Distinct kinetic changes in neurotransmitter release after SNARE protein cleavage

Neurotransmitter release is triggered by calcium ions and depends critically on the correct function of three types of SNARE [soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins. With use of the large calyx of Held presynaptic terminal from rats, we found that cleavage of different SNARE proteins by clostridial neurotoxins caused distinct kinetic changes in neurotransmitter release. When elevating calcium ion concentration directly at the presynaptic terminal with the use of caged calcium, cleavage of SNAP-25 by botulinum toxin A (BoNT/A) produced a strong reduction in the calcium sensitivity for release, whereas cleavage of syntaxin using BoNT/C1 and synaptobrevin using tetanus toxin (TeNT) produced an all-or-nothing block without changing the kinetics of remaining vesicles. When stimulating release by calcium influx through channels, a difference between BoNT/C1 and TeNT emerged, which suggests that cleavage of synaptobrevin modifies the coupling between channels and release-competent vesicles.     

Development of two types of calcium channels in cultured mammalian hippocampal neurons

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.     

全脑缺血模型大鼠再灌后对皮层神经元L型Ca2+通道的动态观察

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。     

Modulation of calcium channels in cardiac and neuronal cells by an endogenous peptide

Calcium channels mediate the generation of action potentials, pacemaking, excitation-contraction coupling, and secretion and signal integration in muscle, secretory, and neuronal cells. The physiological regulation of the L-type calcium channel is thought to be mediated primarily by guanine nucleotide-binding proteins (G proteins). A low molecular weight endogenous peptide has been isolated and purified from rat brain. This peptide regulates up and down the cardiac and neuronal calcium channels, respectively. In cardiac myocytes, the peptide-induced enhancement of the L-type calcium current had a slow onset (half-time approximately 75 seconds), occurred via a G protein-independent mechanism, and could not be inhibited by alpha 1-adrenergic, beta-adrenergic, or angiotensin II blockers. In neuronal cells, on the other hand, the negative effect had a rapid onset (half-time less than 500 milliseconds) and was observed on both T-type and L-type calcium channels.     

Calcium-sensitive inactivation in the gating of single calcium channels

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.     

BKCa-Cav channel complexes mediate rapid and localized Ca2+-activated K+ signaling

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.     

Multiple calcium channels and neuronal function

Recent investigations have demonstrated that neurons have a number of different types of calcium channels, each with their own unique properties and pharmacology. These calcium channels may be important in the control of different aspects of nerve activity. Some of the possibilities can now be discussed.     

Sodium current-induced release of calcium from cardiac sarcoplasmic reticulum

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.     

External calcium ions are required for potassium channel gating in squid neurons

The effects of calcium removal on the voltage-dependent potassium channels of isolated squid neurons were studied with whole cell patch-clamp techniques. When the calcium ion concentration was lowered from 10 to 0 millimolar (that is, no added calcium), potassium channel activity, identified from its characteristic time course, disappeared within a few seconds and there was a parallel increase in resting membrane conductance and in the holding current. The close temporal correlation of the changes in the three parameters suggests that potassium channels lose their ability to close in the absence of calcium and simultaneously lose their selectivity. If potassium channels were blocked by barium ion before calcium ion was removed, the increases in membrane conductance and holding current were delayed or prevented. Thus calcium is an essential cofactor in the gating of potassium channels in squid neurons.     

Inactivation and block of calcium channels by photo-released Ca2+ in dorsal root ganglion neurons

Calcium channels are inactivated by voltage and intracellular calcium. To study the kinetics and the mechanism of calcium-induced inactivation of calcium channels, a "caged" calcium compound, dimethoxy-nitrophen was used to photo-release about 50 microM calcium ion within 0.2 millisecond in dorsal root ganglion neurons. When divalent cations were the charge carriers, intracellular photo-release of calcium inactivated the calcium channel with an invariant rate [time constant (tau) approximately equal to 7 milliseconds]. When the monovalent cation sodium was the charge carrier, photorelease of calcium inside or outside of the cell blocked the channel rapidly (tau approximately equal to 0.4 millisecond), but the block was greater from the external side. Thus the kinetics of calcium-induced calcium channel inactivation depends on the valency of the permeant cation. The data imply that calcium channels exist in either of two conformational states, the calcium- and sodium-permeant forms, or, alternatively, calcium-induced inactivation occurs at a site closely associated with the internal permeating site.     

Calcium and sodium channels in spontaneously contracting vascular muscle cells

Electrophysiological recordings of inward currents from whole cells showed that vascular muscle cells have one type of sodium channel and two types of calcium channels. One of the calcium channels, the transient calcium channel, was activated by small depolarizations but then rapidly inactivated. It was equally permeable to calcium and barium and was blocked by cadmium, but not by tetrodotoxin. The other type, the sustained calcium channel, was activated by larger depolarizations, but inactivated very little; it was more permeable to barium than calcium. The sustained calcium channel was more sensitive to block by cadmium than the transient channel, but also was not blocked by tetrodotoxin. The sodium channel inactivated 15 times more rapidly than the transient calcium channel and at more negative voltages. This sodium channel, which is unusual because it is only blocked by a very high (60 microM) tetrodotoxin concentration but not by cadmium, is the first to be characterized in vascular muscle, and together with the two calcium channels, provides a basis for different patterns of excitation in vascular muscles.     

Snapin蛋白与心肌L型钙离子通道Cav1.3的相互作用

[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。     

Depolarization without calcium can release gamma-aminobutyric acid from a retinal neuron

Calcium influx is often an essential intermediate step for the release of neurotransmitter. However, some retinal neurons appear to release transmitter by a mechanism that does not require calcium influx. It was uncertain whether depolarization released calcium from an intracellular store or released transmitter by a mechanism that does not require calcium. The possibility that voltage, and not calcium, can regulate the release of transmitter was studied with pairs of solitary retinal neurons. Horizontal and bipolar cells were isolated from fish retinas and juxtaposed in culture. Communication between them was studied with electrophysiological methods. A horizontal cell released its neurotransmitter, gamma-aminobutyric acid, when depolarized during conditions that buffered the internal calcium concentration and prohibited calcium entry. The speed and amount of material released were sufficient for a contribution to synaptic transmission.     

Stretch-inactivated ion channels coexist with stretch-activated ion channels

Stretch-activated ion channels of animal, plant, bacterial, and fungal cells are implicated in mechanotransduction and osmoregulation. A new class of channel has now been described that is stretch-inactivated. These channels occur in neurons, where they coexist with stretch-activated channels. Both channels are potassium selective. The differing stretch sensitivities of the two channels minimize potassium conductance over an intermediate range of tension, with the consequence that, over this same range, voltage-gated calcium channels are most readily opened. Thus, by setting the relation between membrane tension and transmembrane calcium fluxes, stretch-sensitive potassium channels may participate in the control of calcium-dependent motility in differentiating, regenerating, or migrating neurons.     

Calcium-mediated reduction of ionic currents: a biophysical memory trace

Learning behavior similar to vertebrate classical conditioning was demonstrated for the mollusc Hermissenda crassicornis. Postsynaptic membrane changes within well-defined neural systems that mediate the learning play a casual role in recording the learned association for later recall. Specific ionic currents in neural tissue undergo transformations lasting days after associative training with physiologic stimuli. During acquisition the intracellular calcium increases; this increase is accompanied by specific potassium current reduction that lasts for days after conditioning. The increase of calcium enhances calmodulin-dependent phosphorylation of proteins that either regulate or are part of ion channels. These currents and the conditions that precede their transformation occur in many types of vertebrate neurons, and hence this biophysical basis of Hermissenda learning could have relevance for species other than the gastropod studied.     

Cytosolic calcium during contraction of isolated mammalian gastric muscle cells

During activation of visceral smooth muscle there is an increase in cytosolic-free calcium, but the source (intracellular calcium release or calcium influx), kinetics, and stoichiometry of this increase have not been determined. Here, the fluorescent indicator, quin2-acetoxymethyl ester, was used to measure directly cytosolic-free calcium during contraction of isolated stomach muscle cells induced by the two neuropeptides cholecystokinin-octapeptide and Met-enkephalin as well as acetylcholine. An increase in cytosolic-free calcium was seen that was (i) dependent on the concentration of contractile agonist, (ii) derived from intracellular sources (that is, not significantly affected by removal of ambient calcium or addition of a calcium channel blocker), and (iii) kinetically and stoichiometrically related to net calcium efflux and contraction. In contrast, the increase in cytosolic-free calcium induced by depolarizing concentrations of potassium was caused by influx of calcium through voltage-dependent calcium channels.     

Functional properties of rat brain sodium channels expressed in a somatic cell line

Transfection of Chinese hamster ovary cells with complementary DNA encoding the RIIA sodium channel alpha subunit from rat brain led to expression of functional sodium channels with the rapid, voltage-dependent activation and inactivation characteristic of sodium channels in brain neurons. The sodium currents mediated by these transfected channels were inhibited by tetrodotoxin, persistently activated by veratridine, and prolonged by Leiurus alpha-scorpion toxin, indicating that neurotoxin receptor sites 1 through 3 were present in functional form. The RIIA sodium channel alpha subunit cDNA alone is sufficient for stable expression of functional sodium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.     

Highly cooperative opening of calcium channels by inositol 1,4,5-trisphosphate

The kinetics of calcium release by inositol 1,4,5-trisphosphate (IP3) in permeabilized rat basophilic leukemia cells were studied to obtain insight into the molecular mechanism of action of this intracellular messenger of the phosphoinositide cascade. Calcium release from intracellular storage sites was monitored with fura-2, a fluorescent indicator. The dependence of the rate of calcium release on the concentration of added IP3 in the 4 to 40 nM range showed that channel opening requires the binding of at least three molecules of IP3. Channel opening occurred in the absence of added adenosine triphosphate, indicating that IP3 acts directly on the channel or on a protein that gates it. The channels were opened by IP3 in less than 4 seconds. The highly cooperative opening of calcium channels by nanomolar concentrations of IP3 enables cells to detect and amplify very small changes in the concentration of this messenger in response to hormonal, sensory, and growth control stimuli.     

Voltage-sensitive calcium channels in normal and transformed 3T3 fibroblasts

Patch clamp recordings of whole-cell and single channel currents revealed the presence of two voltage-sensitive calcium channel types in the membrane of 3T3 fibroblasts. The two calcium channel types were identified by their unitary properties and pharmacological sensitivities. Both calcium channel types were present in all control 3T3 cells, but one type was selectively suppressed in 3T3 cells that had been transformed by activated c-H-ras, EJ-ras, v-fms, or polyoma middle T oncogenes. The presence of voltage-sensitive calcium channels in these nonexcitable cells and the control of their functional expression by transforming oncogenes raises questions about their role in the control of calcium-sensitive processes such as cell motility, cytoskeletal organization, and cell growth.     

拳参-413对大鼠离体胸主动脉环的舒张作用机制研究

[目的]研究拳参-413对大鼠离体胸主动脉血管的舒张作用机制。[方法]采用离体血管环灌流方法观察拳参-413在含Ca+或无Ca+的Krebs液孵育条件下对去甲肾上腺素(NA)引起的血管平滑肌收缩的影响,考察拳参-413舒张血管作用的时间依赖性,并观察拳参-413对浓度40和80 mmol/L的KCl引起的血管平滑肌收缩的影响。[结果]拳参-413能舒张NA引起的血管收缩,且呈浓度依赖性;拳参-413(100μmol/L)在30 min达到最大舒张效应;无Ca+组拳参-413抑制NA所致血管平滑肌收缩效应大于含Ca+组;拳参-413对浓度40和80 mmol/L的KCl引起的血管平滑肌收缩均有抑制作用,且两者量效曲线明显上移。[结论]拳参-413可舒张血管平滑肌,其作用机制可能与该药促进NO合成释放,开放钙激活的钾通道以及抑制血管平滑肌细胞外钙内流和内钙释放有关。