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1.
Relative expression pattern of short and long isoforms of hKv4.3 channels was evaluated by RT-PCR and RPA. Electrophysiological studies were performed in HEK293 cells transfected with short or long hKv4.3 cDNA. The long variant L-hKv4.3 was the only form present in lung, pancreas, and small intestine. The short variant S-hKv4.3 was predominant in brain whereas expression levels of the two isoforms were similar in cardiac and skeletal muscles. Properties of the ionic channels encoded by L-hKv4.3 and S-hKv4.3 cDNAs were essentially similar. Cadmium chloride and verapamil inhibited hKv4.3 current (with EC50s of 0.110 +/- 0.004 mM and 492.9 +/- 15.1 microM, respectively). Verapamil also accelerated current inactivation. Another calcium channel antagonist nicardipine was found inactive. In conclusion, this study confirms that both isoforms underlie the transient outward potassium current. Moreover, calcium channel inhibitors markedly affect hKv4.3 current, an effect which must be considered when evaluating transient outward potassium channel properties in native tissues.  相似文献   

2.
3.
The molecular basis of K+ channel function is universally conserved. K+ channels allow K+ flux and are essential for the generation of electric current across excitable membranes. K+ channels are also the targets of various intracellular control mechanisms, such that the suboptimal regulation of channel function might be related to pathological conditions. Because of the fundamental role of K+ channels in controlling membrane excitability, a structural understanding of their function and regulation will provide a useful framework for understanding neuronal physiology. Many recent physiological and crystallographic studies have led to new insights into the workings of K+ channels.  相似文献   

4.
Spectral analyses of conductance fluctuations produced by monazomycin pores in black lipid membranes show slow (2 s), fast (30 ms) and ultra-fast (2 ms) kinetics. The extremely low level of this noise compared with the cationic permeability to large ions such Tris+ gives support to a model where only a small fraction of the total pore conductance is fluctuating.  相似文献   

5.
Potassium channel regulation   总被引:1,自引:0,他引:1       下载免费PDF全文
The sulphonylurea receptor (SUR) is a member of the ATP-binding cassette (ABC) family of membrane proteins. It functions as the regulatory subunit of the ATP-sensitive potassium (KATP) channel, which comprises SUR and Kir6.x proteins. Here, we review data demonstrating functional differences between the two nucleotide binding domains (NBDs) of SUR1. In addition, to explain the structural basis of these functional differences, we have constructed a molecular model of the NBD dimer of human SUR1. We discuss the experimental data in the context of this model, and show how the model can be used to design experiments aimed at elucidating the relationship between the structure and function of the KATP channel.  相似文献   

6.
离子通道病   总被引:4,自引:0,他引:4  
90年代以来,医学界和生物学界开始注意到细胞膜上电压调控性钠,钙,钾和氯离子通道功能改变与先天性和后天性疾病发生之间的关系,对于离子通道基因缺陷,功能改变与某些疾病关系的研究,将可更新在离子通道生理学,病理学和分子遗传学等方面的知识,有助于开辟通道病的治疗新途径,本文主要介绍离子通道病的分子生物学,遗传学,病理学和生理学的联合研究上取得的最新进展。  相似文献   

7.
We have characterized mechanisms of ionic permeation in the K channel of canine cardiac sarcoplasmic reticulum (SR K channel). Ionic selectivity, as measured by relative permeabilities, followed Eisenman sequence l, a low field strength sequence. Slope conductance measured in symmetrical solutions across the bilayer followed Eisenman sequence V. In all cases, the selectivity characteristics of the prominent subconductance state (O1) were similar to those of the main-state (O2). Further, our studies have revealed that this channel differs in three significant ways from the highly characterized SR K channel of skeletal muscle. First, the ratio of permeabilities Cs+ to K+ was a complex function of ion concentration. Second, the concentration dependence of conductance was not well described by the Michaelis-Menten formalism. Instead, we modeled the observed relations using a more general approach based on classical rate theory. Third, mole fraction experiments (Cs+ with K+) demonstrated a prominent anomalous effect. Certain of our Cs+ data required the Eyring rate theory approach for adequate interpretation. We adopted a symmetrical energy profile incorporating ion-ion interaction and thereby accounted for much of the data. We conclude that the canine cardiac SR K channel is significantly different from that of skeletal muscle, and it may accommodate more than one ion at a time.  相似文献   

8.
9.
Amiloride-sensitive ion channels are formed from homo- or heteromeric combinations of subunits from the epithelial Na+ channel (ENaC)/degenerin superfamily, which also includes the acid-sensitive ion channel (ASIC) family. These channel subunits share sequence homology and topology. In this study, we have demonstrated, using confocal fluorescence resonance energy transfer microscopy and co-immunoprecipitation, that ASIC and ENaC subunits are capable of forming cross-clade intermolecular interactions. We have also shown that combinations of ASIC1 with ENaC subunits exhibit novel electrophysiological characteristics compared with ASIC1 alone. The results of this study suggest that heteromeric complexes of ASIC and ENaC subunits may underlie the diversity of amiloride-sensitive cation conductances observed in a wide variety of tissues and cell types where co-expression of ASIC and ENaC subunits has been observed.  相似文献   

10.
Cholesterol is a major regulator of a variety of ion channels but the mechanisms underlying cholesterol sensitivity of ion channels are still poorly understood. The key question is whether cholesterol regulates ion channels by direct binding to the channel protein or by altering the physical environment of lipid bilayer. In this study, we provide the first direct evidence that cholesterol binds to prokaryotic Kir channels, KirBac1.1, and that cholesterol binding is essential for its regulatory effect. Specifically, we show that cholesterol is eluted together with the KirBac1.1 protein when separated on an affinity column and that the amount of bound cholesterol is proportional to the amount of the protein. We also show that cholesterol binding to KirBac1.1 is saturable with a K(D) of 390μM. Moreover, there is clear competition between radioactive and non-radioactive cholesterol for the binding site. There is no competition, however, between cholesterol and 5-Androsten 3β-17 β-diol, a sterol that we showed previously to have no effect on KirBac1.1 function. Finally, we show that cholesterol-KirBac1.1 binding is significantly inhibited by trifluoperazine, known to inhibit cholesterol binding to other proteins, and that inhibition of cholesterol-KirBac1.1 binding results in full recovery of the channel activity. Collectively, results from this study indicate that cholesterol-induced suppression of KirBac1.1 activity is mediated by direct interaction between cholesterol and the channel protein.  相似文献   

11.
A mechanosensitive Ca(2+)-permeable cation channel was recorded by patch clamp in isolated rat aortic endothelial cells. A low level of channel activity could be observed after seal formation. The channel displayed some inward rectification and had a conductance for inward current of approx. 32 pS in Ca(2+)-free pipette and bath solutions. Negative suction of -10 to -20 mmHg increased the probability of the channel being open. When the negative pressure in the pipette was raised to -35 to -45 mmHg, the channel underwent an abrupt transition to a large conductance substate that was interrupted occasionally by two other low conductance levels. Under this condition, the overwhelming majority of openings and closings were between a main level of 83 pS and the closed level. Compared to the 32 pS substate, the 83 pS large conductance substate had shorter mean open and closed times. The two channel substates had similar ionic selectivity and both were sensitive to the inhibition of cGMP and protein kinase G. This is the first demonstration showing that mechanostress can change the single channel conductance level of an ion channel in eukaryotic cells.  相似文献   

12.
Potassium ion channels are generally believed to have current-voltage (IV) relations which are linearly related to driving force ( V - E(K)), where V is membrane potential and E(K) is the potassium ion equilibrium potential. Consequently, activation curves for K+ channels have often been measured by normalizing voltage-clamp families of macroscopic K+ currents with (V - E(K)), where V is the potential of each successive step in the voltage clamp sequence. However, the IV relation for many types of K+ channels actually has a non-linear dependence upon driving force which is well described by the Goldman-Hodgkin-Katz relation. When the GHK dependence on (V - E(K)) is used in the normalization procedure, a very different voltage dependence of the activation curve is obtained which may more accurately reflect this feature of channel gating. Novel insights into the voltage dependence of the rapidly inactivating I(A) channels Kv1.4 and Kv4.2 have been obtained when this procedure was applied to recently published results.  相似文献   

13.
Mitochondrial potassium channels in the brain have been suggested to have an important role in neuroprotection. The single channel activity of mitochondrial potassium channels was measured after reconstitution of the purified inner membrane from rat brain mitochondria into a planar lipid bilayer. In addition to a large conductance potassium channel that was described previously, we identified a potassium channel that has a mean conductance of 219 ± 15 pS. The activity of this channel was inhibited by ATP/Mg2+ and activated by the potassium channel opener BMS191095. Channel activity was not influenced either by 5-hydroxydecanoic acid, an inhibitor of mitochondrial ATP-regulated potassium channels, or by the plasma membrane ATP-regulated potassium channel blocker HMR1098. Likewise, this mitochondrial potassium channel was unaffected by the large conductance potassium channel inhibitor iberiotoxin or by the voltage-dependent potassium channel inhibitor margatoxin. The amplitude of the conductance was lowered by magnesium ions, but the opening ability was unaffected. Immunological studies identified the Kir6.1 channel subunit in the inner membrane from rat brain mitochondria. Taken together, our results demonstrate for the first time the single channel activity and properties of an ATP-regulated potassium channel from rat brain mitochondria.  相似文献   

14.
15.
The channels of the mitochondrial outer membrane represent a useful model for studies into the mechanisms underlying phenomena of voltage-dependent gating and ion selectivity.  相似文献   

16.
Identification of the minimum number of ways in which open and closed states communicate is a crucial step in defining the gating kinetics of multistate channels. We used certain correlation functions to extract information about the pathways connecting the open and closed states of the cation channel of the purified nicotinic acetylcholine receptor and of the chloride channel of Torpedo californica electroplax membranes. Single channel currents were recorded from planar lipid bilayers containing the membrane channel proteins under investigation. The correlation functions are conveniently computed from single channel current records and yield information on E, the minimum number of entry/exit states into the open or closed aggregates. E gives a lower limit on the numbers of transition pathways between open and closed states. For the acetylcholine receptor, the autocorrelation analysis shows that there are at least two entry/exit states through which the open and closed aggregates communicate. The chloride channel fluctuates between three conductance substates, here indentified as C, M, and H for closed, intermediate, and high conductance, respectively. Correlation analysis shows that E is greater than or equal to 2 for the M aggregate, indicating that there are at least two distinct entry/exit states in the M aggregate. In contrast, there is no evidence for the existence of more than one entry/exit state in the C or H aggregates. Thus, these correlation functions provide a simple and general strategy to extract information on channel gating kinetics.  相似文献   

17.
The genetic disease cystic fibrosis is caused by mutation of the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Controversial studies reported regulation of the epithelial sodium channel (ENaC) by CFTR. We found that uptake of 22Na+ through ENaC is modulated by activation of CFTR in oocytes, coexpressing CFTR and ENaC, depending on extracellular chloride concentration. Furthermore we found that the effect of CFTR activation could be mimicked by other chloride channels. Voltage– and patch–clamp measurements, however, showed neither stimulation nor inhibition of ENaC-mediated conductance by activated CFTR. We conclude that the observed modulation of 22Na+ uptake by activated CFTR is due to the effect of CFTR-mediated chloride conductance on the membrane potential. These findings argue against the notion of a specific influence of CFTR on ENaC and emphasize the chloride channel function of CFTR.  相似文献   

18.
We examined the effects of calcium channel beta subunits upon the recovery from inactivation of alpha(1) subunits expressed in Xenopus oocytes. Recovery of the current carried by the L-type alpha(1) subunit (cyCa(v)1) from the jellyfish Cyanea capillata was accelerated by coexpression of any beta subunit, but the degree of potentiation differed according to which beta isoform was coexpressed. The Cyanea beta subunit was most effective, followed by the mammalian b(3), b(4), and beta(2a) subtypes. Recovery of the human Ca(v)2.3 subunit was also modulated by beta subunits, but was slowed instead. beta(3) was the most potent subunit tested, followed by beta(4), then beta(2a), which had virtually no effect. These results demonstrate that different beta subunit isoforms can affect recovery of the channel to varying degrees, and provide an additional mechanism by which beta subunits can differentially regulate alpha(1) subunits.  相似文献   

19.
Considerable controversy surrounds the location of the closed channel gate in members of the Cys-loop receptor family of neurotransmitter-gated ion channels that includes the GABAA, glycine, acetylcholine, and 5-HT3 receptors. Cysteine-accessibility studies concluded that the gate is near the cytoplasmic end of the channel in acetylcholine and GABAA receptors but in the middle of the 5-HT3A receptor channel. Zn2+ accessibility studies in a chimeric 5-HT3-ACh receptor suggested the gate is near the channel's cytoplasmic end. In the 4-A resolution structure of the acetylcholine receptor closed state determined by cryoelectron microscopy, the narrowest region, inferred to be the gate, is in the channel's midsection from 9' to 14' but the M1-M2 loop residues at the channel's cytoplasmic end were not resolved in that structure. We used blocker trapping experiments with picrotoxin, a GABAA receptor open channel blocker, to determine whether a gate exists at a position more extracellular than the picrotoxin binding site, which is in the vicinity of alpha1Val257 (2') near the channel's cytoplasmic end. We show that picrotoxin can be trapped in the channel after removal of GABA. By using the state-dependent accessibility of engineered cysteines as reporters for the channel's structural state we infer that after GABA washout, with picrotoxin trapped in the channel, the channel appears to be in the closed state. We infer that a gate exists between the picrotoxin binding site and the channel's extracellular end, consistent with a closed channel gate in the middle of the channel. Given the homology with acetylcholine and 5-HT3 receptors there is probably a similar gate in those channels as well. This does not preclude the existence of an additional gate at a more cytoplasmic location.  相似文献   

20.
In pancreatic β-cells, KATP channels consisting of Kir6.2 and SUR1 couple cell metabolism to membrane excitability and regulate insulin secretion. Sulfonylureas, insulin secretagogues used to treat type II diabetes, inhibit KATP channel activity primarily by abolishing the stimulatory effect of MgADP endowed by SUR1. In addition, sulfonylureas have been shown to function as pharmacological chaperones to correct channel biogenesis and trafficking defects. Recently, we reported that carbamazepine, an anticonvulsant known to inhibit voltage-gated sodium channels, has profound effects on KATP channels. Like sulfonylureas, carbamazepine corrects trafficking defects in channels bearing mutations in the first transmembrane domain of SUR1. Moreover, carbamazepine inhibits the activity of KATP channels such that rescued mutant channels are unable to open when the intracellular ATP/ADP ratio is lowered by metabolic inhibition. Here, we investigated the mechanism by which carbamazepine inhibits KATP channel activity. We show that carbamazepine specifically blocks channel response to MgADP. This gating effect resembles that of sulfonylureas. Our results reveal striking similarities between carbamazepine and sulfonylureas in their effects on KATP channel biogenesis and gating and suggest that the 2 classes of drugs may act via a converging mechanism.  相似文献   

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