共查询到20条相似文献,搜索用时 467 毫秒
1.
M Fink F Lesage F Duprat C Heurteaux R Reyes M Fosset M Lazdunski 《Canadian Metallurgical Quarterly》1998,17(12):3297-3308
TWIK-1, TREK-1 and TASK K+ channels comprise a class of pore-forming subunits with four membrane-spanning segments and two P domains. Here we report the cloning of TRAAK, a 398 amino acid protein which is a new member of this mammalian class of K+ channels. Unlike TWIK-1, TREK-1 and TASK which are widely distributed in many different mouse tissues, TRAAK is present exclusively in brain, spinal cord and retina. Expression of TRAAK in Xenopus oocytes and COS cells induces instantaneous and non-inactivating currents that are not gated by voltage. These currents are only partially inhibited by Ba2+ at high concentrations and are insensitive to the other classical K+ channel blockers tetraethylammonium, 4-aminopyridine and Cs+. A particularly salient feature of TRAAK is that they can be stimulated by arachidonic acid (AA) and other unsaturated fatty acids but not by saturated fatty acids. These channels probably correspond to the functional class of fatty acid-stimulated K+ currents that recently were identified in native neuronal cells but have not yet been cloned. These TRAAK channels might be essential in normal physiological processes in which AA is known to play an important role, such as synaptic transmission, and also in pathophysiological processes such as brain ischemia. TRAAK channels are stimulated by the neuroprotective drug riluzole. 相似文献
2.
Xenopus oocytes injected with GIRK1 mRNA express inwardly rectifying K+ channels resembling IKACh. Yet IKACh, the atrial G protein-regulated ion channel, is a heteromultimer of GIRK1 and CIR. Reasoning that an oocyte protein might be substituting for CIR, we cloned XIR, a CIR homolog endogenously expressed by Xenopus oocytes. Coinjecting XIR and GIRK1 mRNAs produced large, inwardly rectifying K+ currents responsive to m2-muscarinic receptor stimulation. The m2-stimulated currents of oocytes expressing GIRK1 alone decreased 80% after injecting antisense oligonucleotides specific to the 5' untranslated region of XIR, but GIRK1/CIR currents were unaffected. Thus, GIRK1 without XIR or CIR only ineffectively produces currents in oocytes. This result suggests that GIRK1 does not form native homomultimeric channels. 相似文献
3.
The product of the Saccharomyces cerevisiae K+-channel gene YKC1 includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarization and is regulated by extracellular K+ concentration ([K+]o) both in the yeast and when expressed in Xenopus oocytes. Analysis of the wild-type current now shows that: (i) [K+]o suppresses a very slowly relaxing component, accelerating activation; (ii) [K+]o slows deactivation in a dose-dependent fashion; and (iii) Rb+, Cs+ and, to a lesser extent, Na+ substitute for K+ in its action on gating. We have identified single residues, L293 and A428, at equivalent positions within the two pore loops that affect the [K+]o sensitivity. Substitution of these residues gave channels with reduced sensitivity to [K+]o in macroscopic current kinetics and voltage dependence, but had only minor effects on selectivity among alkali cations in gating and on single-channel conductance. In some mutants, activation was slowed sufficiently to confer a sigmoidicity to current rise at low [K+]o. The results indicate that these residues are involved in [K+]o sensing. Their situation close to the permeation pathway points to an interaction between gating and permeation. 相似文献
4.
Voltage-gated K+ channel (Kv) pore-forming (alpha) subunits of the Kv1 and Kv4 subfamilies have been cloned from heart cDNA libraries, and are thought to play roles in the generation of the transient outward K+ current, Ito. Heterologous expression of these subunits in Xenopus oocytes, however, reveals K+ currents that are quite distinct from Ito. In the experiments here, the detailed time- and voltage-dependent properties of the currents expressed in mammalian cell lines and in cardiac myocytes by Kv1.4 and Kv4.2 were examined and compared to previous findings in studies of oocytes, as well as to Ito characterized in various myocardial cells. As in oocytes, expression of Kv1.4 in HEK-293, Ltk- or neonatal rat ventricular cells reveals rapidly activating K+ currents. In contrast to the currents in oocytes, however, there are two components of inactivation of the Kv1.4-induced currents in mammalian cells, and both components are significantly slower in myocytes than in either HEK-293 or Ltk- cells. In addition, in all three cell types, recovery of Kv1.4 from steady-state inactivation is very slow, proceeding with mean time constants in the range of 6-8 s. The properties of Kv4.2-induced currents also vary with cell type and, importantly, the rates of activation, inactivation and recovery from inactivation are significantly faster in mammalian cells than in Xenopus oocytes. In HEK-293, Chinese hamster ovary (CHO) and neonatal rat ventricular cells, for example, the currents recover from steady-state inactivation with mean (+/-SD) time constants of 153+/-32 (n=12), 245+/-112 (n=10) and 86+/-38 (n=11) ms, respectively; therefore, recovery proceeds 5-10 times faster than observed for Kv4.2 in oocytes. These results emphasize the importance of the cellular expression environment in efforts to correlate endogenous K+ currents with heterologously expressed K+ channel subunits. In addition, the finding that Kv alpha subunits produce distinct K+ currents in different cells suggests that cell-type-specific associations with endogenous Kv alpha or accessory beta subunits and/or post-translational processing play roles in determining the properties of functional K+ channels. 相似文献
5.
F Duprat E Guillemare G Romey M Fink F Lesage M Lazdunski E Honore 《Canadian Metallurgical Quarterly》1995,92(25):11796-11800
Free radical-induced oxidant stress has been implicated in a number of physiological and pathophysiological states including ischemia and reperfusion-induced dysrhythmia in the heart, apoptosis of T lymphocytes, phagocytosis, and neurodegeneration. We have studied the effects of oxidant stress on the native K+ channel from T lymphocytes and on K+ channels cloned from cardiac, brain, and T-lymphocyte cells and expressed in Xenopus oocytes. The activity of three Shaker K+ channels (Kv1.3, Kv1.4, and Kv1.5), one Shaw channel (Kv3.4), and one inward rectifier K+ channel (IRK3) was drastically inhibited by photoactivation of rose bengal, a classical generator of reactive oxygen species. Other channel types (such as Shaker K+ channel Kv1.2, Shab channels Kv2.1 and Kv2.2, Shal channel Kv4.1, inward rectifiers IRK1 and ROMK1, and hIsK) were completely resistant to this treatment. On the other hand tert-butyl hydroperoxide, another generator of reactive oxygen species, removed the fast inactivation processes of Kv1.4 and Kv3.4 but did not alter other channels. Xanthine/xanthine oxidase system had no effect on all channels studied. Thus, we show that different types of K+ channels are differently modified by reactive oxygen species, an observation that might be of importance in disease states. 相似文献
6.
I Darboux E Lingueglia G Champigny S Coscoy P Barbry M Lazdunski 《Canadian Metallurgical Quarterly》1998,273(16):9424-9429
Amiloride-sensitive sodium channels have been implicated in reproductive and early developmental processes of several species. These include the fast block of polyspermy in Xenopus oocytes that follows the sperm binding to the egg or blastocoel expansion in mammalian embryo. We have now identified a gene called dGNaC1 that is specifically expressed in the gonads and early embryo in Drosophila melanogaster. The corresponding protein belongs to the superfamily of cationic channels blocked by amiloride that includes Caenorhabditis elegans degenerins, the Helix aspersa FMRF-amide ionotropic receptor (FaNaC), the mammalian epithelial Na+ channel (ENaC), and acid-sensing ionic channels (ASIC, DRASIC, and MDEG). Expression of dGNaC1 in Xenopus oocytes generates a constitutive current that does not discriminate between Na+ and Li+, but is selective for Na+ over K+. This current is blocked by amiloride (IC50 = 24 microM), benzamil (IC50 = 2 microM), and ethylisopropyl amiloride (IC50 = 49 microM). These properties are clearly different from those obtained after expression of the previously cloned members of this family, including ENaC and the human alphaENaC-like subunit, deltaNaC. Interestingly, the pharmacology of dGNaC1 is not very different from that found for the Na+ channel characterized in rabbit preimplantation embryos. We postulate that this channel may participate in gametogenesis and early embryonic development in Drosophila. 相似文献
7.
The effects of benzyltetrahydropalmatine (BTHP) on delayed rectified K+ currents (Ik) expressed in Xenopus oocytes and Ik of toad (Bufo bufo gargarizans) oocytes were studied. The Ik expressed in Xenopus oocytes was measured after microinjection of mRNA isolated from carp fish (C anratus L.) brains with double -microelectrode voltage clamp technique. The maximum and mean value of Ik expressed in Xenopus oocytes were 600 nA and 360 +/- 104 nA, respectively. BTHP reduced the current amplitude of Ik expressed in Xenopus oocytes in 10-1000 mumol.L-1 dose-dependently, EC50 was 29 mumol.L-1. Also, the reduction of Ik of toad oocytes was 9.1%, 29.1%, 54.7% and 68.6% by BTHP 10, 30, 100 and 1000 mumol.L-1, respectively, EC50 was 33 mumol.L-1. The results showed that BTHP possesses an inhibitory effect on Ik, the main ion mechanism of antiarrhythmic action of BTHP. 相似文献
8.
In Na(+)- and K(+)-free solution, an inward-directed current can be detected in Xenopus oocytes, which is inhibited by cardiac glycosides and activated by ATP. Therefore, it is assumed to be generated by the Na+,K+ pump. At negative membrane potentials, the pump current increases with more negative potentials and with increasing [H+] in the external medium. This current is not observed when Mg2+ instead of Ba2+ is the only divalent cation present in the bath medium, and it does not depend on whether Na+ or K+ is present internally. At 5 to 10 mM Na+ externally, maximum pump-generated current is obtained while no current can be detected in presence of physiological [Na+]. It is suggested that in low-Na+ and K(+)-free medium the Na+,K+ pump molecule can either form a conductive pathway that is permeable to Ba2+ or protons or operate in its conventional transport mode accepting Ba2+ as a K+ congener. A reversed pump mode or an electrogenic uncoupled Na(+)-efflux mode is excluded. 相似文献
9.
DS Krafte K Davison N Dugrenier K Estep K Josef RL Barchi RG Kallen PJ Silver AM Ezrin 《Canadian Metallurgical Quarterly》1994,266(3):245-254
Pharmacological modulation of human sodium current was examined in Xenopus oocytes expressing human heart Na+ channels. Na+ currents activated near -50 mV with maximum current amplitudes observed at -20 mV. Steady-state inactivation was characterized by a V1/2 value of -57 +/- 0.5 mV and a slope factor (k) of 7.3 +/- 0.3 mV. Sodium currents were blocked by tetrodotoxin with an IC50 value of 1.8 microM. These properties are consistent with those of Na+ channels expressed in mammalian myocardial cells. We have investigated the effects of several pharmacological agents which, with the exception of lidocaine, have not been characterized against cRNA-derived Na+ channels expressed in Xenopus oocytes. Lidocaine, quinidine and flecainide blocked resting Na+ channels with IC50 values of 521 microM, 198 microM, and 41 microM, respectively. Use-dependent block was also observed for all three agents, but concentrations necessary to induce block were higher than expected for quinidine and flecainide. This may reflect differences arising due to expression in the Xenopus oocyte system or could be a true difference in the interaction between human cardiac Na+ channels and these drugs compared to other mammalian Na+ channels. Importantly, however, this result would not have been predicted based upon previous studies of mammalian cardiac Na+ channels. The effects of DPI 201-106, RWJ 24517, and BDF 9148 were also tested and all three agents slowed and/or removed Na+ current inactivation, reduced peak current amplitudes, and induced use-dependent block. These data suggest that the alpha-subunit is the site of interaction between cardiac Na+ channels and Class I antiarrhythmic drugs as well as inactivation modifiers such as DPI 201-106. 相似文献
10.
D Oliver H Hahn C Antz JP Ruppersberg B Fakler 《Canadian Metallurgical Quarterly》1998,74(5):2318-2326
Blocking cloned inward-rectifier potassium (Kir) channels from the cytoplasmic side was analyzed with a rapid application system exchanging the intracellular solution on giant inside-out patches from Xenopus oocytes in <2 ms. Dependence of the pore-block on interaction of the blocking molecule with permeant and impermeant ions on either side of the membrane was investigated in Kir1.1 (ROMK1) channels blocked by ammonium derivatives and in Kir4.1 (BIR10) channels blocked by spermine. The blocking reaction in both systems showed first-order kinetics and allowed separate determination of on- and off-rates. The off-rates of block were strongly dependent on the concentration of internal and external bulk ions, but almost independent of the ion species at the cytoplasmic side of the membrane. With K+ as the only cation on both sides of the membrane, off-rates exhibited strong coupling to the K+ reversal potential (E(K)) and increased and decreased with reduction in intra and extracellular K+ concentration, respectively. The on-rates showed significant dependence on concentration and species of internal bulk ions. This control of rate-constants by interaction of permeant and impermeant internal and external ions governs the steady-state current-voltage relation (I-V) of Kir channels and determines their physiological function under various conditions. 相似文献
11.
Guinea-pig distal colonic mRNA injection into Xenopus laevis oocytes resulted in expression of functional active epithelial Na+ channels in the oocyte plasma membrane. Poly(A)+ RNA was extracted from distal colonic mucosa of animals fed either a high-salt (HS) or a low-salt (LS) diet. The electrophysiological properties of the expressed amiloride-sensitive Na+ conductances were investigated by conventional two-electrode voltage-clamp and patch-clamp measurements. Injection of poly(A)+ RNA from HS-fed animals [from hereon referred to as HS-poly(A)+ RNA] into oocytes induced the expression of amiloride-sensitive Na+ conductances. On the other hand, oocytes injected with poly(A)+ RNA from LS-fed animals [LS-poly(A)+ RNA] expressed a markedly larger amount of amiloride-blockable Na+ conductances. LS-poly(A)+ RNA-induced conductances were completely inhibitable by amiloride with a Ki of 77 nM, and were also blocked by benzamil with a Ki of 1.8 nM. 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA), even in high doses (25 "mu"M), had no detectable effect on the Na+ conductances. Expressed amiloride-sensitive Na+ channels could be further activated by cAMP leading to nearly doubled clamp currents. When Na+ was replaced by K+, amiloride (1 "mu"M) showed no effect on the clamp current. Single-channel analysis revealed slow gating behaviour, open probabilities (Po) between 0.4 and 0.9, and slope conductances of 3. 8 pS for Na+ and 5.6 pS for Li+. The expressed channels showed to be highly selective for Na+ over K+ with a permeability ratio PNa/PK > 20. Amiloride (500 nM) reduced channel Po to values < 0.05. All these features make the guinea-pig distal colon of LS-fed animals an interesting mRNA source for the expression of highly amiloride-sensitive Na+ channels in Xenopus oocytes, which could provide new insights in the regulatory mechanism of these channels. 相似文献
12.
13.
We recently reported that mutations in HERG, a potassium channel gene, cause long QT syndrome. Heterologous expression of HERG in Xenopus oocytes revealed that this channel had biophysical properties nearly identical to a cardiac delayed rectifier K+ current I(Kr), but had dissimilar pharmacological properties. Class III antiarrhythmic drugs such as E-4031 and MK-499 are potent and specific blockers of I (Kr) in cardiac myocytes. Our initial studies indicated that these compounds did not block HERG at a concentration of 1 micromol/L. In the present study, we used standard two-microelectrode voltage-clamp techniques to further characterize the effects of these drugs on HERG channels expressed in oocytes. Consistent with initial findings, 1 micromol/L MK-499 and E-4031 had not effect on HERG when oocytes were voltage clamped at a negative potential and not pulsed during equilibration with the drug. However, MK-499 did block HERG current if oocytes were repetitively pulsed, or clamped at a voltage positive to the threshold potential for channel activation. This finding is in contrast to previous studies that showed significant block of I(Kr) in isolated myocytes by similar drugs, even in the absence of pulsing. This apparent discrepancy may be due to differences in channel characteristics (HERG versus guinea pig and mouse I (Kr)), tissue (oocytes versus myocytes), or specific drugs. Under steady state conditions, block of HERG by MK-499 was half maximal at 123 +/- 12 nmol/L at a test potential of -20 mV. MK-499 (150 nmol/L) did not affect the voltage dependence of activation and rectification nor the kinetics of activation and deactivation of HERG. These data indicate that MK-499 preferentially blocks open HERG channels and further support the conclusion that HERG subunits form I(Kr) channels in cardiac myocytes. 相似文献
14.
RGS8 accelerates G-protein-mediated modulation of K+ currents 总被引:1,自引:0,他引:1
O Saitoh Y Kubo Y Miyatani T Asano H Nakata 《Canadian Metallurgical Quarterly》1997,390(6659):525-529
Transmembrane signal transduction via heterotrimeric G proteins is reported to be inhibited by RGS (regulators of G-protein signalling) proteins. These RGS proteins work by increasing the GTPase activity of G protein alpha-subunits (G alpha), thereby driving G proteins into their inactive GDP-bound form. However, it is not known how RGS proteins regulate the kinetics of physiological responses that depend on G proteins. Here we report the isolation of a full-length complementary DNA encoding a neural-tissue-specific RGS protein, RGS8, and the determination of its function. We show that RGS8 binds preferentially to the alpha-subunits G(alpha)o and G(alpha)i3 and that it functions as a GTPase-activating protein (GAP). When co-expressed in Xenopus oocytes with a G-protein-coupled receptor and a G-protein-coupled inwardly rectifying K+ channel (GIRK1/2), RGS8 accelerated not only the turning off but also the turning on of the GIRK1/2 current upon receptor stimulation, without affecting the dose-response relationship. We conclude that RGS8 accelerates the modulation of G-protein-coupled channels and is not just a simple negative regulator. This property of RGS8 may be crucial for the rapid regulation of neuronal excitability upon stimulation of G-protein-coupled receptors. 相似文献
15.
As metabotropic glutamate receptor type 1 (mGluR1) is known to couple L-type Ca2+ channels and ryanodine receptors (RyR, Chavis et al., 1996) in cerebellar granule cells, we examined if such a coupling could activate a Ca2+-sensitive K+ channel, the big K+ (BK) channel, in cultured cerebellar granule cells. We observed that (+/-)-1-amino-cyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) and quisqualate (QA) stimulated the activity of BK channels. On the other hand, (2S, 3S, 4S)-alpha-carboxycyclopropyl-glycine (L-CCG-I) and L-(+)-2-amino-4-phosphonobutyrate (L-AP4) had no effect on BK channels, indicating a specific activation by group I mGluRs. Group I mGluRs stimulation of the basal BK channel activity was mimicked by caffeine and both effects were blocked by ryanodine and nifedipine. Interestingly, carbachol stimulated BK channel activity but through a pertussis toxin (PTX)-sensitive pathway that was independent of L-type Ca2+ channel activity. Our report indicates that unlike the muscarinic receptors, group I mGluRs activate BK channels by mobilizing an additional pathway involving RyR and L-type Ca2+ channels. 相似文献
16.
DJ Henry DK Grandy HA Lester N Davidson C Chavkin 《Canadian Metallurgical Quarterly》1995,47(3):551-557
Xenopus oocytes expressed kappa-opioid specific binding sites after injection of cRNA prepared from a clone of the rat kappa-opioid receptor. Coinjection of kappa receptor cRNA with cRNA coding for a G protein-linked, inwardly rectifying, K+ channel (GIRK1, or KGA) resulted in oocytes that responded to the kappa agonist U-69593 by activating a large (1.0-1.5-microA) K+ current. U-69593 exhibited an EC50 of 260 +/- 50 nM and was blocked by the opioid antagonists norbinaltorphimine and naloxone. The kappa agonist bremazocine was 200-fold more potent than U-69593 in eliciting K+ current but exhibited a partial agonist profile in this expression system. The present results indicate that stimulation of inwardly rectifying K+ channels may be a potential effector mechanism for kappa-opioid receptors. 相似文献
17.
Injection of total RNA from carp retina induced the formation of a membrane K+ channel in Xenopus oocytes. We found an oligonucleotide complementary to the mRNA coding for a peptide fragment in the K+ channel of drosophila and mice could suppress the K+ channel current in a very specific manner. This provides important information for the cloning of this gene and for studying it's function. 相似文献
18.
K Malminiemi O Kari ML Latvala R Voutilainen A Miettinen A Jauch 《Canadian Metallurgical Quarterly》1996,74(3):280-284
Possible heteromultimer formation between Kv- and Kir-type K+ channels was investigated, in connection with the known functional diversity of K+ channels in vivo. Voltage-clamp experiments were performed on Xenopus oocytes, either injected with concatenated Kir2.1-Kv1.1 mRNA, or co-injected with Kv1.1 and Kir2.1 mRNA. K+ currents could be approximated by the algebraic sum of the 2 K+ current types alone. The tandem construct did not show functional expression, although it could be detected by Western blotting. We conclude that Kv1.1 and Kir2.1 alpha-subunit proteins fail to assemble and do not contribute functional diversity to K+ channels. 相似文献
19.
SD Silberberg A Lagrutta JP Adelman KL Magleby 《Canadian Metallurgical Quarterly》1996,70(6):2640-2651
Cloned large conductance Ca(2+)-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminated a putative cyclic AMP-dependent protein kinase phosphorylation site exhibited large, slow fluctuations in open probability with time. These fluctuations, termed "wanderlust kinetics," occurred with a time course of tens of seconds to minutes and had kinetic properties inconsistent with simple gating models. Wanderlust kinetics was still observed in the presence of 5 mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution was increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics did not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics could be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrated up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels. 相似文献
20.
M Levy J Jing D Chikvashvili WB Thornhill I Lotan 《Canadian Metallurgical Quarterly》1998,273(11):6495-6502
Various brain K+ channels, which may normally exist as complexes of alpha (pore-forming) and beta (auxiliary) subunits, were subjected to regulation by metabotropic glutamate receptors. Kv1.1/Kvbeta1.1 is a voltage-dependent K+ channel composed of alpha and beta proteins that are widely expressed in the brain. Expression of this channel in Xenopus oocytes resulted in a current that had fast inactivating and noninactivating components. Previously we showed that basal and protein kinase A-induced phosphorylation of the alpha subunit at Ser-446 decreases the fraction of the noninactivating component. In this study we investigated the effect of protein kinase C (PKC) on the channel. We showed that a PKC-activating phorbol ester (phorbol 12-myristate 13-acetate (PMA)) increased the noninactivating fraction via activation of a PKC subtype that was inhibited by staurosporine and bisindolylmaleimide but not by calphostin C. However, it was not a PKC-induced phosphorylation but rather a dephosphorylation that mediated the effect. PMA reduced the basal phosphorylation of Ser-446 significantly in plasma membrane channels and failed to affect the inactivation of channels having an alpha subunit that was mutated at Ser-446. Also, the activation of coexpressed mGluR1a known to activate phospholipase C mimicked the effect of PMA on the inactivation via induction of dephosphorylation at Ser-446. Thus, this study identified a potential neuronal pathway initiated by activation of metabotropic glutamate receptor 1a coupled to a signaling cascade that possibly utilized PKC to induce dephosphorylation and thereby to decrease the extent of inactivation of a K+ channel. 相似文献