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1.
A G protein directly regulates mammalian cardiac calcium channels   总被引:45,自引:0,他引:45  
A possible direct effect of guanine nucleotide binding (G) proteins on calcium channels was examined in membrane patches excised from guinea pig cardiac myocytes and bovine cardiac sarcolemmal vesicles incorporated into planar lipid bilayers. The guanosine triphosphate analog, GTP gamma S, prolonged the survival of excised calcium channels independently of the presence of adenosine 3',5'-monophosphate (cAMP), adenosine triphosphate, cAMP-activated protein kinase, and the protein kinase C activator tetradecanoyl phorbol acetate. A specific G protein, activated Gs, or its alpha subunit, purified from the plasma membranes of human erythrocytes, prolonged the survival of excised channels and stimulated the activity of incorporated channels. Thus, in addition to regulating calcium channels indirectly through activation of cytoplasmic kinases, G proteins can regulate calcium channels directly. Since they also directly regulate a subset of potassium channels, G proteins are now known to directly gate two classes of membrane ion channels.  相似文献   

2.
Multiple types of calcium channels have been found in neurons, but uncertainty remains about which ones are involved in stimulus-secretion coupling. Two types of calcium channels in rat sympathetic neurons were described, and their relative importance in controlling norepinephrine release was analyzed. N-type and L-type calcium channels differed in voltage dependence, unitary barium conductance, and pharmacology. Nitrendipine inhibited activity of L-type channels but not N-type channels. Potassium-evoked norepinephrine release was markedly reduced by cadmium and the conesnail peptide toxin omega-Conus geographus toxin VIA, agents that block both N- and L-type channels, but was little affected by nitrendipine at concentrations that strongly reduce calcium influx, as measured by fura-2. Thus N-type calcium channels play a dominant role in the depolarization-evoked release of norepinephrine.  相似文献   

3.
Electrophysiological recordings were used to analyze single calcium channels in planar lipid bilayers after membranes from bovine cardiac sarcolemmal vesicles had been incorporated into the bilayer. In these cell-free conditions, channels in the bilayer showed unitary barium or calcium conductances, gating kinetics, and pharmacological responses that were similar to dihydropyridine-sensitive calcium channels in intact cells. The open channel current varied in a nonlinear manner with voltage under asymmetric (that is, physiological) ionic conditions. However, with identical solutions on both sides of the bilayer, the current-voltage relation was linear. In matched experiments, calcium channels from skeletal muscle T-tubules differed significantly from cardiac calcium channels in their conductance properties and gating kinetics.  相似文献   

4.
目的:对全脑缺血模型大鼠再灌后不同时间点L型C a2 通道的开放和关闭状态进行动态研究,以进一步揭示缺血性神经元损伤的机制。方法:参照改良的Pu lsinelli四血管闭塞法制备全脑缺血大鼠模型,缺血后的大鼠分别在再灌注2、12、24、48、72h后进行皮层神经细胞急性分离,单通道电流经EPC-9膜片钳放大器放大,用Pu lsefit Pu lse采集入计算机,用分析软件TAC进行测量。结果:再灌后2、12、24、48、72 h5个不同时间点,大鼠大脑皮层神经元L型C a2 通道平均开放时间出现两个高峰期,第1次出现在再灌2、12、24h,第2次出现在48、72 h,较第1次更高;大鼠大脑皮层神经元L型C a2 通道开放概率出现两个峰值:第1次出现在再灌后2 h(显著高于正常组),至12 h又回落至接近正常水平;第2次出现在再灌24 h。结论:在脑缺血再灌注的不同时点,缺血性损伤对L型C a2 通道的影响机制即可利用性和开放特性的影响不同。在缺血再灌后的2 h至72 h的各时段,缺血性损伤通过增加L型C a2 通道的可利用性引起神经元胞内C a2 超载;在再灌后期(48 h),缺血性损伤则通过增加L型C a2 通道的开放特性而引起神经元胞内C a2 超载。  相似文献   

5.
[目的]研究Snapin蛋白与心肌L型钙离子通道Cav1.3的相互作用。[方法]利用免疫共沉淀实验确定Snapin蛋白与Cav1.3在体外表达系统和心房组织存在相互作用。[结果]在过表达Snapin和Cav1.3的HEK293细胞及内源性表达二者的心房肌组织中,Snapin蛋白与Cav1.3钙离子通道存在相互作用。  相似文献   

6.
The signaling pathways by which beta-adrenergic agonists modulate voltage-dependent cardiac sodium currents are unknown, although it is likely that adenosine 3'5'-monophosphate (cAMP) is involved. Single-channel and whole-cell sodium currents were measured in cardiac myocytes and the signal transducing G protein Gs was found to couple beta-adrenergic receptors to sodium channels by both cytoplasmic (indirect) and membrane-delimited (direct) pathways. Hence, Gs can act on at least three effectors in the heart: sodium channels, calcium channels, and adenylyl cyclase. The effect on sodium currents was inhibitory and was enhanced by membrane depolarization. During myocardial ischemia the sodium currents of depolarized cells may be further inhibited by the accompanying increase in catecholamine levels.  相似文献   

7.
Large-conductance calcium- and voltage-activated potassium channels (BKCa) are dually activated by membrane depolarization and elevation of cytosolic calcium ions (Ca2+). Under normal cellular conditions, BKCa channel activation requires Ca2+ concentrations that typically occur in close proximity to Ca2+ sources. We show that BKCa channels affinity-purified from rat brain are assembled into macromolecular complexes with the voltage-gated calcium channels Cav1.2 (L-type), Cav2.1 (P/Q-type), and Cav2.2 (N-type). Heterologously expressed BKCa-Cav complexes reconstitute a functional "Ca2+ nanodomain" where Ca2+ influx through the Cav channel activates BKCa in the physiological voltage range with submillisecond kinetics. Complex formation with distinct Cav channels enables BKCa-mediated membrane hyperpolarization that controls neuronal firing pattern and release of hormones and transmitters in the central nervous system.  相似文献   

8.
Calcium-sensitive inactivation in the gating of single calcium channels   总被引:9,自引:0,他引:9  
Voltage-activated calcium channels open and close, or gate, according to molecular transition rates that are regulated by transmembrane voltage and neurotransmitters. Here evidence for the control of gating by calcium was found in electrophysiological records of single, L-type calcium channels in heart cells. Conditional open probability analysis revealed that calcium entry during the opening of a single channel produces alterations in gating transition rates that evolve over the course of hundreds of milliseconds. Such alteration of calcium-channel gating by entry of a favored permeant ion provides a mechanism for the short-term modulation of single-ion channels.  相似文献   

9.
Sodium current-induced release of calcium from cardiac sarcoplasmic reticulum   总被引:52,自引:0,他引:52  
The role of sodium-calcium exchange at the sarcolemma in the release of calcium from cardiac sarcoplasmic reticulum was investigated in voltage-clamped, isolated cardiac myocytes. In the absence of calcium entry through voltage-dependent calcium channels, membrane depolarization elicited release of calcium from ryanodine-sensitive internal stores. This process was dependent on sodium entry through tetrodotoxin-sensitive sodium channels. Calcium release under these conditions was also dependent on extracellular calcium concentration, suggesting a calcium-induced trigger release mechanism that involves calcium entry into the cell by sodium-calcium exchange. This sodium current-induced calcium release mechanism may explain, in part, the positive inotropic effects of cardiac glycosides and the negative inotropic effects of a variety of antiarrhythmic drugs that interact with cardiac sodium channels. In response to a transient rise of intracellular sodium, sodium-calcium exchange may promote calcium entry into cardiac cells and trigger sarcoplasmic calcium release during physiologic action potentials.  相似文献   

10.
The mammalian heart rate is regulated by the vagus nerve, which acts via muscarinic acetylcholine receptors to cause hyperpolarization of atrial pacemaker cells. The hyperpolarization is produced by the opening of potassium channels and involves an intermediary guanosine triphosphate-binding regulatory (G) protein. Potassium channels in isolated, inside-out patches of membranes from atrial cells now are shown to be activated by a purified pertussis toxin-sensitive G protein of subunit composition alpha beta gamma, with an alpha subunit of 40,000 daltons. Thus, mammalian atrial muscarinic potassium channels are activated directly by a G protein, not indirectly through a cascade of intermediary events. The G protein regulating these channels is identified as a potent Gk; it is active at 0.2 to 1 pM. Thus, proteins other than enzymes can be under control of receptor coupling G proteins.  相似文献   

11.
Signal transducing guanine nucleotide binding (G) proteins are heterotrimers with different alpha subunits that confer specificity for interactions with receptors and effectors. Eight to ten such G proteins couple a large number of receptors for hormones and neurotransmitters to at least eight different effectors. Although one G protein can interact with several receptors, a given G protein was thought to interact with but one effector. The recent finding that voltage-gated calcium channels are stimulated by purified Gs, which stimulates adenylyl cyclase, challenged this concept. However, purified Gs may have four distinct alpha-subunit polypeptides, produced by alternative splicing of messenger RNA. By using recombinant DNA techniques, three of the splice variants were synthesized in Escherichia coli and each variant was shown to stimulate both adenylyl cyclase and calcium channels. Thus, a single G protein alpha subunit may regulate more than one effector function.  相似文献   

12.
Regulation of a heart potassium channel by protein kinase A and C   总被引:28,自引:0,他引:28  
The enzymes adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (protein kinase A) and protein kinase C regulate the activity of a diverse group of cellular proteins including membrane ion channel proteins. When protein kinase A was stimulated in cardiac ventricular myocytes with the membrane-soluble cAMP analog 8-chlorphenylthio cAMP (8-CPT cAMP), the amplitude of the delayed-rectifier potassium current (IK) doubled when recorded at 32 degrees C but was not affected at 22 degrees C. In contrast, modulation of the calcium current (ICa) by 8-CPT cAMP was independent of temperature with similar increases in ICa occurring at both temperatures. Stimulation of protein kinase C by phorbol 12,13-dibutyrate also enhanced IK in a temperature-dependent manner but failed to increase ICa at either temperature. Thus, cardiac delayed-rectifier potassium but not calcium channels are regulated by two distinct protein kinases in a similar temperature-dependent fashion.  相似文献   

13.
Apical membrane chloride channels control chloride secretion by airway epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis. In normal intact cells, activation of protein kinase C (PKC) by phorbol ester either stimulated or inhibited chloride secretion, depending on the physiological status of the cell. In cell-free membrane patches, PKC also had a dual effect: at a high calcium concentration, PKC inactivated chloride channels; at a low calcium concentration, PKC activated chloride channels. In cystic fibrosis cells, PKC-dependent channel inactivation was normal, but activation was defective. Thus it appears that PKC phosphorylates and regulates two different sites on the channel or on an associated membrane protein, one of which is defective in cystic fibrosis.  相似文献   

14.
Calcium influx through voltage-gated membrane channels plays a crucial role in a variety of neuronal processes, including long-term potentiation and epileptogenesis in the mammalian cortex. Recent studies indicate that calcium channels in some cell types are heterogeneous. This heterogeneity has now been shown for calcium channels in mammalian cortical neurons. When dissociated embryonic hippocampal neurons from rat were grown in culture they first had only low voltage-activated, fully inactivating somatic calcium channels. These channels were metabolically stable and conducted calcium better than barium. Appearing later in conjunction with neurite outgrowth and eventually predominating in the dendrites, were high voltage-activated, slowly inactivating calcium channels. These were metabolically labile and more selective to barium than to calcium. Both types of calcium currents were reduced by classical calcium channel antagonists, but the low voltage-activated channels were more strongly blocked by the anticonvulsant drug phenytoin. These findings demonstrate the development and coexistence of two distinct types of calcium channels in mammalian cortical neurons.  相似文献   

15.
Calcium signals, pivotal in controlling cell function, can be generated by calcium entry channels activated by plasma membrane depolarization or depletion of internal calcium stores. We reveal a regulatory link between these two channel subtypes mediated by the ubiquitous calcium-sensing STIM proteins. STIM1 activation by store depletion or mutational modification strongly suppresses voltage-operated calcium (Ca(V)1.2) channels while activating store-operated Orai channels. Both actions are mediated by the short STIM-Orai activating region (SOAR) of STIM1. STIM1 interacts with Ca(V)1.2 channels and localizes within discrete endoplasmic reticulum/plasma membrane junctions containing both Ca(V)1.2 and Orai1 channels. Hence, STIM1 interacts with and reciprocally controls two major calcium channels hitherto thought to operate independently. Such coordinated control of the widely expressed Ca(V)1.2 and Orai channels has major implications for Ca(2+) signal generation in excitable and nonexcitable cells.  相似文献   

16.
Interleukin-8 (IL-8) is an inflammatory cytokine that activates neutrophil chemotaxis, degranulation, and the respiratory burst. Neutrophils express receptors for IL-8 that are coupled to guanine nucleotide-binding proteins (G proteins); binding of IL-8 to its receptor induces the mobilization of intracellular calcium stores. A cDNA clone from HL-60 neutrophils, designated p2, has now been isolated that encodes a human IL-8 receptor. When p2 is expressed in oocytes from Xenopus laevis, the oocytes bind 125I-labeled IL-8 specifically and respond to IL-8 by mobilizing calcium stores with an EC50 of 20 nM. This IL-8 receptor has 77% amino acid identity with a second human neutrophil receptor isotype that binds IL-8 with higher affinity. It also exhibits 69% amino acid identity with a protein reported to be an N-formyl peptide receptor from rabbit neutrophils, but less than 30% identity with all other known G protein-coupled receptors, including the human N-formyl peptide receptor.  相似文献   

17.
18.
Calcitonin is a calcium regulating peptide hormone with binding sites in kidney and bone as well as in the central nervous system. The mechanisms of signal transduction by calcitonin receptors were studied in a pig kidney cell line where the hormone was found to regulate sodium pumps. Calcitonin receptors activated the cyclic adenosine monophosphate (cAMP) or the protein kinase C (PKC) pathways. The two transduction pathways required guanosine triphosphate (GTP)-binding proteins (G proteins) (the choleratoxin sensitive Gs and the pertussis toxin sensitive Gi, respectively) and led to opposite biological responses. Moreover, selective activation of one or the other pathway was cell cycle-dependent. Therefore, calcitonin may induce different biological responses in target cells depending on their positions in the cell cycle. Such a modulation of ligand-induced responses could be of importance in rapidly growing cell populations such as during embryogenesis, growth, and tumor formation.  相似文献   

19.
Guanosine 5'-triphosphate (GTP)-binding proteins (G proteins) are involved in exocytosis, endocytosis, and recycling of vesicles in yeast and mammalian secretory cells. However, little is known about their contribution to fast synaptic transmission. We loaded guanine nucleotide analogs directly into a giant nerve terminal in rat brainstem slices. Inhibition of G-protein activity had no effect on basal synaptic transmission, but augmented synaptic depression and significantly slowed recovery from depression. A nonhydrolyzable GTP analog blocked recovery of transmission from activity-dependent depression. Neither effect was accompanied by a change in presynaptic calcium currents. Thus, G proteins contribute to fast synaptic transmission by refilling synaptic vesicles depleted after massive exocytosis.  相似文献   

20.
Lambert-Eaton syndrome, an autoimmune disorder frequently associated with small-cell carcinoma of the lung, is characterized by impaired evoked release of acetylcholine from the motor nerve terminal. Immunoglobulin G (IgG) antibodies from patients with the syndrome, applied to bovine adrenal chromaffin cells, reduced the voltage-dependent calcium channel currents by about 40 percent. When calcium was administered directly into the cytoplasm, however, the IgG-treated cells exhibited normal exocytotic secretion, as assayed by membrane capacitance measurement. Measurement with the fluorescent calcium indicator fura-2 indicated that the IgG treatment reduced potassium-stimulated increase in free intracellular calcium concentration. The pathogenic IgG modified neither kinetics of calcium channel activation nor elementary channel activity, suggesting that a reduction in the number of functional calcium channels underlies the IgG-induced effect. Therefore, Lambert-Eaton syndrome IgG reacts with voltage-dependent calcium channels and blocks their function, a phenomenon that can account for the presynaptic impairment characteristic of this disorder.  相似文献   

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