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1.
Strong depolarization and dihydropyridine agonists potentiate inward currents through native L-type Ca2+ channels, but the effect on outward currents is less clear due to the small size of these currents. Here, we examined potentiation of wild-type alpha1C and two constructs bearing mutations in conserved glutamates in the pore regions of repeats II and IV (E2A/E4A-alpha1C) or repeat III (E3K-alpha1C). With 10 mM Ca2+ in the bath and 110 mM Cs+ in the pipette, these mutated channels, expressed in dysgenic myotubes, produced both inward and outward currents of substantial amplitude. For both the wild-type and mutated channels, we observed strong inward rectification of potentiation: strong depolarization had little effect on outward tail currents but caused the inward tail currents to be larger and to decay more slowly. Similarly, exposure to DHP agonist increased the amplitude of inward currents and decreased the amplitude of outward currents through both E2A/E4A-alpha1C and E3K-alpha1C. As in the absence of drug, strong depolarization in the presence of dihydropyridine agonist had little effect on outward tail currents but increased the amplitude and slowed the decay of inward tail currents. We tested whether cytoplasmic Mg2+ functions as the blocking particle responsible for the rectification of potentiated L-type Ca2+ channels. However, even after complete removal of cytoplasmic Mg2+, (-)BayK 8644 still potentiated inward current and partially blocked outward current via E2A/E4A-alpha1C. Although zero Mg2+ did not reveal potentiation of outward current by DHP agonist, it did have two striking effects, (a) a strong suppression of decay of both inward and outward currents via E2A/E4A-alpha1C and (b) a nearly complete elimination of depolarization-induced potentiation of inward tail currents. These results can be explained by postulating that potentiation exposes a binding site in the pore to which an intracellular blocking particle can bind and produce inward rectification of the potentiated channels. 相似文献
2.
V. A. Bouryi 《Neurophysiology》1998,30(4-5):301-304
Barium currents through ion channels formed by α1-subunit of L-type Ca2+ channel (I
α1) were recorded from cultured chinese hamster ovary (CHO) cells. The cells were stably transfected with either a cardiac or
a smooth muscle (SM) variant of α1-subunit. TheI
α1 in both cases exhibited similar fast voltage-dependent activation kinetics and slow apparent inactivation kinetics. With
10 mM Ba2+ in the bath solution,I
α1 was activated at potentials more positive than −40 mV, peaked between 0 and +10 mV, and reversed at about +50 mV. In addition
to slow apparent inactivation of inward current, both subunits provided an extremely slow voltage-dependent inactivation at
potentials more positive than −100 mV, with half-maximum inactivation at −43.4 mV for cardiac and −41.4 mV for SM α1-subunits.
The onset of inactivation as well as recovery from this process were within a time range of minutes. The voltage dependence
of steady-state inactivation could be fitted by the sum of two Boltzmann's equations with slope factors of about 12 mV and
5 mV. A less sloped component has its midpoints at −75.6 and −63.7 mV, and a steeper component has its midpoints at −42.8
and −37.7 mV for cardiac and SM α1-subunits, respectively. Relative contribution of the steeper component was higher in both
subunits (0.86 and 0.66 for cardiac and SM subunits, respectively). For comparison, the inactivation curves for 5-sec-long
conditioning prepulses could be fitted by single Boltzmann's distribution with a 20 mV more positive midpoint and a slope
factor of about 13 mV. In contrast to the steady-state inactivation curves, they showed considerable overlap with the steady-state
activation curve. Our results reflect functional consequences of known sequence differences between α1-subunits of the cardiac
and SM L-type Ca2+ channels and could be used in structural modeling of Ca2+ channel gating. In addition, they show that depolarization-induced window current has a transient nature and decays with
the development of extremely slow inactivation. This is the first demonstration that slow inactivation of the L-type Ca2+ channel is an intrinsic property of its α1-subunits. 相似文献
3.
Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain 总被引:3,自引:0,他引:3
Based on electrophysiological studies, Ca(2+)-activated K(+) channels and voltage-gated Ca(2+) channels appear to be located in close proximity in neurons. Such colocalization would ensure selective and rapid activation of K(+) channels by local increases in the cytosolic calcium concentration. The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels and precipitated with antibodies against alpha(1C) and alpha(1D) L-type Ca(2+) channels. To confirm the specificity of the interaction, precipitation experiments were carried out also in reverse order. Also, additive precipitation was performed because alpha(1C) and alpha(1D) L-type Ca(2+) channels always refer to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain. 相似文献
4.
Hepcidin is a key player in the regulation of iron homeostasis. Several pathological conditions associated with iron overload are attributed to the depressed expression of hepcidin and are often associated with bone diseases including osteoporosis. Hepcidin was suggested to have anti-osteoporosis effects by preventing iron overload. We recently observed that hepcidin could increase intracellular calcium concentration in cultured osteoblast cells. The present study was designed to elucidate the source of the increased intracellular calcium following hepcidin activation. Cultured hFOB1.19 cells were used to test whether there was a dose dependent effect of hepcidin on increasing intracellular calcium. After finding the optimal concentration in increasing intracellular calcium, Cultured hFOB1.19 cells were then divided into three groups: (1) control group, (2) and (3) groups pretreated with either nimodipine (2 × 10(-5)mol/L) or EDTA (2 × 10(-3)mol/L) for 10 min before incubation with hepcidin (100 nmol/L). All cells were stimulated with hepcidin for 60 min and then stained with fluo-3/AM for 40 min before the intracellular calcium was observed using flow cytometry (FCM). As compared with controls, hepcidin treatment significantly increased intracellular calcium concentration. This effect was blocked by nimodipine and EDTA pretreatments which suggested that hepcidin-mediated calcium inflow was mainly through L-type Ca(2+) channels and that the release of intracellular calcium store was not significant. Hepcidin increases of intracellular calcium may be related to its anti-osteoporosis effect but this hypothesis needs further investigation. 相似文献
5.
Isaev D Solt K Gurtovaya O Reeves JP Shirokov R 《The Journal of general physiology》2004,123(5):555-571
Both intracellular calcium and transmembrane voltage cause inactivation, or spontaneous closure, of L-type (CaV1.2) calcium channels. Here we show that long-lasting elevations of intracellular calcium to the concentrations that are expected to be near an open channel (>/=100 microM) completely and reversibly blocked calcium current through L-type channels. Although charge movements associated with the opening (ON) motion of the channel's voltage sensor were not altered by high calcium, the closing (OFF) transition was impeded. In two-pulse experiments, the blockade of calcium current and the reduction of gating charge movements available for the second pulse developed in parallel during calcium load. The effect depended steeply on voltage and occurred only after a third of the total gating charge had moved. Based on that, we conclude that the calcium binding site is located either in the channel's central cavity behind the voltage-dependent gate, or it is formed de novo during depolarization through voltage-dependent rearrangements just preceding the opening of the gate. The reduction of the OFF charge was due to the negative shift in the voltage dependence of charge movement, as previously observed for voltage-dependent inactivation. Elevation of intracellular calcium concentration from approximately 0.1 to 100-300 microM sped up the conversion of the gating charge into the negatively distributed mode 10-100-fold. Since the "IQ-AA" mutant with disabled calcium/calmodulin regulation of inactivation was affected by intracellular calcium similarly to the wild-type, calcium/calmodulin binding to the "IQ" motif apparently is not involved in the observed changes of voltage-dependent gating. Although calcium influx through the wild-type open channels does not cause a detectable negative shift in the voltage dependence of their charge movement, the shift was readily observable in the Delta1733 carboxyl terminus deletion mutant, which produces fewer nonconducting channels. We propose that the opening movement of the voltage sensor exposes a novel calcium binding site that mediates inactivation. 相似文献
6.
Yamaoka K Yuki T Kawase K Munemori M Seyama I 《American journal of physiology. Heart and circulatory physiology》2002,282(3):H1092-H1101
We examined the concentration-dependent blocking effects of intracellular Mg2+ on L-type Ca2+ channels in cardiac myocytes using the whole cell patch-clamp technique. The increase of L-type Ca2+ channel current (I(Ca)) (due to relief of Mg2+ block) occurred in two temporal phases. The rapid phase (runup) transiently appeared early (<5 min) in dialysis of the low-Mg2+ solution; the slow phase began later in dialysis (>10 min). Runup was not blocked by intracellular GTP (GTP(i)). The late phase of the I(Ca) increase (late I(Ca)) was suppressed by GTP(i) (0.4 mM) and was observed in myocytes of the guinea pig or frog at higher (32 or 24 degrees C, respectively) rather than lower temperatures (24 or 17.5 degrees C, respectively). At pMg = 6.0, raising the temperature from 24 to 32 degrees C evoked late I(Ca) with a Q10 of 14.5. Restoring the temperature to 24 degrees C decreased I(Ca) with a Q10 of only 2.4. The marked difference in the Q10 values indicated that late I(Ca) (pMg = 5-6) is an irreversible phenomenon. Phosphorylation suppressed the intracellular [Mg2+] dependency of late I(Ca). This effect of phosphorylation together with the inhibitory action of GTP(i) on Mg2+-dependent blocking of I(Ca) are common properties of mammalian and amphibian cardiomyocytes. 相似文献
7.
A kinetic model of Ca2+-dependent inactivation (CDI) of L-type Ca2+ channels was developed. The model is based on the hypothesis that postulates the existence of four short-lived modes with lifetimes of a few hundreds of milliseconds. Our findings suggest that the transitions between the modes is primarily determined by the binding of Ca2+ to two intracellular allosteric sites located in different motifs of the CI region, which have greatly differing binding rates for Ca2+ (different k(on)). The slow-binding site is controlled by local Ca2+ near a single open channel that is consistent with the "domain" CDI model, and Ca2+ binding to the fast-binding site(s) depends on Ca2+ arising from distant sources that is consistent with the "shell" CDI model. The model helps to explain numerous experimental findings that are poorly understood so far. 相似文献
8.
Calmodulin is the Ca2+ sensor for Ca2+ -dependent inactivation of L-type calcium channels 总被引:6,自引:0,他引:6
Elevated intracellular Ca2+ triggers inactivation of L-type calcium channels, providing negative Ca2+ feedback in many cells. Ca2+ binding to the main alpha1c channel subunit has been widely proposed to initiate such Ca2+ -dependent inactivation. Here, we find that overexpression of mutant, Ca2+ -insensitive calmodulin (CaM) ablates Ca2+ -dependent inactivation in a "dominant-negative" manner. This result demonstrates that CaM is the actual Ca2+ sensor for inactivation and suggests that CaM is constitutively tethered to the channel complex. Inactivation is likely to occur via Ca2+ -dependent interaction of tethered CaM with an IQ-like motif on the carboxyl tail of alpha1c. CaM also binds to analogous IQ regions of N-, P/Q-, and R-type calcium channels, suggesting that CaM-mediated effects may be widespread in the calcium channel family. 相似文献
9.
《The Journal of general physiology》1996,108(5):363-374
Hydrogen ions are important regulators of ion flux through voltage- gated Ca2+ channels but their site of action has been controversial. To identify molecular determinants of proton block of L-type Ca2+ channels, we combined site-directed mutagenesis and unitary current recordings from wild-type (WT) and mutant L-type Ca2+ channels expressed in Xenopus oocytes. WT channels in 150 mM K+ displayed two conductance states, deprotonated (140 pS) and protonated (45 pS), as found previously in native L-type Ca2+ channels. Proton block was altered in a unique fashion by mutation of each of the four P-region glutamates (EI-EIV) that form the locus of high affinity Ca2+ interaction. Glu(E)-->Gln(Q) substitution in either repeats I or III abolished the high-conductance state, as if the titration site had become permanently protonated. While the EIQ mutant displayed only an approximately 40 pS conductance, the EIIIQ mutant showed the approximately 40 pS conductance plus additional pH-sensitive transitions to an even lower conductance level. The EIVQ mutant exhibited the same deprotonated and protonated conductance states as WT, but with an accelerated rate of deprotonation. The EIIQ mutant was unusual in exhibiting three conductance states (approximately 145, 102, 50 pS, respectively). Occupancy of the low conductance state increased with external acidification, albeit much higher proton concentration was required than for WT. In contrast, the equilibrium between medium and high conductance levels was apparently pH-insensitive. We concluded that the protonation site in L-type Ca2+ channels lies within the pore and is formed by a combination of conserved P-region glutamates in repeats I, II, and III, acting in concert. EIV lies to the cytoplasmic side of the site but exerts an additional stabilizing influence on protonation, most likely via electrostatic interaction. These findings are likely to hold for all voltage-gated Ca2+ channels and provide a simple molecular explanation for the modulatory effect of H+ ions on open channel flux and the competition between H+ ions and permeant divalent cations. The characteristics of H+ interactions advanced our picture of the functional interplay between P-region glutamates, with important implications for the mechanism of Ca2+ selectivity and permeation. 相似文献
10.
Chahine M Sculptoreanu A Varma DR 《Canadian journal of physiology and pharmacology》2003,81(2):135-141
L-type Ca2+ channels are essential in triggering the intracellular Ca2+ release and contraction in heart cells. In this study, we used patch clamp technique to compare the effect of two pure enantiomers of L-type Ca2+ channel agonists: (+)-CGP 48506 and the dihydropyridine (+)-SDZ-202 791 in cardiomyocytes from rats 2-5 days old. The predominant Ca2+ current activated by standard step pulses in these myocytes was L-type Ca2+ current. The dihydropyridine antagonist (+)-PN200-110 (5 microM) blocked over 90% of Ca2+ currents in most cells tested. CGP 48506 lead to a maximum of 200% increase in currents. The threshold concentration for the CGP effect was at 1 microM and the maximum was reached at 20 microM. SDZ-202 791 had effects in nanomolar concentrations and a maximum effect at about 2 microM. The maximal effect of (+)-SDZ-202 791 was a 400% increase in the amplitude of Ca2+ currents and was accompanied by a 10-15 mV leftward shift in the voltage dependence of activation. CGP 48506 increased the currents equally at all voltages tested. Both compounds slowed the deactivation of tail currents and lead to the appearance of slowly activating and slowly deactivating current components. However, SDZ-202 791 had larger effects on deactivation and CGP 48506 had larger effect on the rate of Ca2+ current activation. The effect of SDZ-202 791 was fully additive to that of CGP 48506 even after maximum concentrations of CGP. This observation suggests that the two Ca2+ channel agonists may act at two different sites on the L-type Ca2+ channel. We suggest that CGP 48506 would be a potential cardiotonic agent without the deleterious proarrhythmic effects attributable to the dihydropyridine agonists. 相似文献
11.
Díaz-Prieto N Herrera-Peco I de Diego AM Ruiz-Nuño A Gallego-Sandín S López MG García AG Cano-Abad MF 《Cell calcium》2008,44(4):339-352
Altered calcium homeostasis and increased cytosolic calcium concentrations ([Ca(2+)](c)) are linked to neuronal apoptosis in epilepsy and in cerebral ischemia, respectively. Apoptotic programmed cell death is regulated by the antiapoptotic Bcl2 family of proteins. Here, we investigated the role of Bcl2 on calcium (Ca(2+)) homeostasis in PC12 cells, focusing on L-type voltage-dependent calcium channels (VDCC). Cytosolic Ca(2+) transients ([Ca(2+)](c)) and changes of mitochondrial Ca(2+) concentrations ([Ca(2+)](m)) were monitored using cytosolic and mitochondrially targeted aequorins of control PC12 cells and PC12 cells stably overexpressing Bcl2. We found that: (i) the [Ca(2+)](c) and [Ca(2+)](m) elevations elicited by K(+) pulses were markedly depressed in Bcl2 cells, with respect to control cells; (ii) such depression of [Ca(2+)](m) was not seen either in digitonin-permeabilized cells or in intact cells treated with ionomycin; (iii) the [Ca(2+)](c) transient depression seen in Bcl2 cells was reversed by shRNA transfection, as well as by the Bcl2 inhibitor HA14-1; (iv) the L-type Ca(2+) channel agonist Bay K 8644 enhanced K(+)-evoked [Ca(2+)](m) peak fourfold in Bcl2, and twofold in control cells; (v) in current-clamped cells the depolarization evoked by K(+) generated a more hyperpolarized voltage step in Bcl2, as compared to control cells. Taken together, our experiments suggest that the reduction of the [Ca(2+)](c) and [Ca(2+)](m) transients elicited by K(+), in PC12 cells overexpressing Bcl2, is related to the reduction of Ca(2+) entry through L-type Ca(2+) channels. This may be due to the fact that Bcl2 mitigates cell depolarization, thus diminishing the recruitment of L-type Ca(2+) channels, the subsequent Ca(2+) entry, and mitochondrial Ca(2+) overload. 相似文献
12.
Molecular basis of calmodulin tethering and Ca2+-dependent inactivation of L-type Ca2+ channels. 总被引:6,自引:0,他引:6
G S Pitt R D Zühlke A Hudmon H Schulman H Reuter R W Tsien 《The Journal of biological chemistry》2001,276(33):30794-30802
Ca(2+)-dependent inactivation (CDI) of L-type Ca(2+) channels plays a critical role in controlling Ca(2+) entry and downstream signal transduction in excitable cells. Ca(2+)-insensitive forms of calmodulin (CaM) act as dominant negatives to prevent CDI, suggesting that CaM acts as a resident Ca(2+) sensor. However, it is not known how the Ca(2+) sensor is constitutively tethered. We have found that the tethering of Ca(2+)-insensitive CaM was localized to the C-terminal tail of alpha(1C), close to the CDI effector motif, and that it depended on nanomolar Ca(2+) concentrations, likely attained in quiescent cells. Two stretches of amino acids were found to support the tethering and to contain putative CaM-binding sequences close to or overlapping residues previously shown to affect CDI and Ca(2+)-independent inactivation. Synthetic peptides containing these sequences displayed differences in CaM-binding properties, both in affinity and Ca(2+) dependence, leading us to propose a novel mechanism for CDI. In contrast to a traditional disinhibitory scenario, we suggest that apoCaM is tethered at two sites and signals actively to slow inactivation. When the C-terminal lobe of CaM binds to the nearby CaM effector sequence (IQ motif), the braking effect is relieved, and CDI is accelerated. 相似文献
13.
14.
15.
Intracellular pH modulates the availability of vascular L-type Ca2+ channels 总被引:5,自引:1,他引:4 下载免费PDF全文
《The Journal of general physiology》1994,103(4):647-663
L-type Ca2+ channel currents were recorded from myocytes isolated from bovine pial and porcine coronary arteries to study the influence of changes in intracellular pH (pHi). Whole cell ICa fell when pHi was made more acidic by substituting HEPES/NaOH with CO2/bicarbonate buffer (pHo 7.4, 36 degrees C), and increased when pHi was made more alkaline by addition of 20 mM NH4Cl. Peak ICa was less pHi sensitive than late ICa (170 ms after depolarization to 0 mV). pHi-effects on single Ca2+ channel currents were studied with 110 mM BaCl2 as the charge carrier (22 degrees C, pHo 7.4). In cell-attached patches pHi was changed by extracellular NH4Cl or through the opened cell. In inside-out patches pHi was controlled through the bath. Independent of the method used the following results were obtained: (a) Single channel conductance (24 pS) and life time of the open state were not influenced by pHi (between pHi 6 and 8.4). (b) Alkaline pHi increased and acidic pHi reduced the channel availability (frequency of nonblank sweeps). (c) Alkaline pHi increased and acidic pHi reduced the frequency of late channel re- openings. The effects are discussed in terms of a deprotonation (protonation) of cytosolic binding sites that favor (prevent) the shift of the channels from a sleepy to an available state. Changes of bath pHo mimicked the pHi effects within 20 s, suggesting that protons can rapidly permeate through the surface membrane of vascular smooth muscle cells. The role of pHi in Ca2+ homeostases and vasotonus is discussed. 相似文献
16.
A role of pertussis toxin (PTX)-sensitive pathway in regulation of glucose-stimulated Ca2+ signaling in rat islet beta-cells was investigated by using clonidine as a selective agonist to alpha2-adrenoceptors which link to the pathway. An elevation of extracellular glucose concentration from 5.5 to 22.2 mM (glucose stimulation) increased the levels of [Ca2+]i of beta-cells, and clonidine reversibly reduced the elevated levels of [Ca2+]i. This clonidine effect was antagonized by yohimbine, and abolished in beta-cells pre-treated with PTX. Clonidine showed little effect on membrane currents including those through ATP-sensitive K+ channels induced by voltage ramps from -90 to -50 mV. Clonidine showed little effect on the magnitude of whole-cell currents through L-type Ca2+ channels (ICa(L)), but increased the inactivation process of the currents. Clonidine increased the magnitude of the voltage-dependent K+ currents (IVK). These clonidine effects on ICa(L) and IVK were abolished in beta-cells treated with PTX or GDP-betaS. These results suggest that the PTX-sensitive pathway increases IVK activity and decreases ICa(L) activity of islet beta-cells, resulting in a decrease in the levels of [Ca2+]i elevated by depolarization-induced Ca2+ entry. This mechanism seems responsible at least in part for well-known inhibitory action of PTX-sensitive pathway on glucose-stimulated insulin secretion from islet beta-cells. 相似文献
17.
18.
Gong Q Kakei M Koriyama N Nakazaki M Morimitsu S Yaekura K Tei C 《Cell structure and function》2000,25(5):279-289
We used the patch-clamp technique to study the effects of extracellular ATP on the activity of ion channels recorded in rat pancreatic beta-cells. In cell-attached membrane patches, action currents induced by 8.3 mM glucose were inhibited by 0.1 mM ATP, 0.1 mM ADP or 15 microM ADPbetaS but not by 0.1 mM AMP or 0.1 mM adenosine. In perforated membrane patches, action potentials were measured in current clamp, induced by 8.3 mM glucose, and were also inhibited by 0.1 mM ATP with a modest hyperpolarization to -43 mV. In whole-cell clamp experiments, ATP dose-dependently decreased the amplitudes of L-type Ca2+ channel currents (ICa) to 56.7+/-4.0% (p<0.001) of the control, but did not influence ATP-sensitive K+ channel currents observed in the presence of 0.1 mM ATP and 0.1 mM ADP in the pipette. Agonists of P2Y purinoceptors, 2-methylthio ATP (0.1 mM) or ADPbetaS (15 microM) mimicked the inhibitory effect of ATP on ICa, but PPADS (0.1 mM) and suramin (0.2 mM), antagonists of P2 purinoceptors, counteracted this effect. When we used 0.1 mM GTPgammaS in the pipette solution, ATP irreversibly reduced ICa to 58.4+/-6.6% of the control (p<0.001). In contrast, no inhibitory effect of ATP was observed when 0.2 mM GDPbetaS was used in the pipette solution. The use of either 20 mM BAPTA instead of 10 mM EGTA, or 0.1 mM compound 48/80, a blocker of phospholipase C (PLC), in the pipette solution abolished the inhibitory effect of ATP on ICa, but 1 microM staurosporine, a blocker of protein kinase C (PKC), did not. When the beta-cells were pretreated with 0.4 microM thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca2+ pump, ATP lost the inhibitory effect on ICa. These results suggest that extracellular ATP inhibits action potentials by Ca2+-induced ICa inhibition in which an increase in cytosolic Ca2+ released from thapsigargin-sensitive store sites was brought about by a P2Y purinoceptor-coupled G-protein, PI-PLC and IP3 pathway. 相似文献
19.
Lemmens R Larsson O Berggren PO Islam MS 《The Journal of biological chemistry》2001,276(13):9971-9977
Stimulus-secretion coupling in pancreatic beta-cells involves membrane depolarization and Ca(2+) entry through voltage-gated L-type Ca(2+) channels, which is one determinant of increases in the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)). We investigated how the endoplasmic reticulum (ER)-associated Ca(2+) apparatus further modifies this Ca(2+) signal. When fura-2-loaded mouse beta-cells were depolarized by KCl in the presence of 3 mm glucose, [Ca(2+)](i) increased to a peak in two phases. The second phase of the [Ca(2+)](i) increase was abolished when ER Ca(2+) stores were depleted by thapsigargin. The steady-state [Ca(2+)](i) measured at 300 s of depolarization was higher in control cells compared with cells in which the ER Ca(2+) pools were depleted. The amount of Ca(2+) presented to the cytoplasm during depolarization as estimated from the integral of the increment in [Ca(2+)](i) over time (integralDelta[Ca(2+)](i).dt) was approximately 30% higher compared with that in the Ca(2+) pool-depleted cells. neo-thapsigargin, an inactive analog, did not affect [Ca(2+)](i) response. Using Sr(2+) in the extracellular medium and exploiting the differences in the fluorescence properties of Ca(2+)- and Sr(2+)-bound fluo-3, we found that the incoming Sr(2+) triggered Ca(2+) release from the ER. Depolarization-induced [Ca(2+)](i) response was not altered by, an inhibitor of phosphatidylinositol-specific phospholipase C, suggesting that stimulation of the enzyme by Ca(2+) is not essential for amplification of Ca(2+) signaling. [Ca(2+)](i) response was enhanced when cells were depolarized in the presence of 3 mm glucose, forskolin, and caffeine, suggesting involvement of ryanodine receptors in the amplification process. Pretreatment with ryanodine (100 microm) diminished the second phase of the depolarization-induced increase in [Ca(2+)](i). We conclude that Ca(2+) entry through L-type voltage-gated Ca(2+) channels triggers Ca(2+) release from the ER and that such a process amplifies depolarization-induced Ca(2+) signaling in beta-cells. 相似文献
20.
Growth factor-induced cell migration underlies various physiological and pathological processes. The mechanisms by which growth factors regulate cell migration are not completely understood. Although intracellular elevation of Ca2+ is known to be critical in cell migration, the source of this Ca2+ elevation and the mechanism by which Ca2+ modulates this process in fibroblast cells are not well defined. Here we show that increase of cellular Ca2+ through Ca2+ influx, rather than Ca2+ release from intracellular stores, is essential for growth factor-induced fibroblast cell migration. Voltage-gated L-type Ca2+ channels, previously known to exist in excitable cells such as neurons and muscle cells, are shown here to be present in fibroblasts as well. Furthermore, these channels are responsible for the Ca2+ influx. L-type Ca2+ channel inhibitors block growth factor-induced Ca2+ influx and fibroblast cell migration. One mechanism by which Ca2+ signals control cell migration is to regulate the contraction of the trailing edge of migrating fibroblasts; this process is controlled by the small GTPase Rho in fast migrating cells such as leukocytes. Downstream of Ca2+, both calmodulin and myosin light chain kinase, but not calcineurin, are involved leading to phosphorylation of the myosin light chain at the trailing end. Thus, trailing edge contraction is critically regulated by Ca2+ influx through L-type Ca2+ channels in growth factor-induced fibroblast cell migration. 相似文献