Methacholine and PDGF activate store-operated calcium entry in neuronal precursor cells via distinct calcium entry channels

Neurons are a diverse cell type exhibiting hugely different morphologies and neurotransmitter specifications. Their distinctive phenotypes are established during differentiation from pluripotent precursor cells. The signalling pathways that specify the lineage down which neuronal precursor cells differentiate remain to be fully elucidated. Among the many signals that impinge on the differentiation of neuronal cells, cytosolic calcium (Ca2+) has an important role. However, little is known about the nature of the Ca2+ signals involved in fate choice in neuronal precursor cells, or their sources. In this study, we show that activation of either muscarinic or platelet-derived growth factor (PDGF) receptors induces a biphasic increase in cytosolic Ca2+ that consists of release from intracellular stores followed by sustained entry across the plasma membrane. For both agonists, the prolonged Ca2+ entry occurred via a store-operated pathway that was pharmacologically indistinguishable from Ca2+ entry initiated by thapsigargin. However, muscarinic receptor-activated Ca2+ entry was inhibited by siRNA-mediated knockdown of TRPC6, whereas Ca2+ entry evoked by PDGF was not. These data provide evidence for agonist-specific activation of molecularly distinct store-operated Ca2+ entry pathways, and raise the possibility of privileged communication between these Ca2+ entry pathways and downstream processes.     

Overexpression of T-type calcium channels in HEK-293 cells increases intracellular calcium without affecting cellular proliferation

Increased expression of low voltage-activated, T-type Ca(2+) channels has been correlated with a variety of cellular events including cell proliferation and cell cycle kinetics. The recent cloning of three genes encoding T-type alpha(1) subunits, alpha(1G), alpha(1H) and alpha(1I), now allows direct assessment of their involvement in mediating cellular proliferation. By overexpressing the human alpha(1G) and alpha(1H) subunits in human embryonic kidney (HEK-293) cells, we describe here that, although T-type channels mediate increases in intracellular Ca(2+) concentrations, there is no significant change in bromodeoxyuridine incorporation and flow cytometric analysis. These results demonstrate that expressions of T-type Ca(2+) channels are not sufficient to modulate cellular proliferation of HEK-293 cells.     

Functional properties of endogenous receptor- and store-operated calcium influx channels in HEK293 cells

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: I(CRAC), Imin, Imax, and I(NS); 2) I(CRAC) channels are highly Ca2+-selective (P(Ca/Cs)>1000) and I(CRAC) single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity P(Ba/K)=20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (P(Ba/K)=4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) I(NS) channels are non-selective (P(Ba/K)=0.4) and display a single channel conductance of 5 pS; and 8) I(NS) channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.     

Endogenous TRPC1, TRPC3, and TRPC7 proteins combine to form native store-operated channels in HEK-293 cells

Endogenously expressed canonical transient receptor potential (TRPC) homologs were investigated for their role in forming store-operated, 1-oleoyl-2-acetyl-sn-glycerol-stimulated, or carbachol (CCh)-stimulated calcium entry pathways in HEK-293 cells. Measurement of thapsigargin-stimulated Ba(2+) entry indicated that the individual suppression of TRPC1, TRPC3, or TRPC7 protein levels, by small interfering RNA (siRNA) techniques, dramatically inhibited (52-68%) store-operated calcium entry (SOCE), whereas suppression of TRPC4 or TRPC6 had no effect. Combined suppression of TRPC1-TRPC3, TRPC1-TRPC7, TRPC3-TRPC7, or TRPC1-TRPC3-TRPC7 gave only slightly more inhibition of SOCE (74-78%) than seen with suppression of TRPC1 alone (68%), suggesting that these three TRPC homologs work in tandem to mediate a large component of SOCE. Evidence from co-immunoprecipitation experiments indicates that a TRPC1-TRPC3-TRPC7 complex, predicted from siRNA results, does exist. The suppression of either TRPC3 or TRPC7, but not TRPC1, induced a high Ba(2+) leak flux that was inhibited by 2-APB and SKF96365, suggesting that the influx is via leaky store-operated channels. The high Ba(2+) leak flux is eliminated by co-suppression of TRPC1-TRPC3 or TRPC1-TRPC7. For 1-oleoyl-2-acetyl-sn-glycerol-stimulated cells, siRNA data indicate that TRPC1 plays no role in mediating Ba(2+) entry, which appears to be mediated by the participation of TRPC3, TRPC4, TRPC6, and TRPC7. CCh-stimulated Ba(2+) entry, on the other hand, could be inhibited by suppression of any of the five endogenously expressed TRPC homologs, with the degree of inhibition being consistent with CCh stimulation of both store-operated and receptor-operated channels. In summary, endogenous TRPC1, TRPC3, and TRPC7 participate in forming heteromeric store-operated channels, whereas TRPC3 and TRPC7 can also participate in forming heteromeric receptor-operated channels.     

Two distinct calcium pools in the endoplasmic reticulum of HEK-293T cells

Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating agonists, carbachol and ATP, mobilized this Ca2+ pool. We find in HEK-293T, but not in HeLa cells, a distinct agonist-releasable Ca2+ pool insensitive to the SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) inhibitor TBH [2,5-di-(t-butyl)-benzohydroquinone]. TG (thapsigargin) and CPA (cyclopiazonic acid) completely emptied this pool, whereas lysosomal disruption or manoeuvres collapsing endomembrane pH gradients did not. Our results indicate that SERCA3d is important for filling the TBH-resistant store as: (i) SERCA3d is more abundant in HEK-293T than in HeLa cells; (ii) the SERCA 3 ATPase activity of HEK-293T cells is not fully blocked by TBH; and (iii) the expression of SERCA3d in HeLa cells generated a TBH-resistant agonist-mobilizable compartment in the ER. Therefore the distribution of SERCA isoforms may originate the heterogeneity of the ER Ca2+ stores and this may be the basis for store specialization in diverse functions. This adds to recent evidence indicating that SERCA3 isoforms may subserve important physiological and pathophysiological mechanisms.     

Release of calcium from endolysosomes increases calcium influx through N-type calcium channels: Evidence for acidic store-operated calcium entry in neurons

Neurons possess an elaborate system of endolysosomes. Recently, endolysosomes were found to have readily releasable stores of intracellular calcium; however, relatively little is known about how such ‘acidic calcium stores’ affect calcium signaling in neurons. Here we demonstrated in primary cultured neurons that calcium released from acidic calcium stores triggered calcium influx across the plasma membrane, a phenomenon we have termed “acidic store-operated calcium entry (aSOCE)”. aSOCE was functionally distinct from store-operated calcium release and calcium entry involving endoplasmic reticulum. aSOCE appeared to be governed by N-type calcium channels (NTCCs) because aSOCE was attenuated significantly by selectively blocking NTCCs or by siRNA knockdown of NTCCs. Furthermore, we demonstrated that NTCCs co-immunoprecipitated with the lysosome associated membrane protein 1 (LAMP1), and that aSOCE is accompanied by increased cell-surface expression levels of NTCC and LAMP1 proteins. Moreover, we demonstrated that siRNA knockdown of LAMP1 or Rab27a, both of which are key proteins involved in lysosome exocytosis, attenuated significantly aSOCE. Taken together our data suggest that aSOCE occurs in neurons, that aSOCE plays an important role in regulating the levels and actions of intraneuronal calcium, and that aSOCE is regulated at least in part by exocytotic insertion of N-type calcium channels into plasma membranes through LAMP1-dependent lysosome exocytosis.     

Making sense with TRP channels: store-operated calcium entry and the ion channel Trpm5 in taste receptor cells

The sense of taste plays a critical role in the life and nutritional status of organisms. During the last decade, several molecules involved in taste detection and transduction have been identified, providing a better understanding of the molecular physiology of taste receptor cells. However, a comprehensive catalogue of the taste receptor cell signaling machinery is still unavailable. We have recently described the occurrence of calcium signaling mechanisms in taste receptor cells via apparent store-operated channels and identified Trpm5, a novel candidate taste transduction element belonging to the mammalian family of transient receptor potential channels. Trpm5 is expressed in a tissue-restricted manner, with high levels in gustatory tissue. In taste cells, Trpm5 is co-expressed with taste-signaling molecules such as alpha-gustducin, Ggamma(13), phospholipase C beta(2) and inositol 1,4,5-trisphosphate receptor type III. Biophysical studies of Trpm5 heterologously expressed in Xenopus oocytes and mammalian CHO-K1 cells indicate that it functions as a store-operated channel that mediates capacitative calcium entry. The role of store-operated channels and Trpm5 in capacitative calcium entry in taste receptor cells in response to bitter compounds is discussed.     

Voltage-dependent facilitation of cardiac L-type Ca channels expressed in HEK-293 cells requires beta-subunit

The activity of native L-type Ca channels can be facilitated by strong depolarizations. The cardiac Ca channel alpha(1C)-subunit was transiently expressed in human embryonic kidney (HEK-293) cells, but these channels did not exhibit voltage-dependent facilitation. Coexpression of the Ca channel beta(1a)- or beta(2a)-subunit with the alpha(1C)-subunit enabled voltage-dependent facilitation in 40% of cells tested. The onset of facilitation in alpha(1C) + beta(1a)-expressing HEK-293 cells was rapid after a depolarization to +100 mV (tau = 7.0 ms). The kinetic features of the facilitated currents were comparable to those observed for voltage-dependent relief of G protein inhibition demonstrated for many neuronal Ca channels; however, intracellular dialysis with guanosine 5'-O-(2-thiodiphosphate) and guanosine 5'-O-(3-thiotriphosphate) in the patch pipette had no effect on facilitation. Stimulation of G protein-coupled receptors, either endogenous (somatostatin receptors) or coexpressed (adenosine A(1) receptors), did not affect voltage-dependent facilitation. These results indicate that the cardiac Ca channel alpha(1C)-subunit can exhibit voltage-dependent facilitation in HEK-293 cells only when coexpressed with an auxiliary beta-subunit and that this facilitation is independent of G protein pathways.     

Activation of muscarinic receptors reduces store-operated Ca2+ entry in HEK293 cells

In many cell types membrane receptors for hormones or neurotransmitters activate a signal transduction pathway which releases Ca2+ from intracellular Ca2+ stores by the second messenger inositol 1,4,5-trisphosphate. As a consequence store-operated Ca2+ entry (SOCE) becomes activated. In the present study we addressed the question if receptor/agonist binding can modulate Ca2+ entry by mechanisms different from the store-operated one. Therefore SOCE was examined in HEK293 cells microscopically with the fura-2 technique and with patch clamp. We found that maximally preactivated SOCE could, concentration dependently, be reduced up to 80% by the muscarinic agonist acetylcholine when the cytoplasmic Ca2+ concentration was used as a measure. Muscarinic receptors seem to mediate this decrease since atropine blocked the effect completely and cell types without muscarinic receptors (BHK21, CHO) did not show acetylcholine-induced decrease of Ca2+ entry. Moreover expression of muscarinic receptor subtypes M1 and M3 in BHK21 cells established the muscarinic decrease of SOCE. Electrical measurements revealed that the membrane potential of HEK293 cells did not show any response to ACh, excluding that changes of driving forces are responsible for the block of Ca2+ entry. In contrast the electrical current which is responsible for SOCE in HEK293 cells (Ca2+ release-activated Ca2+ current (I(CRAC)) was inhibited (maximally 55%) by 10 microM ACh. From these data we conclude that in HEK293 cells a muscarinic signal transduction pathway exists which decreases the cytoplasmic Ca2+ concentration by an inhibition of I(CRAC). This mechanism may serve as a modulator of Ca2+ entry preventing a Ca2+ overload of the cytoplasm after Ca2+ store depletion.     

Overexpression of TRPC3 reduces the content of intracellular calcium stores in HEK-293 cells

The mammalian canonical transient receptor channels (TRPCs) are considered to be candidates for store-operated calcium channels (SOCCs). Many studies have addressed how TRPC3 channels are affected by depletion of intracellular calcium stores. Conflicting results have been shown for TRPC3 regarding its function, and this has been linked to its level of expression in various systems. In the present study, we have investigated how overexpression of TRPC3 interferes with the regulation of intracellular calcium stores. We demonstrate that overexpression of TRPC3 reduces the mobilization of calcium in response to stimulation of the cells with thapsigargin (TG) or the G-protein coupled receptor agonist sphingosine-1-phosphate (S1P). Our results indicate that this is the result of the expression of TRPC3 channels in the endoplasmic reticulum (ER), thus depleting ER calcium stores. OAG evoked calcium entry in cells overexpressing TRPC3, indicating that functional TRPC3 channels were also expressed in the plasma membrane. Taken together, our results show that overexpression of the putative SOCC, TRPC3, actually reduces the calcium content of intracellular stores, but does not enhance agonist-evoked or store-dependent calcium entry. Our results may, in part, explain the conflicting results obtained in previous studies on the actions of TRPC3 channels.     

Tetrapandins, a new class of scorpion toxins that specifically inhibit store-operated calcium entry in human embryonic kidney-293 cells

Venoms from 14 snakes and four scorpions were screened for inhibitory activities toward store-operated Ca2+ entry (SOCE) in human embryonic kidney-293 cells. An inhibitory activity was found in venom from the African scorpion Pandinus imperator. The active agent of this venom was purified by gel filtration and reverse-phase high pressure liquid chromatography methods. Sequence information on the purified fraction, by automatic Edman degradation and mass spectrometry analysis, identified the activity as being contained in two tetrapeptides, which we have named tetrapandins. We demonstrate that synthesized tetrapandins have inhibitory activity for SOCE in human embryonic kidney-293 cells while having no effect on either thapsigargin- or carbachol-stimulated release of Ca2+ stores. These toxins should be extremely useful in future studies to determine downstream events regulated by SOCE as well as to determine whether multiple pathways exist for thapsigargin-stimulated Ca2+ entry.     

The over-expression of TRPC6 channels in HEK-293 cells favours the intracellular accumulation of zinc

TRPC6 are plasma membrane cation channels. By means of live-cell imaging and spectroscopic methods, we found that HEK cells expressing TRPC6 channels (HEK-TRPC6) are enriched in zinc and sulphur and have a reduced copper content when compared to HEK cells and HEK cells expressing TRPC3 channels (HEK-TRPC3). Hence, HEK-TRPC6 cells have larger pools of mobilizable Zn2+ and are more sensitive to an oxidative stress. Synchrotron X-ray fluorescence experiments showed a higher zinc content in the nuclear region indicating that the intracellular distribution of this metal was influenced by the over-expression of TRPC6 channels. Their properties were investigated with the diacylglycerol analogue SAG and the plant extract hyperforin. Electrophysiological recordings and imaging experiments with the fluorescent Zn2+ probe FluoZin-3 demonstrated that TRPC6 channels form Zn2+-conducting channels. In cortical neurons, hyperforin-sensitive channels co-exist with voltage-gated channels, AMPA and NMDA receptors, which are known to transport Zn2+. The ability of these channels to regulate the size of the mobilizable pools of Zn2+ was compared. The data collected indicate that the entry of Zn2+ through TRPC6 channels can up-regulate the size of the DTDP-sensitive pool of Zn2+. By showing that TRPC6 channels constitute a Zn2+ entry pathway, our study suggests that they could play a role in zinc homeostasis.     

Canonical transient receptor potential TRPC7 can function as both a receptor- and store-operated channel in HEK-293 cells

Previous studies on the activation mechanism of canonical transient receptor potential (TRPC) channels have often produced conflicting conclusions. All seven have been shown to be activated by phospholipase C (PLC)-coupled receptors, but TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, and TRPC7 have also been proposed to function as store-operated channels.¹ In the case of TRPC3, the expression environment and the expression level appear to determine the mode of regulation. Evidence of a close structural relative of TRPC3, TRPC7, has been presented that this channel is activated by receptor activation or by store depletion. On the basis of previous findings for TRPC3, we reasoned that subtle differences in structure or expression conditions might account for the apparent distinct gating mechanisms of TRPC7. To reexamine the mode of activation of TRPC7, we stably and transiently transfected human embryonic kidney (HEK)-293 cells with cDNA encoding for human TRPC7. We examined the ability of a PLC-activating agonist and an intracellular Ca²⁺ store-depleting agent to activate these channels. Our findings demonstrate that when transiently expressed in HEK-293 cells, TRPC7 forms channels that are activated by PLC-stimulating agonists, but not by Ca²⁺ store depletion. However, when stably expressed in HEK-293 cells, TRPC7 can be activated by either Ca²⁺ store depletion or PLC activation. To our knowledge, this is the first demonstration of a channel protein that can be activated by both receptor- and store-operated modes in the same cell. In addition, the results reconcile the apparently conflicting findings of other laboratories regarding TRPC7 regulation. calcium signaling; nonselective cation channels     

The role of STIM1 in the receptor- and store-operated calcium influx in HEK293 cells

The possible role of STIM1 protein in the regulation of activity of receptor- and store-operated Ca²⁺ channels in non-excitable cells has been studied. Receptor- and store-operated Ca²⁺ influxes have been measured using the fluorescent method of detection of cytosolic Ca²⁺ concentration and the electrophysiological methods of whole-cell and single-channel current recordings in the control HEK293 cells and in HEK293 cells with suppressed expression of STIM1. The experiments have shown that STIM1 suppression results in a reduction of the amplitudes of both receptor- and store-operated inward calcium currents. The decrease of total Ca²⁺ influx of in response to an agonist or to passive depletion of calcium stores upon STIM1 suppression was due to the decrease or total absence of the activity of high-conductance channels I_max and non-selective channels I_ns in HEK293 cells. A decrease in the STIM1 amount also altered the activity regulation of low-conductance I_min channels that changed from exclusively agonist-operated into store-dependent channels in HEK293 cells.     

TRPC1 protein forms only one type of native store-operated channels in HEK293 cells

TRPC1 is a major component of store-operated calcium entry in many cell types. In our previous studies, three types of endogenous store-operated calcium channels have been described in HEK293 cells, but it remained unknown which of these channels are composed of TRPC1 proteins. Here, this issue has been addressed by performing single-channel analysis in HEK293 cells transfected with anti-TRPC1 siRNA (siTPRC1) or a TPRC1-encoding plasmid. The results show that thapsigargin-or agonist-induced calcium influx is significantly attenuated in siTRPC1-transfected HEK293 cells. TRPC1 knockdown by siRNA results in the disappearance of store-operated I_max channels, while the properties of I_min and I_NS channels are unaffected. In HEK293 cells with overexpressed TRPC1 protein, the unitary current–voltage relationship of exogenous TRPC1 channels is almost linear, with a slope conductance of about 17 pS. The extrapolated reversal potential of expressed TRPC1 channels is +30 mV. Therefore, the main electrophysiological and regulatory properties of expressed TRPC1 and native I_max channels are identical. Moreover, TRPC1 overexpression in HEK293 cells results in an increased number of store-operated I_max channels. All these data allow us to conclude that TRPC1 protein forms native store-operated I_max channels but is not an essential subunit for other store-operated channel types in HEK293 cells.     

Overexpression of Bax induces down-regulation of store-operated calcium entry in prostate cancer cells

Store-operated Ca2+ channels control homeostasis between extracellular Ca2+ reservoir and intracellular Ca2+ storage and play important roles in apoptosis in a wide variety of cells, including prostate epithelia. Recent studies have shown that the acquired apoptosis-resistant nature of androgen-independent prostate cancer is associated with reduced function of store-operated Ca2+ entry (SOCE). This study investigates the functional interaction between Bax and SOCE in the apoptosis signaling cascade in prostate cancer. Our previous findings show that NRP-154, an androgen-independent prostate cancer cell line, could sustain overexpression of exogenous Bax without undergoing apoptosis. Here we show that sustained overexpression of Bax in NRP-154 cells leads to down-regulation of SOCE and reduced Ca2+ storage inside the endoplasmic reticulum. While reduced SOCE may represent an adaptive mechanism for cell survival, increased levels of Bax in the latent state enhances the sensitivity of NRP-154 cells to TGF-beta and thapsigargin-induced apoptosis. This enhanced apoptosis can be reduced by 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of SOCE, or reversed under conditions where SOCE is only partially activated. Our results demonstrate a functional interaction between Bax and SOCE in apoptosis of prostate cancer, and support the concept that improving this interaction has therapeutic implications for prostate cancer.     

Effectors of the frequency of calcium oscillations in HEK-293 cells: wavelet analysis and a computer model

Oscillations of the intracellular concentration of Ca²⁺ in cultured HEK-293 cells, which heterologously expressed the calcium-sensing receptor, were recorded with the fluorophore Fura-2 using fluorescence microscopy. HEK-293 cells are extremely sensitive to small perturbations in extracellular calcium concentrations. Resting cells were attached to cover slips and perifused with saline solution containing physiologically relevant extracellular Ca²⁺ concentrations in the range 0.5–5 mM. Acquired digitized images of the cells showed oscillatory fluctuations in the intracellular Ca²⁺ concentration over the time course, and were processed as a function of the change in Fura-2 excitation ratio and frequency at 12–37°C. Newly developed data processing techniques with wavelet analysis were used to estimate the frequency at which the rectified sinusoidal oscillations occurred; we estimated ~4 min⁻¹ under normal conditions. Temperature variations revealed an Arrhenius relationship in oscillation frequency. A critical Ca²⁺ concentration of ~2 mM was estimated, below which oscillations did not occur. These data were used to develop a kinetic model of the system that was simulated using Mathematica; kinetic parameter values were adjusted to match the experimentally observed oscillations of intracellular Ca²⁺ concentration as a function of extracellular Ca²⁺ concentration, and temperature; and from these, limit cycles were obtained and control coefficients were estimated for all parameters.