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1.
By mediating the Ca 2+ influx that triggers exocytotic fusion, Ca 2+ channels play a central role in a wide range of secretory processes. Ca 2+ channels consist of a complex of protein subunits, including an 1 subunit that constitutes the voltage-dependent Ca 2+-selective membrane pore, and a group of auxiliary subunits, including β, γ, and 2–δ subunits, which modulate channel properties such as inactivation and channel targeting. Subtypes of Ca 2+ channels are constituted by different combinations of 1 subunits (of which 10 have been identified) and auxiliary subunits, particularly β (of which 4 have been identified). Activity-secretion coupling is determined not only by the biophysical properties of the channels involved, but also by the relationship between channels and the exocytotic apparatus, which may differ between fast and slow types of secretion. Colocalization of Ca 2+ channels at sites of fast release may depend on biochemical interactions between channels and exocytotic proteins. The aim of this article is to review recent work on Ca 2+ channel structure and function in exocytotic secretion. We discuss Ca 2+ channel involvement in selected types of secretion, including central neurotransmission, endocrine and neuroendocrine secretion, and transmission at graded potential synapses. Several different Ca 2+ channel subtypes are involved in these types of secretion, and their function is likely to involve a variety of relationships with the exocytotic apparatus. Elucidating the relationship between Ca 2+ channel structure and function is central to our understanding of the fundamental process of exocytotic secretion. 相似文献
2.
Highly Ca 2+ selective Ca 2+ channels activated by store depletion have been recently described in several cell types and have been termed CRAC channels (for calcium release-activated calcium). The present study shows that following store depletion in mast and RBL-1 cells, monovalent outward currents could be recorded if the internal solution contained K + but not Cs +. The activation of the outward K + current correlated with the activation of I CRAC, in both time and amplitude, suggesting that the K + current might be carried by CRAC channels. The amplitude of the outward current was increased if external Ca 2+ was reduced or replaced by external Ba 2+. The outward K + conductance might have a physiological role in maintaining the driving force for Ca 2+ entry during the activation of CRAC channels. 相似文献
3.
Measurements of Ca 2+ influx and [Ca 2+] i changes in Fura-2/AM-loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, were used to identify Ca 2+ as the actual second messenger of the prothoracicotropic hormone (PTTH) of this insect. Dose-dependent increases of [Ca 2+] i in PG cells were recorded in the presence of recombinant PTTH (rPTTH) within 5 minutes. The rPTTH-mediated increases of [Ca 2+] i levels were dependent on extracellular Ca 2+. They were not blocked by the dihydropyridine derivative, nitrendipine, an antagonist of high-voltage-activated (HVA) Ca 2+ channels, and by bepridil, an antagonist of low-voltage-activated (LVA) Ca 2+ channels. The trivalent cation La 3+, a non-specific blocker of plasma membrane Ca 2+ channels, eliminated the rPTTH-stimulated increase of [Ca 2+] i levels in PG cells and so did amiloride, an inhibitor of T-type Ca 2+ channels. Incubation of PG cells with thapsigargin resulted in an increase of [Ca 2+] i levels, which was also dependent on extracellular Ca 2+ and was quenched by amiloride, suggesting the existence of store-operated plasma membrane Ca 2+ channels, which can also be inhibited by amiloride. Thapsigargin and rPTTH did not operate independently in stimulating increases of [Ca 2+] i levels and one agent’s mediated increase of [Ca 2+] i was eliminated in the presence of the other. TMB-8, an inhibitor of intracellular Ca 2+ release from inositol 1,4,5 trisphosphate (IP 3)-sensitive Ca 2+ stores, blocked the rPTTH-stimulated increases of [Ca 2+] i levels, suggesting an involvement of IP 3 in the initiation of the rPTTH signaling cascade, whereas ryanodine did not influence the rPTTH-stimulated increases of [Ca 2+] i levels. The combined results indicate the presence of a cross-talk mechanism between the [Ca 2+] i levels, filling state of IP 3-sensitive intracellular Ca 2+ stores and the PTTH-receptor’s-mediated Ca 2+ influx. 相似文献
4.
Ca 2+ efflux from the sarcoplasmic reticulum (SR) is routed primarily through SR Ca 2+ release channels (ryanodine receptors, RyRs). When clusters of RyRs are activated by trigger Ca 2+ influx through L-type Ca 2+ channels (dihydropyridine receptors, DHPR), Ca 2+ sparks are observed. Close spatial coupling between DHPRs and RyR clusters and the relative insensitivity of RyRs to be triggered by Ca 2+ together ensure the stability of this positive-feedback system of Ca 2+ amplification. Despite evidence from single channel RyR gating experiments that phosphorylation of RyRs by protein kinase A (PKA) or calcium-calmodulin dependent protein kinase II (CAMK II) causes an increase in the sensitivity of the RyR to be triggered by [Ca 2+] i there is little clear evidence to date showing an increase in Ca 2+ spark rate. Indeed, there is some evidence that the SR Ca 2+ content may be decreased in hyperadrenergic disease states. The question is whether or not these observations are compatible with each other and with the development of arrhythmogenic extrasystoles that can occur under these conditions. Furthermore, the appearance of an increase in the SR Ca 2+ “leak” under these conditions is perplexing. These and related complexities are analyzed and discussed in this report. Using simple mathematical modeling discussed in the context of recent experimental findings, a possible resolution to this paradox is proposed. The resolution depends upon two features of SR function that have not been confirmed directly but are broadly consistent with several lines of indirect evidence: (1) the existence of unclustered or “rogue” RyRs that may respond differently to local [Ca 2+] i in diastole and during the [Ca 2+] i transient; and (2) a decrease in cooperative or coupled gating between clustered RyRs in response to physiologic phosphorylation or hyper-phosphorylation of RyRs in disease states such as heart failure. Taken together, these two features may provide a framework that allows for an improved understanding of cardiac Ca 2+ signaling. 相似文献
5.
The adjustment of Ca 2+ entry in cardiac cells is critical to the generation of the force necessary for the myocardium to meet the physiological needs of the body. In this review, we present the concept that Ca 2+ can promote its own entry through Ca 2+ channels by different mechanisms. We refer to it under the general term of ‘Ca2+-induced Ca2+ entry’ (CICE). We review short-term mechanisms (usually termed facilitation) that involve a stimulating effect of Ca 2+ on the L-type Ca 2+ current ( ICa-L) amplitude (positive staircase) or a lessening of Ca 2+-dependent inactivation of ICa-L. This latter effect is related to the amount of Ca 2+ released by ryanodine receptors (RyR2) of the sarcoplasmic reticulum (SR). Both effects are involved in the control of action potential (AP) duration. We also describe a long-term mechanism based on Ca 2+-dependent down-regulation of the Kv4.2 gene controlling functional expression of the repolarizing transient outward K + current ( Ito) and, thereby, AP duration. This mechanism, which might occur very early during the onset of hypertrophy, enhances Ca 2+ entry by maintaining Ca 2+ channel activation during prolonged AP. Both Ca 2+-dependent facilitation and Ca 2+-dependent down-regulation of Ito expression favour AP prolongation and, thereby, promote sustained voltage-gated Ca 2+ entry used to enhance excitation–contraction (EC) coupling (with no change in the density of Ca 2+ channels per se). These self-maintaining mechanisms of Ca 2+ entry have significant functions in remodelling Ca 2+ signalling during the cardiac AP. They might support a prominent role of Ca 2+ channels in the establishment and progression of abnormal Ca 2+ signalling during cardiac hypertrophy and congestive heart failure. 相似文献
6.
The study of Ca 2+ sparks has led to extensive new information regarding the gating of the Ca 2+ release channels underlying these events in skeletal, cardiac and smooth muscle cells, as well as the possible roles of these local Ca 2+ release events in muscle function. Here we review basic procedures for studying Ca 2+sparks in skeletal muscle, primarily from frog, as well as the basic results concerning the properties of these events, their pattern and frequency of occurrence during fiber depolarization and the mechanisms underlying their termination. Finally, we also consider the contribution of different ryanodine receptor (RyR) isoforms to Ca 2+ sparks and the number of RyR Ca 2+ release channels that may contribute to the generation of a Ca 2+ spark. Over the decade since their discovery, Ca 2+ sparks have provided a wealth of information concerning the function of Ca 2+ release channels within their intracellular environment. 相似文献
7.
Excitation-contraction coupling in both skeletal and cardiac muscle depends on structural and functional interactions between the voltage-sensing dihydropyridine receptor L-type Ca 2+ channels in the surface/transverse tubular membrane and ryanodine receptor Ca 2+ release channels in the sarcoplasmic reticulum membrane. The channels are targeted to either side of a narrow junctional gap that separates the external and internal membrane systems and are arranged so that bi-directional structural and functional coupling can occur between the proteins. There is strong evidence for a physical interaction between the two types of channel protein in skeletal muscle. This evidence is derived from studies of excitation–contraction coupling in intact myocytes and from experiments in isolated systems where fragments of the dihydropyridine receptor can bind to the ryanodine receptors in sarcoplasmic reticulum vesicles or in lipid bilayers and alter channel activity. Although micro-regions that participate in the functional interactions have been identified in each protein, the role of these regions and the molecular nature of the protein–protein interaction remain unknown. The trigger for Ca 2+ release through ryanodine receptors in cardiac muscle is a Ca 2+ influx through the L-type Ca 2+ channel. The Ca 2+ entering through the surface membrane Ca 2+ channels flows directly onto underlying ryanodine receptors and activates the channels. This was thought to be a relatively simple system compared with that in skeletal muscle. However, complexities are emerging and evidence has now been obtained for a bi-directional physical coupling between the proteins in cardiac as well as skeletal muscle. The molecular nature of this coupling remains to be elucidated. 相似文献
8.
Cyclic ADP-ribose (cADPR) is the most potent Ca 2+-mobilizing agent known. It has been found in many different cell types, where it is synthesized from its precursor NAD + by ADP-ribosyl cyclases. cADPR binds to Ca 2+ channels in the endoplasmic reticulum membrane to activate a Ca 2+-release mechanism. This release is itself potentiated by elevated cytoplasmic Ca 2+ concentrations. Thus, cADPR may function as an endogenous regulator of Ca 2+-induced Ca 2+ release, and there is excitement that it may also function as a Ca 2+-mobilizing second messenger. 相似文献
9.
We investigated the effect of lysophosphatidic acid (LPA), a bioactive phospholipid, on the response in cytosolic free Ca 2+ concentration ([Ca 2+] i) to mechanical stress in cultured bovine lens epithelial cells. Spritzing of bath solution onto cells as mechanical stress caused marked increase in [Ca 2+] i in the presence of LPA and this increase was concentration-dependent (1–10 μM), whereas neither addition of LPA alone nor the mechanical stress in the absence of LPA affected [Ca 2+] i. The mechanical stress-induced increase in [Ca 2+] i in the presence of LPA was inhibited by removing extracellular Ca 2+ or by addition of Gd 3+, a blocker of mechanosensitive cation channels, but not by nicardipine, thapsigargin, an inhibitor of endoplasmic reticulum-ATPase pump, or U73122, a phospholipase C inhibitor. These results show that LPA sensitises Ca 2+ influx through cation-selective mechanosensitive channels, but does not sensitise Ca 2+ release from intracellular stores, triggered by changes in mechanical stress. On the other hand, phosphatidic acid had less of a sensitising effect than LPA, and neither lysophosphatidylcholine nor chlorpromazine had any effect. Also Ca 2+ mobilising agonists, ATP, histamine and carbachol, did not sensitise Ca 2+ response to the mechanical stress. These results show that LPA sensitises mechanoreceptor-linked response in lens epithelial cells, suggesting that it plays a role in the development of cataracts due to increases in [Ca 2+] i induced by mechanical stress. 相似文献
10.
Large-conductance Ca 2+-activated K + channels (BK channels) constitute an key physiological link between cellular Ca 2+ signaling and electrical signaling at the plasma membrane. Thus these channels are critical to the control of action potential firing and neurotransmitter release in several types of neurons, as well as the dynamic control of smooth muscle tone in resistance arteries, airway, and bladder. Recent advances in our understanding of K + channel structure and function have led to new insight toward the molecular mechanisms of opening and closing (gating) of these channels. Here we will focus on mechanisms of BK channel gating by Ca 2+, transmembrane voltage, and auxiliary subunit proteins. 相似文献
11.
Using a new fluorescence imaging technique, LAMP, we recently reported that Ca 2+ influx through store operated Ca 2+ channels (SOCs) strongly inhibits cell coupling in primary human fibroblasts (HF) expressing Cx43. To understand the mechanism of inhibition, we studied the involvement of cytosolic pH (pH i) and Ca 2+([Ca 2+] i) in the process by using fluorescence imaging and ion clamping techniques. During the capacitative Ca 2+ influx, there was a modest decline of pH i measured by BCECF. Decreasing pH i below neutral using thioacetate had little effect by itself on cell coupling, and concomitant pH i drop with thioacetate and bulk [Ca 2+i rise with ionomycin was much less effective in inhibiting cell coupling than Ca 2+ influx. Moreover, clamping pH i with a weak acid and a weak base during Ca 2+ influx largely suppressed bulk pH i drop, yet the inhibition of cell coupling was not affected. In contrast, buffering [Ca 2+i with BAPTA, but not EGTA, efficiently prevented cell uncoupling by Ca 2+ influx. We concluded that local Ca 2+ elevation subjacent to the plasma membrane is the primary cause for closing Cx43 channels during capacitative Ca 2+ influx. To assess how Ca 2+ influx affects junctional coupling mediated by other types of connexins, we applied the LAMP assay to Hela cells expressing Cx26. Capacitative Ca 2+ influx also caused a strong reduction of cell coupling, suggesting that the inhibitory effect by Ca 2+ influx may be a more general phenomenon. 相似文献
12.
Glucose-induced insuline release, glucose-induced rises in intracellular free Ca 2+ concentration ([Ca 2+] i), and voltage-dependent Ca 2+ channel activity were assessed in monolayer cultures of β-vells 3–5 day-old rats. The glucose-stimulated insulin secretory responses and [Ca 2+] i rises were like those in adult rat β-cells rather than fetal rat β-cells. Voltage-dependent Ca 2+ channel antagonists decreased glucose-induced insulin secretion, aborted the [Ca 2+] 2 rise and, like deprivation of extracellular Ca 2+, prevented the glucose-induced rise in [Ca 2+] i when added before the glucose challenge. The presence of nifedipine-sensitive, voltage-dependent Ca 2+ channels was demonstrated directly by measuring Ca 2+ currents using the whole-cell configuration of the patch-clamp technique and indirectly by measuring [Ca 2+] 1 after membrane depolarization by 45 mMm K + or 200 μM tolbutamide. Thus, in cultured β-cells of 3–5 day-old rats the coupling of glucose stimulation to Ca 2+ influx is essentially mature, in contrast to what has been reported for fetal or very early neonatal cells. 相似文献
13.
The store-mediated Ca 2+ entry was detected in single and cluster of rat submandibular acinar cells by measuring the Ca 2+ activated ionic membrane currents. In the cells where intracellular Ca 2+ was partly depleted by stimulation with submaximal concentration of acetylcholine (ACh) under a Ca 2+-free extracellular condition, an employment of external Ca 2+ in the absence of ACh caused a sustained increase of the K + current without affecting the Cl − current. A renewed ACh challenge without external Ca 2+ caused repetitive spikes of both K + and Cl − currents due to the Ca 2+ release. SK & F 96365 inhibited the generation of the sustained K + current and refilling of the Ca 2+ store following the Ca 2+ readmission. It is suggested that the Ca 2+ enters the cell through the store-mediated pathway near the K + channels and is taken up by the store. Thus, only Ca 2+ released from the store can activate both the K + and Cl − currents. 相似文献
14.
Fluoxetine, a selective 5-HT uptake inhibitor, inhibited 15 mM K +-induced [ 3H] 5-HT release from rat spinal cord and cortical synaptosomes at concentrations > 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K + used to depolarize the synaptosomes and the concentration of external Ca 2+. Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of [ 3H] 5-HT release induced by the Ca 2+-ionophore A 23187 or Ca 2+-independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K +-induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca 2+ channels and Ca 2+ entry. Whereas fluoxetine and paroxetine inhibited binding of [ 3H] nitrendipine to the dihydropyridine-sensitive L-type Ca 2+ channel, the less selective uptake inhibitors did not alter binding. The dihydropyridine antagonist nimodipine partially blocked fluoxetine-induced inhibition of release. Moreover enhanced K +-stimulated release due to the dihydropyridine agonist Bay K 8644 was reversed by fluoxetine. Fluoxetine also inhibited the K +-induced increase in intracellular free Ca 2+ in fura-2 loaded synaptosomes. These data are consistent with the suggestion that fluoxetine inhibits K +-induced [ 3H] 5-HT release by antagonizing voltage-dependent Ca 2+ entry into nerve terminals. 相似文献
15.
Influence of hypotonic swelling on Ca 2+ ( 45Ca 2+) uptake in rat brain synaptosomes was studied. A decrease in medium osmolality from 310 to 260-180 mOsm led to a progressive stimulation of 45Ca 2+ accumulation. The effect was blocked by verapamil (IC 50 = 5 μM), CoCl 50 = 58 μM) and retained at a fixed concentration of external sodium indicating the involvement of Ca 2+ channels rather than Na +/Ca 2+ exchange in swelling-induced Ca 2+ influx. The populations of calcium channels observed in hypoosmotic and depolarizing conditions are different in three aspects: (i) kinetics of 45Ca 2+ entry; (ii) insensitivity to dihydropyridines and ω-conotoxin GVIA; (iii) insensitivity to preliminary depolarization by high potassium. The effects of swelling and depolarization on Ca 2+ uptake were additive. No change in membrane potential monitored with diS-C 3-(5) was recorded during synaptosome hypotonic swelling. The results suggest the existence in synaptosomal plasma membrane of volume-dependent calcium-permeable channels with properties distinct from those of the voltage-dependent calcium channels. Activation of these channels may constitute an early event in volume regulation of nerve terminals in anisoosmotic conditions. 相似文献
16.
The role of Ca 2+ in glycerol dissimilation under hypoosmotic stress in the halotolerant alga Dunaliella tertiolecta was investigated using a pharmacological approach. A stretch-activated Ca 2+ channel blocker, GdCl 3, inhibited glycerol dissimilation under hypoosmotic stress. However, addition of voltage-dependent Ca 2+ channel blockers and inhibitors of mitochondrial and endoplasmic reticulum Ca 2+ channels did not affect the glycerol dissimilation under hypoosmotic stress. The results of the present study suggest that the influx of Ca 2+ from the extracellular space via the stretch-activated Ca 2+ channels localized in the plasma membrane is required for the transduction of osmotic signal of D. tertiolecta. 相似文献
17.
Stretch of the myocardium influences the shape and amplitude of the intracellular Ca 2+([Ca 2+] i) transient. Under isometric conditions stretch immediately increases myofilament Ca 2+ sensitivity, increasing force production and abbreviating the time course of the [Ca 2+] i transient (the rapid response). Conversely, muscle shortening can prolong the Ca 2+ transient by decreasing myofilament Ca 2+ sensitivity. During the cardiac cycle, increased ventricular dilation may increase myofilament Ca 2+ sensitivity during diastolic filling and the isovolumic phase of systole, but enhance the decrease in myofilament Ca 2+ sensitivity during the systolic shortening of the ejection phase. If stretch is maintained there is a gradual increase in the amplitude of the Ca 2+ transient and force production, which takes several minutes to develop fully (the slow response). The rapid and slow responses have been reported in whole hearts and single myocytes. Here we review stretch-induced changes in [Ca 2+] i and the underlying mechanisms. Myocardial stretch also modifies electrical activity and the opening of stretch-activated channels (SACs) is often used to explain this effect. However, the myocardium has many ionic currents that are regulated by [Ca2+]i and in this review we discuss how stretch-induced changes in [Ca2+]i can influence electrical activity via the modulation of these Ca2+-dependent currents. Our recent work in single ventricular myocytes has shown that axial stretch prolongs the action potential. This effect is sensitive to either SAC blockade by streptomycin or the buffering of [Ca2+]i with BAPTA, suggesting that both SACs and [Ca2+]i are important for the full effects of axial stretch on electrical activity to develop. 相似文献
18.
In this study we investigated the release of Ca 2+ in brain microsomes after Ca 2+ loading by the Ca 2+-ATPase or by the Na +/Ca 2+ exchanger. The results show that in microsomes loaded with Ca 2+ by the Ca 2+-ATPase, Ins(1,4,5)P 3 (5 μM) release 21±2% of the total Ca 2+ accumulated, and that in the microsomes loaded with Ca 2+ by the Na 2+/Ca 2+ exchanger, Ins(1,4,5)P 3 released 28±3% of the total Ca 2+ accumulated. These results suggest that receptors of Ins(1,4,5)P 3 may be co-localized with the Na 2+/Ca 2+ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P 3 receptors in the plasma membrane where the Na 2+/Ca 2+ exchanger is normally present, or both. We also found that Ins(1,4,5)P 3 inhibited the Ca 2+-ATPase by 33.7%, but that it had no significant effect on the Na 2+/Ca 2+ exchanger. 相似文献
19.
The role of stretch-activated channels (SACs) on the stretch-induced changes of rat atrial myocytes was studied using a computer model that incorporated various ion channels and transporters including SACs. A relationship between the extent of the stretch and the activation of SACs was formulated in the model based on experimental findings to reproduce changes in electrical activity and Ca 2+ transients by stretch. Action potentials (APs) were significantly changed by the activation of SACs in the model simulation. The duration of the APs decreased at the initial fast phase and increased at the late slow phase of repolarisation. The resting membrane potential was depolarised from −82 to −70 mV. The Ca 2+ transients were also affected. A prolonged activation of SACs in the model gradually increased the amplitude of the Ca 2+ transients. The removal of Ca 2+ permeability through SACs, however, had little effect on the stretch-induced changes in electrical activity and Ca 2+ transients in the control condition. In contrast, the removal of the Na + permeability nearly abolished these stretch-induced changes. Plotting the peaks of the Ca 2+ transients during the activation of the SACs along a time axis revealed that they follow the time course of the Na i+ concentration. The Ca 2+ transients were not changed when the Na i+ concentration was fixed to a control value (5.4 mM). These results predicted by the model suggest that the influx of Na + rather than Ca 2+ through SACs is more crucial to the generation of stretch-induced changes in the electrical activity and associated Ca 2+ transients of rat atrial myocytes. 相似文献
20.
Mechanical compression of cartilage is associated with a rise in the interstitial osmotic pressure, which can alter cell volume and activate volume recovery pathways. One of the early events implicated in regulatory volume changes and mechanotransduction is an increase of intracellular calcium ion ([Ca 2+] i). In this study, we tested the hypothesis that osmotic stress initiates intracellular Ca 2+ signaling in chondrocytes. Using laser scanning microscopy and digital image processing, [Ca 2+] i and cell volume were monitored in chondrocytes exposed to hyper-osmotic solutions. Control experiments showed that exposure to hyper-osmotic solution caused significant decreases in cell volume as well as transient increases in [Ca 2+] i. The initial peak in [Ca 2+] i was generally followed by decaying oscillations. Pretreatment with gadolinium, a non-specific blocker of mechanosensitive ion channels, inhibited this [Ca 2+] i increase. Calcium-free media eliminated [Ca 2+] i increases in all cases. Pretreatment with U73122, thapsigargin, or heparin (blockers of the inositol phosphate pathway), or pertussis toxin (a blocker of G-proteins) significantly decreased the percentage of cells responding to osmotic stress and nearly abolished all oscillations. Cell volume decreased with hyper-osmotic stress and recovered towards baseline levels throughout the duration of the control experiments. The peak volume change with 550 mOsm osmotic stress, as well as the percent recovery of cell volume, was dependent on [Ca 2+] i. These findings indicate that osmotic stress causes significant volume change in chondrocytes and may activate an intracellular second messenger signal by inducing transient increases in [Ca 2+] i. 相似文献
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