Ca2+ entry through plasma membrane IP3 receptors

Inositol 1,4,5-trisphosphate receptors (IP3Rs) release calcium ions, Ca2+, from intracellular stores, but their roles in mediating Ca2+ entry are unclear. IP3 stimulated opening of very few (1.9 +/- 0.2 per cell) Ca2+-permeable channels in whole-cell patch-clamp recording of DT40 chicken or mouse B cells. Activation of the B cell receptor (BCR) in perforated-patch recordings evoked the same response. IP3 failed to stimulate intracellular or plasma membrane (PM) channels in cells lacking IP3R. Expression of IP3R restored both responses. Mutations within the pore affected the conductances of IP3-activated PM and intracellular channels similarly. An impermeant pore mutant abolished BCR-evoked Ca2+ signals, and PM IP3Rs were undetectable. After introduction of an alpha-bungarotoxin binding site near the pore, PM IP3Rs were modulated by extracellular alpha-bungarotoxin. IP(3)Rs are unusual among endoplasmic reticulum proteins in being also functionally expressed at the PM, where very few IP3Rs contribute substantially to the Ca2+ entry evoked by the BCR.     

Odor stimuli trigger influx of calcium into olfactory neurons of the channel catfish

Olfactory transduction is thought to be mediated by a G protein-coupled increase in intracellular adenosine 3',5'-monophosphate (cAMP) that triggers the opening of cAMP-gated cation channels and results in depolarization of the plasma membrane of olfactory neurons. In olfactory neurons isolated from the channel catfish, Ictalurus punctatus, stimulation with olfactory stimuli (amino acids) elicits an influx of calcium that leads to a rapid increase in intracellular calcium. In addition, in a reconstitution assay a plasma membrane calcium channel has been identified that is gated by inositol-1,4,5-trisphosphate (IP3), which could mediate this calcium influx. Together with previous studies indicating that stimulation with olfactory stimuli leads to stimulation of phosphoinositide turnover in olfactory cilia, these data suggest that an influx of calcium triggered by odor stimulation of phosphoinositide turnover may be an alternate or additional mechanism of olfactory transduction.     

IP3 receptor types 2 and 3 mediate exocrine secretion underlying energy metabolism

Type 2 and type 3 inositol 1,4,5-trisphosphate receptors (IP3R2 and IP3R3) are intracellular calcium-release channels whose physiological roles are unknown. We show exocrine dysfunction in IP3R2 and IP3R3 double knock-out mice, which caused difficulties in nutrient digestion. Severely impaired calcium signaling in acinar cells of the salivary glands and the pancreas in the double mutants ascribed the secretion deficits to a lack of intracellular calcium release. Despite a normal caloric intake, the double mutants were hypoglycemic and lean. These results reveal IP3R2 and IP3R3 as key molecules in exocrine physiology underlying energy metabolism and animal growth.     

IgG from patients with Lambert-Eaton syndrome blocks voltage-dependent calcium channels

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.     

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.     

Calcium as a coagonist of inositol 1,4,5-trisphosphate-induced calcium release

Inositol 1,4,5-trisphosphate (IP3)-induced calcium release from intracellular stores is a regulator of cytosolic-free calcium levels. The subsecond kinetics and regulation of IP3-induced calcium-45 release from synaptosome-derived microsomal vesicles were resolved by rapid superfusion. Extravesicular calcium acted as a coagonist, potentiating the transient IP3-induced release of calcium-45. Thus, rapid elevation of cytosolic calcium levels may trigger IP3-induced calcium release in vivo. Extravesicular calcium also produced a more slowly developing, reversible inhibition of IP3-induced calcium-45 release. Sequential positive and negative feedback regulation by calcium of IP3-induced calcium release may contribute to transients and oscillations of cytosolic-free calcium in vivo.     

IP_3敏感的钙离子通透性通道参与茉莉酸诱导的钙动员

[目的]研究茉莉酸诱导的钙动员通往IP3敏感的钙离子通透性通道的作用。[方法]以低温导入法将钙离子荧光探针Fluo-3/AM导入拟南芥叶片细胞中,利用LASAF（Leica Application Suite-Advanced Fluorescence）软件记录肝素对茉莉酸（JA）诱导的胞内钙离子荧光强度的变化。[结果]经不同浓度的肝素预处理后,拟南芥叶细胞中胞内钙离子的荧光强度降低,再用100μmol/LJA处理时,其荧光强度升高,但仅与未经肝素处理的荧光强度相当。[结论]肝素预处理可抑制JA诱导的胞内钙离子浓度的升高。     

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.     

Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3

A wide variety of nonexcitable cells generate repetitive transient increases in cytosolic calcium ion concentration ([Ca2+]i) when stimulated with agonists that engage the phosphoinositide signalling pathway. Current theories regarding the mechanisms of oscillation disagree on whether Ca2+ inhibits or stimulates its own release from internal stores and whether inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) also undergo oscillations linked to the Ca2+ spikes. In this study, Ca2+ was found to stimulate its own release in REF52 fibroblasts primed by mitogens plus depolarization. However, unlike Ca2+ release in muscle and nerve cells, this amplification was insensitive to caffeine or ryanodine and required hormone receptor occupancy and functional IP3 receptors. Oscillations in [Ca2+]i were accompanied by oscillations in IP3 concentration but did not require functional protein kinase C. Therefore, the dominant feedback mechanism in this cell type appears to be Ca2+ stimulation of phospholipase C once this enzyme has been activated by hormone receptors.     

The Role of Extracellular Ca2+Influx, Intracellular Ca2+ Release and Calmodulin in Mouse Egg Fertilization

The effects of various Ca^2 -modifying drugs on moue egg fertilization were studied.Ca^2 chelator,ethylen glycol-bis-(2-aminoethyl)-tetracetic acid(EGTA),and calmodulin(CaM) antagonist,trifluoperzaine (TFP),inhibited fertilization in a dose-dependent manner,whild Ca^2 channel bolcker,verspamil,did not have any effect.When intracellular Ca^2 release was blocked by 8-(N,N-diethylamino) octy 1-3,4,5-trimethoxy-benzonate(TME-8) or the Ca^2 oscillations were inhibited by an inhibitor of endoplasmic reticulum Ca^2 -At-Pase,thapsigargin,the second polar body emission and pronuclear formation were significantly decreased.In contrast,inhibition of intracellular Ca^2 release via bolckage of inositol 1,4,5-triphosphate (IP3) production by neomycin or lithium did not affect fertilization.The results sugest that both extracellular influx,intracellular Ca^2 release and CaM activation are required for mormal fertilization.However,extracellular influx through voltage-gated Ca^2 channel and intracellular release induced by IP3 and not the only pathways for producing Ca^2 transients in moue eggs.     

A bitter substance induces a rise in intracellular calcium in a subpopulation of rat taste cells

The sense of taste permits animals to discriminate between foods that are safe and those that are toxic. Because most poisonous plant alkaloids are intensely bitter, bitter taste warns animals of potentially hazardous foods. To investigate the mechanism of bitter taste transduction, a preparation of dissociated rat taste cells was developed that can be studied with techniques designed for single-cell measurements. Denatonium, a very bitter substance, caused a rise in the intracellular calcium concentration due to release from internal stores in a small subpopulation of taste cells. Thus, the transduction of bitter taste may occur via a receptor-second messenger mechanism leading to neurotransmitter release and may not involve depolarization-mediated calcium entry.     

不同钙效应剂对长春花光合特性的影响

研究了细胞质膜钙通道阻断剂氯化镧(LaCl3)、胞内IP3通道阻断剂肝素(Heparin)、胞内CaM活性抑制剂三氟啦嗪(TFP)对长春花光合作用的影响,结果发现:Ca2+通道阻断剂处理后长春花叶片净光合速率(Pn)、PSII最大量子产量(Fv/Fm)、PSⅡ实际量子产量(Yield)、电子传递速率(ETR)、光化学淬灭系数(qP)均下降,非光化学淬灭系数(qN)及Chla、Chlb、Chla+Chlb含量上升,表明Ca2+通道阻断剂对长春花的光合作用有较大影响,且不是通过加速叶片光合色素降解或抑制其合成来实现抑制叶片的光合能力;其中Heparin处理的长春花叶片相关参数变化幅度最大,表明胞内钙库通过IP3通道释放的Ca2+在长春花叶片光合作用过程中发挥了更积极的作用.     

Inositol 1,3,4,5-tetrakisphosphate induces Ca2+ sequestration in rat liver cells

Inositol 1,4,5-trisphosphate [I(1,4,5)P3] is a second messenger generated along with diacylglycerol upon the binding of various physiological agents with their cell surface receptors. I(1,4,5)P3 mobilizes Ca2+ from intracellular storage sites through a receptor-coupled mechanism, and the subsequent increased intracellular free calcium ion concentration [( Ca2+]i) activates a multitude of cellular responses. Electropermeabilized neoplastic rat liver epithelial (261B) cells were used to study Ca2+ sequestration, a process that reverses the elevated [Ca2+]i to resting levels and replenishes intracellular Ca2+ pools. Although I (1,4,5)P3-mobilized Ca2+ is readily sequestered into storage pools by the action of Ca2+-adenosine triphosphatases, Ca2+ mobilized by addition of the nonmetabolized inositol trisphosphate isomer I(2,4,5)P3 is not sequestered, suggesting that metabolism is necessary to eliminate the stimulus for Ca2+ release. Several inositol phosphate compounds were examined for their ability to lower the buffer [Ca2+] to determine if a specific I(1,4,5)P3 metabolite might be involved in stimulating Ca2+ sequestration; of these, I(1,3,4,5)P4 alone was found to induce Ca2+ sequestration, demonstrating a physiological role for this inositol trisphosphate metabolite.     

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.     

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.     

Divalent cations directly affect the conductance of excised patches of rod photoreceptor membrane

Phototransduction in rod cells is likely to involve an intracellular messenger system that links the absorption of light by rhodopsin to a change in membrane conductance. The direct effect of guanosine 3',5'-monophosphate (cGMP) on excised patches of rod outer segment membrane strongly supports a role for cGMP as an intracellular messenger in phototransduction. It is reported here that magnesium and calcium directly affect the conductance of excised patches of rod membrane in the absence of cGMP and that magnesium, applied to intact rod cells, blocks a component of the cellular light response. The divalent cation-suppressed conductance in excised patches showed outward rectification and cation-selective permeability resembling those of the light-suppressed conductance measured from the intact rod cell. The divalent cation-suppressed conductance was partly blocked by a concentration of the pharmacological agent L-cis-diltiazem that blocked all of the cGMP-activated conductance. Divalent cations may act, together with cGMP, as an intracellular messenger system that mediates the light response of the rod photoreceptor cell.     

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.     

细胞内钙离子形成的波形图案

目的利用包含缓冲和扩散影响的钙离子Shuai—Jung振荡模型，研究细胞内和钙库上不同的释放位置分布．方法研究细胞内钙离子振荡形成的波形结构，得到细胞内的钙波图案和细胞内一点的钙信号发放．结果细胞内的钙波信号具有各类实验出现的波形结构，细胞内固定点的钙波变化显示钙信号具有传递信使的特性．结论钙离子是细胞内重要的第二信使，其振荡传播可直接传导生理信息．     

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.