduced inhibition of barium currents (IBa) by (−)D600 upon depolarization by 100 ms steps at 0.1 Hz. This apparent reduction in drug sensitivity was only evident if test pulses were applied at a low frequency of 0.1 Hz and almost disappeared at the frequency of 1 Hz. (−)D600 slowed IBa recovery after maintained membrane depolarization (1–3 sec) to a comparable extent in all channel constructs. A drug-induced delay in the onset of IBa recovery from inactivation suggests that PAAs promote the transition to a deep inactivated channel conformation. These findings indicate that apparent PAA sensitivity of Ca2+ channels is not only defined by drug interaction with its receptor site but also crucially dependent on intrinsic gating properties of the channel molecule. A molecular model for PAA-Ca2+ channel interaction that accounts for the relationship between drug induced inactivation and channel block by PAA is proposed. 相似文献
6.
E. Palma L. Maggi R. Miledi F. Eusebi 《Proceedings of the National Academy of Sciences of the United States of America》1998,95(17):10246-10250
Zn2+ is a key structural/functional component of many proteins and is present at high concentrations in the brain and retina, where it modulates ligand-gated receptors. Therefore, a study was made of the effects of zinc on homomeric neuronal nicotinic receptors expressed in Xenopus oocytes after injection of cDNAs encoding the chicken wild or mutant α7 subunits. In oocytes expressing wild-type receptors, Zn2+ alone did not elicit appreciable membrane currents. Acetylcholine (AcCho) elicited large currents (IAcCho) that were reduced by Zn2+ in a reversible and dose-dependent manner, with an IC50 of 27 μM and a Hill coefficient of 0.4. The inhibition of IAcCho by Zn2+ was competitive and voltage-independent, a behavior incompatible with a channel blockade mechanism. In sharp contrast, in oocytes expressing a receptor mutant, with a threonine-for-leucine 247 substitution (L247Tα7), subnanomolar concentrations of Zn2+ elicited membrane currents (IZn) that were reversibly inhibited by the nicotinic receptor blockers methyllycaconitine and α-bungarotoxin. Cell-attached single-channel recordings showed that Zn2+ opened channels that had a mean open time of 5 ms and a conductance of 48 pS. At millimolar concentrations Zn2+ reduced IAcCho and the block became stronger with cell hyperpolarization. Thus, Zn2+ is a reversible blocker of wild-type α7 receptors, but becomes an agonist, as well as an antagonist, following mutation of the highly conserved leucine residue 247 located in the M2 channel domain. We conclude that Zn2+ is a modulator as well as an activator of homomeric nicotinic α7 receptors. 相似文献
7.
Association of calcium channel α1S and β1a subunits is required for the targeting of β1a but not of α1S into skeletal muscle triads
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Birgit Neuhuber Uli Gerster Frank Dring Hartmut Glossmann Tsutomu Tanabe Bernhard E. Flucher 《Proceedings of the National Academy of Sciences of the United States of America》1998,95(9):5015-5020
The skeletal muscle L-type Ca2+ channel is a complex of five subunits that is specifically localized in the triad. Its primary function is the rapid activation of Ca2+ release from cytoplasmic stores in a process called excitation-contraction coupling. To study the role of α1S–β1a interactions in the incorporation of the functional channel complex into the triad, α1S and β1a [or a β1a-green fluorescent protein (GFP) fusion protein] were expressed alone and in combination in myotubes of the dysgenic cell line GLT. βGFP expressed in dysgenic myotubes that lack the skeletal muscle α1S subunit was diffusely distributed in the cytoplasm. On coexpression with the α1S subunit βGFP distribution became clustered and colocalized with α1S immunofluorescence. Based on the colocalization of βGFP and α1S with the ryanodine receptor the clusters were identified as T-tubule/sarcoplasmic reticulum junctions. Expression of α1S with and without β1a restored Ca2+ currents and depolarization-induced Ca2+ release. The translocation of βGFP from the cytoplasm into the junctions failed when βGFP was coexpressed with α1S mutants in which the β interaction domain had been altered (α1S-Y366S) or deleted (α1S-Δ351–380). Although α1S-Y366S did not associate with βGFP it was incorporated into the junctions, and it restored Ca2+ currents and depolarization-induced Ca2+ release. Thus, β1a requires the association with the β interaction domain in the I–II cytoplasmic loop of α1S for its own incorporation into triad junctions, but stable α1S–β1a association is not necessary for the targeting of α1S into the triads or for its normal function in Ca2+ conductance and excitation-contraction coupling. 相似文献
8.
Decay of prepulse facilitation of N type calcium channels during G protein inhibition is consistent with binding of a single Gβγ subunit
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Gerald W. Zamponi Terry P. Snutch 《Proceedings of the National Academy of Sciences of the United States of America》1998,95(7):4035-4039
We have examined the modulation of cloned and stably expressed rat brain N type calcium channels (α1B + β1b + α2δ subunits) by exogenously applied purified G protein βγ subunits. In the absence of Gβγ, barium currents through N type channels are unaffected by application of strong depolarizing prepulses. In contrast, inclusion of purified Gβγ in the patch pipette results in N type currents that initially facilitated upon application of positive prepulses followed by rapid reinhibition. Examination of the kinetics of Gβγ-dependent reinhibition showed that as the duration between the test pulse and the prepulse was increased, the degree of facilitation was attenuated in a monoexponential fashion. The time constant τ for the recovery from facilitation was sensitive to exogenous Gβγ, so that the inverse of τ linearly depended on the Gβγ concentration. Overall, the data are consistent with a model whereby a single Gβγ molecule dissociates from the channel during the prepulse, and that reassociation of Gβγ with the channel after the prepulse occurs as a bimolecular reaction. 相似文献
9.
Streaming potential measurements in αβγ-rat epithelial Na+ channel in planar lipid bilayers
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Iskander I. Ismailov Vadim Gh. Shlyonsky Dale J. Benos 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(14):7651-7654
Streaming potentials across cloned epithelial Na+ channels (ENaC) incorporated into planar lipid bilayers were measured. We found that the establishment of an osmotic pressure gradient (Δπ) across a channel-containing membrane mimicked the activation effects of a hydrostatic pressure differential (ΔP) on αβγ-rENaC, although with a quantitative difference in the magnitude of the driving forces. Moreover, the imposition of a Δπ negates channel activation by ΔP when the Δπ was directed against ΔP. A streaming potential of 2.0 ± 0.7 mV was measured across αβγ-rat ENaC (rENaC)-containing bilayers at 100 mM symmetrical [Na+] in the presence of a 2 Osmol/kg sucrose gradient. Assuming single file movement of ions and water within the conduction pathway, we conclude that between two and three water molecules are translocated together with a single Na+ ion. A minimal effective pore diameter of 3 Å that could accommodate two water molecules even in single file is in contrast with the 2-Å diameter predicted from the selectivity properties of αβγ-rENaC. The fact that activation of αβγ-rENaC by ΔP can be reproduced by the imposition of Δπ suggests that water movement through the channel is also an important determinant of channel activity. 相似文献
10.
INAUGURAL ARTICLE by a Recently Elected Academy Member:Ca2+-sensitive inactivation of L-type Ca2+ channels depends on multiple cytoplasmic amino acid sequences of the α1C subunit
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Roger D. Zühlke Harald Reuter 《Proceedings of the National Academy of Sciences of the United States of America》1998,95(6):3287-3294
Ca2+-dependent inactivation of Ca2+ currents is a physiological phenomenon widely associated with L-type Ca2+ channels. Although the pore-forming α1C subunit of the channel is the target for Ca2+ binding, the amino acid sequences involved in the binding and/or in the coordination of Ca2+-dependent inactivation are still unclear. Based on previous experiments, we have prepared truncation mutants of a human α1C subunit by systematically deleting an EF-hand motif and sequences in a segment of 80 amino acids in the carboxyl-terminal tail. We found that the rate as well as the Ca2+ dependence of inactivation of currents through these mutated channels were very different. We have identified three amino acid sequences, the presence of which is important for Ca2+-dependent inactivation: (i) a putative Ca2+-binding EF-hand motif, (ii) two hydrophilic residues (asparagine and glutamic acid) 77–78 amino acids downstream of the EF-hand motif, and (iii) a putative IQ calmodulin binding motif. We suggest that Ca2+-dependent inactivation is a cooperative process involving several amino acid sequences in cytoplasmic segments of the α1C subunit. 相似文献
11.
Human autoantibodies specific for the α1A calcium channel subunit reduce both P-type and Q-type calcium currents in cerebellar neurons
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Ashwin Pinto Samantha Gillard Fraser Moss Kathryn Whyte Paul Brust Mark Williams Ken Stauderman Michael Harpold Bethan Lang John Newsom-Davis David Bleakman David Lodge John Boot 《Proceedings of the National Academy of Sciences of the United States of America》1998,95(14):8328-8333
The pharmacological properties of voltage-dependent calcium channel (VDCC) subtypes appear mainly to be determined by the α1 pore-forming subunit but, whether P-and Q-type VDCCs are encoded by the same α1 gene presently is unresolved. To investigate this, we used IgG antibodies to presynaptic VDCCs at motor nerve terminals that underlie muscle weakness in the autoimmune Lambert–Eaton myasthenic syndrome (LEMS). We first studied their action on changes in intracellular free Ca2+ concentration [Ca2+]i in human embryonic kidney (HEK293) cell lines expressing different combinations of human recombinant VDCC subunits. Incubation for 18 h with LEMS IgG (2 mg/ml) caused a significant dose-dependent reduction in the K+-stimulated [Ca2+]i increase in the α1A cell line but not in the α1B, α1C, α1D, and α1E cell lines, establishing the α1A subunit as the target for these autoantibodies. Exploiting this specificity, we incubated cultured rat cerebellar neurones with LEMS IgG and observed a reduction in P-type current in Purkinje cells and both P- and Q-type currents in granule cells. These data are consistent with the hypothesis that the α1A gene encodes for the pore-forming subunit of both P-type and Q-type VDCCs. 相似文献
12.
Targeted disruption of the Ca2+ channel β3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltagedependent activation of P/Q-type Ca2+ channels in neurons
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Yoon Namkung Stephen M. Smith Seong Beom Lee Nataliya V. Skrypnyk Hyung-Lae Kim Hemin Chin Richard H. Scheller Richard W. Tsien Hee-Sup Shin 《Proceedings of the National Academy of Sciences of the United States of America》1998,95(20):12010-12015
In comparison to the well characterized role of the principal subunit of voltage-gated Ca2+ channels, the pore-forming, antagonist-binding α1 subunit, considerably less is understood about how β subunits contribute to neuronal Ca2+ channel function. We studied the role of the Ca2+ channel β3 subunit, the major Ca2+ channel β subunit in neurons, by using a gene-targeting strategy. The β3 deficient (β3−/−) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic β3−/− neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca2+ channels was described by two Boltzmann components with different voltage dependence, analogous to the “reluctant” and “willing” states reported for N-type channels. The absence of the β3 subunit was associated with a hyperpolarizing shift of the “reluctant” component of activation. Norepinephrine inhibited wt and β3−/− neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca2+ channels. The reduction in the expression of N-type Ca2+ channels in the β3−/− mice may be expected to impair Ca2+ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels. 相似文献
13.
Experimental support for a β-propeller domain in integrin α-subunits and a calcium binding site on its lower surface
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Claus Oxvig Timothy A. Springer 《Proceedings of the National Academy of Sciences of the United States of America》1998,95(9):4870-4875
Integrins are large, heterodimeric surface molecules of wide importance in cell adhesion. The N-terminal half of all integrin α-subunits contains seven weak sequence repeats of ≈60 amino acids that are important in ligand binding and have been predicted to fold cooperatively into a single β-propeller domain with seven β-sheets. We provide evidence supporting this model with a mouse mAb to human Mac-1 (αMβ2, CD11b/CD18). This antibody, CBRM1/20, binds to amino acid residues that are in different repeats and are 94 residues apart in the primary structure in the loop between strands 1 and 2 of β-sheet 5 and in the loop between strands 3 and 4 of β-sheet 6. The 1–2 loops of β-sheets 5–7 in integrins have EF hand-like Ca2+-binding motifs. CBRM1/20 binds to Mac-1 in the presence of Ca2+ or Sr2+ with an EC50 of 0.2 mM. Mg2+ or Mn2+ cannot substitute. Antibodies to other epitopes on the Mac-1 β-propeller domain bind in the absence of calcium. mAb CBRM1/20 does not block ligand binding. Thus, the region on the lower surface of the β-propeller domain to which mAb CBRM1/20 binds does not bind ligand and, furthermore, cannot bind other integrin domains, such as those of the β-subunit. 相似文献
14.
Na+ pump low and high ouabain affinity α subunit isoforms are differently distributed in cells
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Magdalena Juhaszova Mordecai P. Blaustein 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(5):1800-1805
Three isoforms (α1, α2, and α3) of the catalytic (α) subunit of the plasma membrane (PM) Na+ pump have been identified in the tissues of birds and mammals. These isoforms differ in their affinities for ions and for the Na+ pump inhibitor, ouabain. In the rat, α1 has an unusually low affinity for ouabain. The PM of most rat cells contains both low (α1) and high (α2 or α3) ouabain affinity isoforms, but precise localization of specific isoforms, and their functional significance, are unknown. We employed high resolution immunocytochemical techniques to localize α subunit isoforms in primary cultured rat astrocytes, neurons, and arterial myocytes. Isoform α1 was ubiquitously distributed over the surfaces of these cells. In contrast, high ouabain affinity isoforms (α2 in astrocytes, α3 in neurons and myocytes) were confined to a reticular distribution within the PM that paralleled underlying endoplasmic or sarcoplasmic reticulum. This distribution is identical to that of the PM Na/Ca exchanger. This raises the possibility that α1 may regulate bulk cytosolic Na+, whereas α2 and α3 may regulate Na+ and, indirectly, Ca2+ in a restricted cytosolic space between the PM and reticulum. The high ouabain affinity Na+ pumps may thereby modulate reticulum Ca2+ content and Ca2+ signaling. 相似文献
15.
A transient outward-rectifying K+ channel current down-regulated by cytosolic Ca2+ in Arabidopsis thaliana guard cells
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Zhen-Ming Pei Victor M. Baizabal-Aguirre Gethyn J. Allen Julian I. Schroeder 《Proceedings of the National Academy of Sciences of the United States of America》1998,95(11):6548-6553
Sustained (noninactivating) outward-rectifying K+ channel currents have been identified in a variety of plant cell types and species. Here, in Arabidopsis thaliana guard cells, in addition to these sustained K+ currents, an inactivating outward-rectifying K+ current was characterized (plant A-type current: IAP). IAP activated rapidly with a time constant of 165 ms and inactivated slowly with a time constant of 7.2 sec at +40 mV. IAP was enhanced by increasing the duration (from 0 to 20 sec) and degree (from +20 to −100 mV) of prepulse hyperpolarization. Ionic substitution and relaxation (tail) current recordings showed that outward IAP was mainly carried by K+ ions. In contrast to the sustained outward-rectifying K+ currents, cytosolic alkaline pH was found to inhibit IAP and extracellular K+ was required for IAP activity. Furthermore, increasing cytosolic free Ca2+ in the physiological range strongly inhibited IAP activity with a half inhibitory concentration of ≈ 94 nM. We present a detailed characterization of an inactivating K+ current in a higher plant cell. Regulation of IAP by diverse factors including membrane potential, cytosolic Ca2+ and pH, and extracellular K+ and Ca2+ implies that the inactivating IAP described here may have important functions during transient depolarizations found in guard cells, and in integrated signal transduction processes during stomatal movements. 相似文献
16.
Regulation of the human ether-a-gogo related gene (HERG) K+ channels by reactive oxygen species
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Maurizio Taglialatela Pasqualina Castaldo Silvana Iossa Anna Pannaccione Angela Fresi Eckhard Ficker Lucio Annunziato 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(21):11698-11703
Human ether-a-gogo related gene (HERG) K+ channels are key elements in the control of cell excitability in both the cardiovascular and the central nervous systems. For this reason, the possible modulation by reactive oxygen species (ROS) of HERG and other cloned K+ channels expressed in Xenopus oocytes has been explored in the present study. Exposure of Xenopus oocytes to an extracellular solution containing FeSO4 (25–100 μM) and ascorbic acid (50–200 μM) (Fe/Asc) increased both malondialdehyde content and 2′,7′-dichlorofluorescin fluorescence, two indexes of ROS production. Oocyte perfusion with Fe/Asc caused a 50% increase of the outward K+ currents carried by HERG channels, whereas inward currents were not modified. This ROS-induced increase in HERG outward K+ currents was due to a depolarizing shift of the voltage-dependence of channel inactivation, with no change in channel activation. No effect of Fe/Asc was observed on the expressed K+ currents carried by other K+ channels such as bEAG, rDRK1, and mIRK1. Fe/Asc-induced stimulation of HERG outward currents was completely prevented by perfusion of the oocytes with a ROS scavenger mixture (containing 1,000 units/ml catalase, 200 ng/ml superoxide dismutase, and 2 mM mannitol). Furthermore, the scavenger mixture also was able to reduce HERG outward currents in resting conditions by 30%, an effect mimicked by catalase alone. In conclusion, the present results seem to suggest that changes in ROS production can specifically influence K+ currents carried by the HERG channels. 相似文献
17.
Shigeomi Shimizu Yutaka Eguchi Wataru Kamiike Yoshimitsu Funahashi Alexandre Mignon Virginia Lacronique Hikaru Matsuda Yoshihide Tsujimoto 《Proceedings of the National Academy of Sciences of the United States of America》1998,95(4):1455-1459
We and others have recently shown that loss of the mitochondrial membrane potential (Δψ) precedes apoptosis and chemical-hypoxia-induced necrosis and is prevented by Bcl-2. In this report, we examine the biochemical mechanism used by Bcl-2 to prevent Δψ loss, as determined with mitochondria isolated from a cell line overexpressing human Bcl-2 or from livers of Bcl-2 transgenic mice. Although Bcl-2 had no effect on the respiration rate of isolated mitochondria, it prevented both Δψ loss and the permeability transition (PT) induced by various reagents, including Ca2+, H2O2, and tert-butyl hydroperoxide. Even under conditions that did not allow PT, Bcl-2 maintained Δψ, suggesting that the functional target of Bcl-2 is regulation of Δψ but not PT. Bcl-2 also maintained Δψ in the presence of the protonophore SF6847, which induces proton influx, suggesting that Bcl-2 regulates ion transport to maintain Δψ. Although treatment with SF6847 in the absence of Ca2+ caused massive H+ influx in control mitochondria, the presence of Bcl-2 induced H+ efflux after transient H+ influx. In this case, Bcl-2 did not enhance K+ efflux. Furthermore, Bcl-2 enhanced H+ efflux but not K+ flux after treatment of mitochondria with Ca2+ or tert-butyl hydroperoxide. These results suggest that Bcl-2 maintains Δψ by enhancing H+ efflux in the presence of Δψ-loss-inducing stimuli. 相似文献
18.
Ca2+-induced rebound potentiation of γ-aminobutyric
acid-mediated currents requires activation of
Ca2+/calmodulin-dependent kinase II
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Masanobu Kano Misao Kano Kohji Fukunaga Arthur Konnerth 《Proceedings of the National Academy of Sciences of the United States of America》1996,93(23):13351-13356
In cerebellar Purkinje neurons, γ-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission undergoes a long-lasting “rebound potentiation” after the activation of excitatory climbing fiber inputs. Rebound potentiation is triggered by the climbing-fiber-induced transient elevation of intracellular Ca2+ concentration and is expressed as a long-lasting increase of postsynaptic GABAA receptor sensitivity. Herein we show that inhibitors of the Ca2+/calmodulin-dependent protein kinase II (CaM-KII) signal transduction pathway effectively block the induction of rebound potentiation. These inhibitors have no effect on the once established rebound potentiation, on voltage-gated Ca2+ channel currents, or on the basal inhibitory transmission itself. Futhermore, a protein phosphatase inhibitor and the intracellularly applied CaM-KII markedly enhanced GABA-mediated currents in Purkinje neurons. Our results demonstrate that CaM-KII activation and the following phosphorylation are key steps for rebound potentiation. 相似文献
19.
Phosphorylation of photolyzed rhodopsin is calcium-insensitive in retina permeabilized by α-toxin
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Annie E. Otto-Bruc Robert N. Fariss J. Preston Van Hooser Krzysztof Palczewski 《Proceedings of the National Academy of Sciences of the United States of America》1998,95(25):15014-15019
Light triggers the phototransduction cascade by activating the visual pigment rhodopsin (Rho → Rho*). Phosphorylation of Rho* by rhodopsin kinase (RK) is necessary for the fast recovery of sensitivity after intense illumination. Ca2+ ions, acting through Ca2+-binding proteins, have been implicated in the desensitization of phototransduction. One such protein, recoverin, has been proposed to regulate RK activity contributing to adaptation to background illumination in retinal photoreceptor cells. In this report, we describe an in vitro assay system using isolated retinas that is well suited for a variety of biochemical assays, including assessing Ca2+ effects on Rho* phosphorylation. Pieces of bovine retina with intact rod outer segments were treated with pore-forming staphylococcal α-toxin, including an α-toxin mutant that forms pores whose permeability is modulated by Zn2+. The pores formed through the plasma membranes of rod cells permit the diffusion of small molecules <2 kDa but prevent the loss of proteins, including recoverin (25 kDa). The selective permeability of these pores was confirmed by using the small intracellular tracer N-(2-aminoethyl) biotinamide hydrochloride. Application of [γ-32P]ATP to α-toxin-treated, isolated retina allowed us to monitor and quantify phosphorylation of Rho*. Under various experimental conditions, including low and high [Ca2+]free, the same level of Rho* phosphorylation was measured. No differences were observed between low and high [Ca2+]free conditions, even when rods were loaded with ATP and the pores were closed by Zn2+. These results suggest that under physiological conditions, Rho* phosphorylation is insensitive to regulation by Ca2+ and Ca2+-binding proteins, including recoverin. 相似文献
20.
Antibodies to several conformation-dependent epitopes of gp120/gp41 inhibit CCR-5-dependent cell-to-cell fusion mediated by the native envelope glycoprotein of a primary macrophage-tropic HIV-1 isolate
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Florence C. Verrier Pierre Charneau Ralf Altmeyer Stephanie Laurent Andrew M. Borman Marc Girard 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(17):9326-9331
The β-chemokine receptor CCR-5 is essential for the efficient entry of primary macrophage-tropic HIV-1 isolates into CD4+ target cells. To study CCR-5-dependent cell-to-cell fusion, we have developed an assay system based on the infection of CD4+ CCR-5+ HeLa cells with a Semliki Forest virus recombinant expressing the gp120/gp41 envelope (Env) from a primary clade B HIV-1 isolate (BX08), or from a laboratory T cell line-adapted strain (LAI). In this system, gp120/gp41 of the “nonsyncytium-inducing,” primary, macrophage-tropic HIV-1BX08 isolate, was at least as fusogenic as that of the “syncytium-inducing” HIV-1LAI strain. BX08 Env-mediated fusion was inhibited by the β-chemokines RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory proteins 1β (MIP-1β) and by antibodies to CD4, whereas LAI Env-mediated fusion was insensitive to these β-chemokines. In contrast soluble CD4 significantly reduced LAI, but not BX08 Env-mediated fusion, suggesting that the primary isolate Env glycoprotein has a reduced affinity for CD4. The domains in gp120/gp41 involved in the interaction with the CD4 and CCR-5 molecules were probed using monoclonal antibodies. For the antibodies tested here, the greatest inhibition of fusion was observed with those directed to conformation-dependent, rather than linear epitopes. Efficient inhibition of fusion was not restricted to epitopes in any one domain of gp120/gp41. The assay was sufficiently sensitive to distinguish between antibody- and β-chemokine-mediated fusion inhibition using serum samples from patient BX08, suggesting that the system may be useful for screening human sera for the presence of biologically significant antibodies. 相似文献