Assessment of the role of the inositol 1,4,5-trisphosphate receptor in the activation of transient receptor potential channels and store-operated Ca2+ entry channels

The mechanism for coupling between Ca(2+) stores and store-operated channels (SOCs) is an important but unresolved question. Although SOCs have not been molecularly identified, transient receptor potential (TRP) channels share a number of operational parameters with SOCs. The question of whether activation of SOCs and TRP channels is mediated by the inositol 1,4,5-trisphosphate receptor (InsP(3)R) was examined using the permeant InsP(3)R antagonist, 2-aminoethoxydiphenyl borate (2-APB) in both mammalian and invertebrate systems. In HEK293 cells stably transfected with human TRPC3 channels, the actions of 2-APB to block carbachol-induced InsP(3)R-mediated store release and carbachol-induced Sr(2+) entry through TRPC3 channels were both reversed at high agonist levels, suggesting InsP(3)Rs mediate TRPC3 activation. However, electroretinogram recordings of the light-induced current in Drosophila revealed that the TRP channel-mediated responses in wild-type as well as trp and trpl mutant flies were all inhibited by 2-APB. This action of 2-APB is likely InsP(3)R-independent since InsP(3)Rs are dispensable for the light response. We used triple InsP(3)R knockout DT40 chicken B-cells to further assess the role of InsP(3)Rs in SOC activation. (45)Ca(2+) flux analysis revealed that although DT40 wild-type cells retained normal InsP(3)Rs mediating 2-APB-sensitive Ca(2+) release, the DT40InsP(3)R-k/o cells were devoid of functional InsP(3)Rs. Using intact cells, all parameters of Ca(2+) store function and SOC activation were identical in DT40wt and DT40InsP(3)R-k/o cells. Moreover, in both cell lines SOC activation was completely blocked by 2-APB, and the kinetics of action of 2-APB on SOCs (time dependence and IC(50)) were identical. The results indicate that (a) the action of 2-APB on Ca(2+) entry is not mediated by the InsP(3)R and (b) the effects of 2-APB provide evidence for an important similarity in the function of invertebrate TRP channels, mammalian TRP channels, and mammalian store-operated channels.     

Modification of store-operated channel coupling and inositol trisphosphate receptor function by 2-aminoethoxydiphenyl borate in DT40 lymphocytes.

Store-operated channels (SOCs) provide an important means for mediating longer-term Ca(2+) signals and replenishment of Ca(2+) stores in a multitude of cell types. However, the coupling mechanism between endoplasmic reticulum stores to activate plasma membrane SOCs remains unknown. In DT40 chicken B lymphocytes, the permeant inositol trisphosphate receptor (InsP(3)R) modifier, 2-aminoethoxydiphenyl borate (2-APB), was a powerful activator of store-operated Ca(2+) entry between 1-10 microm. 2-APB activated authentic SOCs because the entry was totally selective for Ca(2+) (no detectable entry of Ba(2+) or Sr(2+) ions), and highly sensitive to La(3+) ions (IC(50) 30-100 nm). To assess the role of InsP(3)Rs in this response, we used the DT40 triple InsP(3)R-knockout (ko) cell line, DT40InsP(3)R-ko, in which the absence of full-length InsP(3)Rs or InsP(3)R fragments was verified by Western analysis using antibodies cross-reacting with N-terminal epitopes of all three chicken InsP(3)R subtypes. The 2-APB-induced activation of SOCs was identical in the DT40InsP(3)R-ko, cells indicating InsP(3)Rs were not involved. With both wild type (wt) and ko DT40 cells, 2-APB had no effect on Ca(2+) entry in store-replete cells, indicating that its action was restricted to SOCs in a store-coupled state. 2-APB induced a robust activation of Ca(2+) release from stores in intact DT40wt cells but not in DT40InsP(3)R-ko cells, indicating an InsP(3)R-mediated effect. In contrast, 2-APB blocked InsP(3)Rs in permeabilized DT40wt cells, suggesting that the stimulatory action of 2-APB was restricted to functionally coupled InsP(3)Rs in intact cells. Uncoupling of ER/PM interactions in intact cells by calyculin A-induced cytoskeletal rearrangement prevented SOC activation by store-emptying and 2-APB; this treatment completely prevented 2-APB-induced InsP(3)R activation but did not alter InsP(3)R activation mediated by phospholipase C-coupled receptor stimulation. The results indicate that the robust bifunctional actions of 2-APB on both SOCs and InsP(3)Rs are dependent on the coupled state of these channels and suggest that 2-APB may target the coupling machinery involved in mediating store-operated Ca(2+) entry.     

The endoplasmic reticulum and neuronal calcium signalling

The endoplasmic reticulum (ER) is a multifunctional signalling organelle regulating a wide range of neuronal functional responses. The ER is intimately involved in intracellular Ca(2+) signalling, producing local or global cytosolic calcium fluctuations via Ca(2+)-induced Ca(2+) release (CICR) or inositol-1,4,5-trisphosphate-induced Ca(2+) release (IICR). The CICR and IICR are controlled by two subsets of Ca(2+) release channels residing in the ER membrane, the Ca(2+)-gated Ca(2+) release channels, generally known as ryanodine receptors (RyRs) and InsP(3)-gated Ca(2+) release channels, referred to as InsP(3)-receptors (InsP(3)Rs). Both types of Ca(2+) release channels are expressed abundantly in nerve cells and their activation triggers cytoplasmic Ca(2+) signals important for synaptic transmission and plasticity. The RyRs and InsP(3)Rs show heterogeneous localisation in distinct cellular sub-compartments, conferring thus specificity in local Ca(2+) signals. At the same time, the ER Ca(2+) store emerges as a single interconnected pool fenced by the endomembrane. The continuity of the ER Ca(2+) store could play an important role in various aspects of neuronal signalling. For example, Ca(2+) ions may diffuse within the ER lumen with comparative ease, endowing this organelle with the capacity for "Ca(2+) tunnelling". Thus, continuous intra-ER Ca(2+) highways may be very important for the rapid replenishment of parts of the pool subjected to excessive stimulation (e.g. in small compartments within dendritic spines), the facilitated removal of localised Ca(2+) loads, and finally in conveying Ca(2+) signals from the site of entry towards the cell interior and nucleus.     

Inositol 1,4,5-trisphosphate receptors in the heart

Inositol 1,4,5-trisphosphate (InsP3) is an established calcium-mobilizing messenger, which is well-known to activate Ca2+ signaling in many cell types. Contractile cardiomyocytes express hormone receptors that are coupled to the production of InsP3. Such cardioactive hormones, including endothelin, may have profound inotropic and arrhythmogenic actions, but it is unclear whether InsP3 underlies any of these effects. We have examined the expression and localization of InsP3 receptors (InsP3Rs), and the potential role of InsP3 in modulating cardiac excitation-contraction coupling (EC coupling). Stimulation of electrically-paced atrial and ventricular myocytes with a membrane-permeant InsP3 ester was found to evoke an increase in the amplitudes of action potential-evoked Ca2+ transients and to cause pro-arrhythmic diastolic Ca2+ transients. All the effects of the InsP3 ester could be blocked using a membrane-permeant antagonist of InsP3Rs (2-aminoethoxydiphenyl borate; 2-APB). Furthermore, 2-APB blocked arrhythmias evoked by endothelin and delayed the onset of positive inotropic responses. Our data indicate that atrial and ventricular cardiomyocytes express functional InsP3Rs, and these channels have the potential to influence EC coupling.     

Metabotropic Ca2+ channel-induced calcium release in vascular smooth muscle

Contraction of vascular smooth muscle cells (VSMCs) depends on the rise of cytosolic [Ca(2+)] owing to either Ca(2+) influx through voltage-gated Ca(2+) channels of the plasmalemma or to receptor-mediated Ca(2+) release from the sarcoplasmic reticulum (SR). Although the ionotropic role of L-type Ca(2+) channels is well known, we review here data suggesting a new role of these channels in arterial myocytes. After sensing membrane depolarization Ca(2+) channels activate G proteins and the phospholipase C/inositol 1,4,5-trisphosphate (InsP(3)) pathway. Ca(2+) released through InsP(3)-dependent channels of the SR activates ryanodine receptors to amplify the cytosolic Ca(2+) signal, thus triggering arterial cerebral vasoconstriction in the absence of extracellular calcium influx. This metabotropic action of L-type Ca(2+) channels, denoted as calcium channel-induced Ca(2+) release, could have implications in cerebral vascular pharmacology and pathophysiology, because it can be suppressed by Ca(2+) channel antagonists and potentiated with small concentrations of extracellular vasoactive agents as ATP.     

Functional InsP3 receptors that may modulate excitation-contraction coupling in the heart

The roles of the Ca2+-mobilising messenger inositol 1,4,5-trisphosphate (InsP3) in heart are unclear, although many hormones activate InsP3 production in cardiomyocytes and some of their inotropic, chronotropic and arrhythmogenic effects may be due to Ca2+ release mediated by InsP3 receptors (InsP3Rs) [1-3]. In the present study, we examined the expression and subcellular localisation of InsP3R isoforms, and investigated their potential role in modulating excitation-contraction coupling (EC coupling). Western, PCR and InsP3-binding analysis indicated that both atrial and ventricular myocytes expressed mainly type II InsP3Rs, with approximately sixfold higher levels of InsP3Rs in atrial cells. Co-immunostaining of atrial myocytes with antibodies against type II ryanodine receptors (RyRs) and type II InsP3Rs revealed that the latter were arranged in the subsarcolemmal space where they largely co-localised with the junctional RyRs. Stimulation of quiescent or electrically paced atrial myocytes with a membrane-permeant InsP3 ester, which enters cells and directly activates InsP3Rs, caused the appearance of spontaneous Ca2+-release events. In addition, in paced cells, the InsP3 ester evoked an increase in the amplitudes of action potential-evoked Ca2+ transients. These data indicate that atrial cardiomyocytes express functional InsP3Rs, and that these channels could modulate EC coupling.     

Ca2+ entry mediated by store depletion, S-nitrosylation, and TRP3 channels. Comparison of coupling and function

The mechanism for coupling between Ca(2+) stores and store-operated channels (SOCs) is an important but unresolved question. SOC-mediated Ca(2+) entry is complex and may reflect more than one type of channel and coupling mechanism. To assess such possible divergence the function and coupling of SOCs was compared with two other distinct yet related Ca(2+) entry mechanisms. SOC coupling in DDT(1)MF-2 smooth muscle cells was prevented by the permeant inositol 1,4,5-trisphosphate (InsP(3)) receptor blockers, 2-aminoethoxydiphenyl borate (2-APB) and xestospongin C. In contrast, Ca(2+) entry induced by S-nitrosylation and potentiated by store depletion (Ma, H-T., Favre, C. J., Patterson, R. L., Stone, M. R., and Gill, D. L. (1999) J. Biol. Chem. 274, 35318-35324) was unaffected by 2-APB, suggesting that this entry mechanism is independent of InsP(3) receptors. The cycloalkyl lactamimide, MDL-12, 330A (MDL), prevented SOC activation (IC(50) 10 micrometer) and similarly completely blocked S-nitrosylation-mediated Ca(2+) entry. Ca(2+) entry mediated by the TRP3 channel stably expressed in HEK293 cells was activated by phospholipase C-coupled receptors but independent of Ca(2+) store depletion (Ma, H.-T., Patterson, R. L., van Rossum, D. B., Birnbaumer, L., Mikoshiba, K., and Gill, D. L. (2000) Science 287, 1647-1651). Receptor-induced TRP3 activation was 2-APB-sensitive and fully blocked by MDL. Direct stimulation of TRP3 channels by the permeant diacylglycerol derivative, 1-oleoyl-2-acetyl-sn-glycerol, was not blocked by 2-APB, but was again prevented by MDL. The results indicate that although the activation and coupling processes for each of the three entry mechanisms are distinct, sensitivity to MDL is a feature shared by all three mechanisms, suggesting there may be a common structural feature in the channels themselves or an associated regulatory component.     

Regulation of InsP3 receptor activity by neuronal Ca2+-binding proteins

Inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) were recently demonstrated to be activated independently of InsP(3) by a family of calmodulin (CaM)-like neuronal Ca(2+)-binding proteins (CaBPs). We investigated the interaction of both naturally occurring long and short CaBP1 isoforms with InsP(3)Rs, and their functional effects on InsP(3)R-evoked Ca(2+) signals. Using several experimental paradigms, including transient expression in COS cells, acute injection of recombinant protein into Xenopus oocytes and (45)Ca(2+) flux from permeabilised COS cells, we demonstrated that CaBPs decrease the sensitivity of InsP(3)-induced Ca(2+) release (IICR). In addition, we found a Ca(2+)-independent interaction between CaBP1 and the NH(2)-terminal 159 amino acids of the type 1 InsP(3)R. This interaction resulted in decreased InsP(3) binding to the receptor reminiscent of that observed for CaM. Unlike CaM, however, CaBPs do not inhibit ryanodine receptors, have a higher affinity for InsP(3)Rs and more potently inhibited IICR. We also show that phosphorylation of CaBP1 at a casein kinase 2 consensus site regulates its inhibition of IICR. Our data suggest that CaBPs are endogenous regulators of InsP(3)Rs tuning the sensitivity of cells to InsP(3).     

2-Aminoethoxydiphenyl borate inhibits inositol 1,4,5-trisphosphate receptor function,ubiquitination and downregulation,but acts with variable characteristics in different cell types

2-Aminoethoxydiphenyl borate (2-APB) is a putative, membrane-permeable inhibitor of inositol 1,4,5-trisphosphate (InsP(3)) receptors, but it is the case that little is known about its action at the InsP(3) receptor level. Thus, we examined the effects of 2-APB on InsP(3) receptor-mediated effects in a range of cell types expressing different complements of InsP(3) receptor types. In experiments with permeabilized cells we found that 2-APB could inhibit InsP(3)-induced release of stored Ca(2+), but also that it released Ca(2+), and that the prevalence of these two effects varied between different cell types and did not correlate with the expression of a particular receptor type. These effects of 2-APB reflected an interaction distal to the ligand binding site of InsP(3) receptors, since InsP(3) binding was unaffected by 2-APB. In intact cells, we found only inhibitory effects of 2-APB on Ca(2+) mobilization, and that variation between cell types in the characteristics of this inhibition appeared to be due to differential entry of 2-APB. 2-APB also inhibited InsP(3) receptor ubiquitination and proteasomal degradation, which again was cell type dependent. In total, these data reveal a remarkable degree of variation between cell types in the effects of 2-APB, showing that its usefulness as a specific and universal inhibitor of InsP(3) receptors is limited. However, the ability of 2-APB to inhibit InsP(3) receptor ubiquitination and degradation indicates that 2-APB may block InsP(3)-induced conformational changes in the receptor, resulting in perturbation of multiple regulatory events.     

Inhibition of the type 1 inositol 1,4,5-trisphosphate receptor by 2-aminoethoxydiphenylborate

2-Aminoethoxydiphenylborate (2-APB) inhibits the extent of inositol 1,4,5-trisphosphate (InsP(3))-induced Ca(2+) release from cerebellar microsomes with a potency that is dependent upon the InsP(3) concentration used. At high InsP(3) concentrations (10 microM), the concentration of 2-APB required to cause half-maximal InsP(3)-induced Ca(2+) release (IC(50)) was greater than 1 mM, while at 0.25 microM InsP(3) this reduced to 220 microM. The fact that the inhibition of the extent of InsP(3)-induced Ca(2+) release (IICR) by 2-APB was not restored to control levels by high concentrations of InsP(3), in addition to the fact 2-APB did not substantially inhibit [3H]InsP(3) binding to its receptor, indicates that the inhibition is not competitive in nature. Since the cooperativity of IICR as a function of InsP(3) was reduced in the presence of 2-APB (Hill coefficient changing from 1.9 in the absence of 2-APB to 1.4 in the presence of 1 mM 2-APB), this suggests that it is acting as an allosteric inhibitor. 2-APB also reduces the rate constants for IICR. In cerebellar microsomes this release process is biphasic in nature, with a fast and slow phase. 2-APB appears particularly to affect the fast-phase component. Although 2-APB does not inhibit the ryanodine receptor, it does inhibit the Ca(2+) ATPase activity as well store-operated Ca(2+) entry channels, which may limit its use as a specific membrane permeant InsP(3) receptor inhibitor.     

Bcl-2 suppresses Ca release through inositol 1,4,5-trisphosphate receptors and inhibits Ca uptake by mitochondria without affecting ER calcium store content

Cell survival is promoted by the oncoprotein Bcl-2. Previous studies have established that one of the pro-survival actions of Bcl-2 is to reduce cellular fluxes of Ca(2+) within cells. In particular, Bcl-2 has been demonstrated to inhibit the release of Ca(2+) from the endoplasmic reticulum. However, the mechanism by which Bcl-2 causes reduced Ca(2+) release is unclear. In the accompanying paper [C.J. Hanson, M.D. Bootman, C.W. Distelhorst, T. Maraldi, H.L. Roderick, The cellular concentration of Bcl-2 determines its pro- or anti-apoptotic effect, Cell Calcium (2008)], we described that only stable expression of Bcl-2 allowed it to work in a pro-survival manner whereas transient expression did not. In this study, we have employed HEK-293 cells that stably express Bcl-2, and which are, therefore, protected from pro-apoptotic stimuli, to examine the effect of Bcl-2 on Ca(2+) homeostasis and signalling. We observed that Bcl-2 expression decreased the Ca(2+) responses of cells induced by application of submaximal agonist concentrations. Whereas, decreasing endogenous Bcl-2 concentration using siRNA potentiated Ca(2+) responses. Furthermore, we found that Bcl-2 expression reduced mitochondrial Ca(2+) uptake by raising the threshold cytosolic Ca(2+) concentration required to activate sequestration. Using a number of different assays, we did not find any evidence for reduction of endoplasmic reticulum luminal Ca(2+) in our Bcl-2-expressing cells. Indeed, we observed that Bcl-2 served to preserve the content of the agonist-sensitive Ca(2+) pool. Endogenous Bcl-2 was found to interact with inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) in our cells, and to modify the profile of InsP(3)R expression. Our data suggest that the presence of Bcl-2 in the proteome of cells has multiple effects on agonist-mediated Ca(2+) signals, and can abrogate responses to submaximal levels of stimulation through direct control of InsP(3)Rs.     

Temperature-dependent calcium-induced calcium release via InsP3 receptors in mouse olfactory ensheathing glial cells

Cooling can induce Ca(2+) signaling via activation of temperature-sensitive ion channels such as TRPM8, TRPA1 and ryanodine receptor channels. Here we have studied the mechanism of cooling-evoked Ca(2+) signaling in mouse olfactory ensheathing cells (OECs), a specialized type of glial cells in the olfactory nerve layer of the olfactory bulb. Reducing the temperature from above 30°C to 28°C and below triggered Ca(2+) transients that persisted in the absence of external Ca(2+), but were suppressed after Ca(2+) store depletion by cyclopiazonic acid. Cooling-evoked Ca(2+) transients were present in mice deficient of TRPM8 and TRPA1, and were not inhibited by ryanodine receptor antagonists. Inhibition of InsP(3) receptors with 2-APB and caffeine entirely blocked cooling-evoked Ca(2+) transients. Moderate Ca(2+) increases, as evoked by flash photolysis of NP-EGTA (caged Ca(2+)) and cyclopiazonic acid, triggered InsP(3) receptor-mediated Ca(2+) release at 22°C, but not at 31°C. The results suggest that InsP(3) receptors mediate Ca(2+)-induced Ca(2+) release in OECs, and that this Ca(2+) release is temperature-sensitive and can be suppressed at temperatures above 28°C.     

Characterization of Ca(2+) signaling pathways in human mesenchymal stem cells

Human mesenchymal stem cells (HMSC) have the potential to differentiate into many cell types. The physiological properties of HMSCs including their Ca(2+) signaling pathways, however, are not well understood. We investigated Ca(2+) influx and release functions in HMSCs. In Ca(2+) imaging experiments, spontaneous Ca(2+) oscillations were observed in 36 of 50 HMSCs. The Ca(2+) oscillations were completely blocked by the application of 10 micro M cyclopiazonic acid (CPA) or 1 micro M thapsigargin (TG). A brief application of 1 micro M acetylcholine (ACh) induced a transient increase of [Ca(2+)](i) but the application of caffeine (10 mM) did not induce any Ca(2+) transient. When the stores were depleted with Ca(2+)-ATPase blockers (CPA or TG) or muscarinic agonists (ACh), store-operated Ca(2+) (SOC) entry was observed. Using the patch-clamp technique, store-operated Ca(2+) currents (I(SOC)) could be recorded in cells treated with ACh or CPA, but voltage-operated Ca(2+) currents (VOCCs) were not elicited in most of the cells (17/20), but in 15% of cells examined, small dihydropyridine (DHP)-sensitive Ca(2+) currents were recorded. Using RT-PCR, mRNAs were detected for inositol 1,4,5-trisphosphate receptor (InsP(3)R) type I, II, and III and DHP receptors alpha1A and alpha1H were detected, but mRNA was not detected for ryanodine receptor (RyR) or N-type Ca(2+) channels. These results suggest that in undifferentiated HMSCs, Ca(2+) release is mediated by InsP(3)Rs and Ca(2+) entry through plasma membrane is mainly mediated by the SOCs channels with a little contribution of VOCCs.     

Homer proteins and InsP(3) receptors co-localise in the longitudinal sarcoplasmic reticulum of skeletal muscle fibres

Striated muscle represents one of the best models for studies on Ca(2+) signalling. However, although much is known on the localisation and molecular interactions of the ryanodine receptors (RyRs), far less is known on the localisation and on the molecular interactions of the inositol trisphosphate receptors (InsP(3)Rs) in striated muscle cells. Recently, members of the Homer protein family have been shown to cluster type 1 metabotropic glutamate receptors (mGluR1) in the plasma membrane and to interact with InsP(3)R in the endoplasmic reticulum of neurons. Thus, these scaffolding proteins are good candidates for organising plasma membrane receptors and intracellular effector proteins in signalosomes involved in intracellular Ca(2+) signalling. Homer proteins are also expressed in skeletal muscle, and the type 1 ryanodine receptor (RyR1) contains a specific Homer-binding motif. We report here on the relative sub-cellular localisation of InsP(3)Rs and Homer proteins in skeletal muscle cells with respect to the localisation of RyRs. Immunofluorescence analysis showed that both Homer and InsP(3)R proteins present a staining pattern indicative of a localisation at the Z-line, clearly distinct from that of RyR1. Consistent herewith, in sub-cellular fractionation experiments, Homer proteins and InsP(3)R were both found in the fractions enriched in longitudinal sarcoplasmic reticulum (LSR) but not in fractions of terminal cisternae that are enriched in RyRs. Thus, in skeletal muscle, Homer proteins may play a role in the organisation of a second Ca(2+) signalling compartment containing the InsP(3)R, but are apparently not involved in the organisation of RyRs at triads.     

Temporal changes in atrial EC-coupling during prolonged stimulation with endothelin-1

Endothelin-1 (ET-1) is a potent G(q)-coupled agonist with important physiological effects on the heart. In the present study, we characterised the effect of prolonged ET-1 stimulation on Ca(2+) signalling within acutely isolated atrial myocytes. ET-1 induced a reproducible and complex sequence of effects, including negative inotropy, positive inotropy and pro-arrhythmic spontaneous Ca(2+) transients (SCTs). The negative and positive inotropic effects correlated with the ability of Ca(2+) to propagate from the subsarcolemmal sites where EC-coupling initiates into the centre of the atrial cells. We examined the spatial and temporal properties of the SCTs and observed them to range from elementary Ca(2+) sparks, flurries of Ca(2+) sparks, to Ca(2+) waves and action potential-evoked global Ca(2+) transients. The positive inotropic effect of ET-1 and its ability to trigger SCTs were mimicked by direct stimulation of InsP(3)Rs. An antagonist of InsP(3)Rs prevented the generation of SCTs and partially reduced the positive inotropy evoked by ET-1. Our data suggest that ET-1 engages multiple signal transduction pathways to provoke a plethora of different responses within an atrial myocyte. Some of the actions of ET-1 appear to be due to stimulation of InsP(3)Rs.     

Activation of the inositol 1,4,5-trisphosphate receptor by the calcium storage protein chromogranin A

Secretory granules of neuroendocrine cells are inositol 1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) stores in which the Ca(2+) storage protein, chromogranin A (CGA), couples with InsP(3)-gated Ca(2+) channels (InsP(3)R) located in the granule membrane. The functional aspect of this coupling has been investigated via release studies and planar lipid bilayer experiments in the presence and absence of CGA. CGA drastically increased the release activity of the InsP(3)R by increasing the channel open probability by 9-fold and the mean open time by 12-fold. Our results show that CGA-coupled InsP(3)Rs are more sensitive to activation than uncoupled receptors. This modulation of InsP(3)R channel activity by CGA appears to be an essential component in the control of intracellular Ca(2+) concentration by secretory granules and may regulate the rate of vesicle fusion and exocytosis.     

Sequential opening of IP(3)-sensitive Ca(2+) channels and SOC during alpha-adrenergic activation of rabbit vena cava

Alpha(1)-aderenoceptor-mediated constriction of rabbit inferior vena cava (IVC) is signaled by asynchronous wavelike Ca(2+) oscillations in the in situ smooth muscle. We have shown previously that a putative nonselective cationic channel (NSCC) is required for these oscillations. In this report, we show that the application of 2-aminoethoxyphenyl borate (2-APB) to antagonize inositol 1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) release channels (IP(3)R channels) can prevent the initiation and abolish ongoing alpha(1)-aderenoceptor-mediated tonic constriction of the venous smooth muscle by inhibiting the generation of these intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations. The observed effects of 2-APB can only be attributed to its selective inhibition on the IP(3)R channels, not to its slight inhibition of the L-type voltage-gated Ca(2+) channel and the sarco(endo)plasmic reticulum Ca(2+) ATPase. Furthermore, 2-APB had no effect on the ryanodine-sensitive Ca(2+) release channel and the store-operated channel (SOC) in the IVC. These results indicate that the putative NSCC involved in refilling the sarcoplasmic reticulum (SR) and maintaining the tonic contraction is most likely an SOC-type channel because it appears to be activated by IP(3)R-channel-mediated SR Ca(2+) release or store depletion. This is in accordance with its sensitivity to Ni(2+) and La(3+) (SOC blockers). More interestingly, RT-PCR analysis indicates that transient receptor potential (Trp1) mRNA is strongly expressed in the rabbit IVC. The Trp1 gene is known to encode a component of the store-operated NSCC. These new data suggest that the activation of both the IP(3)R channels and the SOC are required for PE-mediated [Ca(2+)](i) oscillations and constriction of the rabbit IVC.     

Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry

Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.     

Single-channel recordings of recombinant inositol trisphosphate receptors in mammalian nuclear envelope.

Inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)Rs) are intracellular Ca(2+) channels gated by the second messenger InsP(3). Here we describe a novel approach for recording single-channel currents through recombinant InsP(3)Rs in mammalian cells that applies patch-clamp electrophysiology to nuclei isolated from COS-7 cells transiently transfected with the neuronal (SII(+)) and peripheral (SII(-)) alternatively-spliced variants of the rat type 1 InsP(3)R. Single channels that were activated by InsP(3) and inhibited by heparin were observed in 45% of patches from nuclei prepared from transfected cells overexpressing recombinant InsP(3)Rs. In contrast, nuclei from cells transfected with the vector alone had InsP(3)-dependent channel activity in only 1.5% of patches. With K(+) (140 mM) as the permeant ion, recombinant SII(+) and SII(-) channels had slope conductances of 370 pS and 390 pS, respectively. The recombinant channels were 4-fold more selective for Ca(2+) over K(+), and their open probabilities were biphasically regulated by cytoplasmic [Ca(2+)]. This approach provides a powerful new methodology to study the permeation and gating properties of recombinant mammalian InsP(3)Rs in a native mammalian membrane environment at the single-channel level.     

alpha(1)-adrenergic stimulation mediates Ca(2+)-dependent inositol phosphate formation through the alpha(1B)-like adrenoceptor subtype in adult rat cardiac myocytes.

We studied the effects of increased Ca(2+) influx on alpha(1)-adrenoceptor-stimulated InsP formation in adult rat cardiac myocytes. We further examined if such effects could be mediated through a specific alpha(1)-adrenoceptor subtype. [(3)H]InsP responses to adrenaline were dependent on extracellular Ca(2+) concentration, from 0.1 microM to 2 mM, and were completely blocked by Ca(2+) removal. However, in cardiac myocytes preloaded with BAPTA, a highly selective calcium chelating agent, Ca(2+) concentrations higher than 1 microM had no effect on adrenaline-stimulated [(3)H]InsP formation. Taken together these results suggest that [(3)H]InsP formation induced by alpha(1)-adrenergic stimulation is in part mediated by increased Ca(2+) influx. Consistent with this, ionomycin, a calcium ionophore, stimulated [(3)H]InsP formation. This response was additive with the response to adrenaline stimulation implying that different signaling mechanisms may be involved. In cardiac myocytes treated with the alpha(1B)-adrenoceptor alkylating agent, CEC, [(3)H]InsP formation remained unaffected by increased Ca(2+) concentrations, a pattern similar to that observed when intracellular Ca(2+) was chelated with BAPTA. In contrast, addition of the alpha(1A)-subtype antagonist, 5'-methyl urapidil, did not affect the Ca(2+) dependence of [(3)H]InsP formation. Neither nifedipine, a voltage-dependent Ca(2+) channel blocker nor the inorganic Ca(2+) channel blockers, Ni(2+) and Co(2+), had any effect on adrenaline stimulated [(3)H]InsP, at concentrations that inhibit Ca(2+) channels. The results suggest that in adult rat cardiac myocytes, in addition to G protein-mediated response, alpha(1)-adrenergic-stimulated [(3)H]InsP formation is activated by increased Ca(2+) influx mediated by the alpha(1B)-subtype.