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.     

G protein-independent activation of an inward Na(+) current by muscarinic receptors in mouse pancreatic beta-cells

Depolarization of pancreatic beta-cells is critical for stimulation of insulin secretion by acetylcholine but remains unexplained. Using voltage-clamped beta-cells, we identified a small inward current produced by acetylcholine, which was suppressed by atropine or external Na(+) omission, but was not mimicked by nicotine, and was insensitive to nicotinic antagonists, tetrodotoxin, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DiDS), thapsigargin pretreatment, and external Ca(2+) and K(+) removal. This suggests that muscarinic receptor stimulation activates voltage-insensitive Na(+) channels distinct from store-operated channels. No outward Na(+) current was produced by acetylcholine when the electrochemical Na(+) gradient was reversed, indicating that the channels are inward rectifiers. No outward K(+) current occurred either, and the reversal potential of the current activated by acetylcholine in the presence of Na(+) and K(+) was close to that expected for a Na(+)-selective membrane, suggesting that the channels opened by acetylcholine are specific for Na(+). Overnight pretreatment with pertussis toxin or the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) or guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S) instead of GTP to the pipette solution did not alter this current, excluding involvement of G proteins. Injection of a current of a similar amplitude to that induced by acetylcholine elicited electrical activity in beta-cells perifused with a subthreshold glucose concentration. These results demonstrate that muscarinic receptor activation in pancreatic beta-cells triggers, by a G protein-independent mechanism, a selective Na(+) current that explains the plasma membrane depolarization.     

Muscarinic effects on ion channels in smooth muscle cells

Acetylcholine is the most important excitatory neurotransmitter providing depolarization of the membrane and contraction of different smooth muscle cells due to activation of the muscarinic receptors. In our review, we analyze and summarize the published data on the effects of activation of acetylcholine muscarinic receptors on ion channels expressed in smooth muscle cells of different organs and the results of our own studies of this topic. Special attention is paid to the mechanisms of depolarizing effects of acetylcholine mediated by activation of non-selective cationic channels. Intracellular mechanisms underlying modulating influences on calcium, potassium, and chloride channels are also analyzed. Physiological roles of activation and regulation of different ion channels and possible interactions within this complicated system are discussed.     

Extracellular calcium participates in responses to acetylcholine in Xenopus oocytes

We tested the contribution of extracellular calcium (Ca2+) to membrane electrical responses to acetylcholine (ACh) in native Xenopus oocytes. Removal of Cao caused a decrease in both the rapid (D1) and the slow (D2) chloride currents that comprise the common depolarizing response to ACh in native oocyte. The effect of Ca2+o removal on the muscarinic response was mimicked by the addition of 1 mM Mn2+, an effective antagonist of calcium influx, though not by antagonists of voltage-sensitive calcium channels. When oocytes were challenged with ACh in Ca2(+)-free medium, subsequent addition of 1.8 mM CaCl2 resulted in a rapid, often transient, depolarizing current. Similarly to the Ca2+o-dependent component of membrane electrical responses, the Ca2(+)-evoked current was reversibly abolished by Mn2+, though not by antigonists of voltage-sensitive calcium channels. Depletion of cellular calcium potentiated the Ca2(+)-evoked current, implying negative feedback of calcium channels by calcium. Injection of 10-100 fmol of inositol 1,4,5-trisphosphate (IP3) resulted in a two-component depolarizing current. IP3 injection promoted the appearance of Ca2+o-evoked current that was significantly potentiated by previous calcium depletion. We suggest that activation of cell-membrane muscarinic receptors causes opening of apparently voltage-insensitive and verapamil or diltiazem-resistant calcium channels. These channels may be activated by IP3 or its metabolites, which increase following the activation of cell membrane receptors coupled to a phospholipase C. The channels may be identical to receptor-operated channels described in other model systems.     

Effects of Muscarinic Agonists and Depolarizing Agents on Inositol Monophosphate Accumulation in the Rabbit Vagus Nerve

The effects of muscarinic agonists and depolarizing agents on inositol phospholipid hydrolysis in the rabbit vagus nerve were assessed by the measurement of [3H]inositol monophosphate production in nerves that had been preincubated with [3H]inositol. After 1 h of drug action, carbachol, oxotremorine, and arecoline increased the inositol monophosphate accumulation, though the maximal increase induced by these agonists differed. Addition of the muscarinic antagonists atropine or pirenzepine shifted the carbachol dose-response curves to the right, without decreasing the carbachol maximal stimulatory effects. The KB for pirenzepine was 35 nM, which is characteristic of muscarinic high-affinity binding sites coupled to phosphoinositide turnover and often associated with the M1 receptor subtype. On the other hand, agents known to depolarize or to increase the intracellular Ca2+ concentration, e.g., elevated extracellular K+, ouabain, Ca2+, and the Ca2+ ionophore A23187, also increased inositol monophosphate accumulation. These effects were not mediated by the release of acetylcholine, as suggested by the fact that they could not be potentiated by the addition of physostigmine nor inhibited by the addition of atropine. The Ca(2+)-channel antagonist Cd2+, also known to inhibit the Na+/Ca2+ exchanger, was able to block the effects of K+ and ouabain, but did not alter those of carbachol. These results suggest that depolarizing agents increase inositol monophosphate accumulation in part through elevation of the intracellular Ca2+ concentration and that muscarinic receptors coupled to phosphoinositide turnover are present along the trunk of the rabbit vagus nerve.     

Cholinergic regulation of pericyte-containing retinal microvessels

The aim of this study was to test the hypothesis that the neurotransmitter acetylcholine regulates the function of pericyte-containing retinal microvessels. A vasoactive role for acetylcholine is suggested by the presence of muscarinic receptors on pericytes, which are abluminally positioned contractile cells that may regulate capillary perfusion. However, little is known about the response of retinal microvessels to this neurotransmitter. Here we assessed the effects of cholinergic agonists on microvessels freshly isolated from the adult rat retina. Ionic currents were monitored via perforated patch pipettes; intracellular Ca(2+) levels were quantified with the use of fura 2, and microvascular contractions were visualized with the aid of time-lapse photography. We found that activation of muscarinic receptors elevated pericyte calcium levels, increased depolarizing Ca(2+)-activated chloride currents and caused pericytes to contract in a Ca(2+)-dependent manner. Most contracting pericytes were near capillary bifurcations. Contraction of a pericyte caused the adjacent capillary lumen to constrict. Thus acetylcholine may serve as a vasoactive signal by regulating pericyte contractility and thereby capillary perfusion in the retina.     

Modulation of the bursting properties of single mouse pancreatic beta-cells by artificial conductances

Glucose triggers bursting activity in pancreatic islets, which mediates the Ca2+ uptake that triggers insulin secretion. Aside from the channel mechanism responsible for bursting, which remains unsettled, it is not clear whether bursting is an endogenous property of individual beta-cells or requires an electrically coupled islet. While many workers report stochastic firing or quasibursting in single cells, a few reports describe single-cell bursts much longer (minutes) than those of islets (15-60 s). We studied the behavior of single cells systematically to help resolve this issue. Perforated patch recordings were made from single mouse beta-cells or hamster insulinoma tumor cells in current clamp at 30-35 degrees C, using standard K+-rich pipette solution and external solutions containing 11.1 mM glucose. Dynamic clamp was used to apply artificial KATP and Ca2+ channel conductances to cells in current clamp to assess the role of Ca2+ and KATP channels in single cell firing. The electrical activity we observed in mouse beta-cells was heterogeneous, with three basic patterns encountered: 1) repetitive fast spiking; 2) fast spikes superimposed on brief (<5 s) plateaus; or 3) periodic plateaus of longer duration (10-20 s) with small spikes. Pattern 2 was most similar to islet bursting but was significantly faster. Burst plateaus lasting on the order of minutes were only observed when recordings were made from cell clusters. Adding gCa to cells increased the depolarizing drive of bursting and lengthened the plateaus, whereas adding gKATP hyperpolarized the cells and lengthened the silent phases. Adding gCa and gKATP together did not cancel out their individual effects but could induce robust bursts that resembled those of islets, and with increased period. These added currents had no slow components, indicating that the mechanisms of physiological bursting are likely to be endogenous to single beta-cells. It is unlikely that the fast bursting (class 2) was due to oscillations in gKATP because it persisted in 100 microM tolbutamide. The ability of small exogenous currents to modify beta-cell firing patterns supports the hypothesis that single cells contain the necessary mechanisms for bursting but often fail to exhibit this behavior because of heterogeneity of cell parameters.     

Modulatory action of acetylcholine on the Na-dependent action potentials in Kenyon cells isolated from the mushroom body of the cricket brain

Kenyon cells, intrinsic neurons of the insect mushroom body, have been assumed to be a site of conditioning stimulus (CS) and unconditioned stimulus (US) association in olfactory learning and memory. Acetylcholine (ACh) has been implicated to be a neurotransmitter mediating CS reception in Kenyon cells, causing rapid membrane depolarization via nicotinic ACh receptors. However, the long-term effects of ACh on the membrane excitability of Kenyon cells are not fully understood. In this study, we examined the effects of ACh on Na⁺ dependent action potentials (Na⁺ spikes) elicited by depolarizing current injection and on net membrane currents under the voltage clamp condition in Kenyon cells isolated from the mushroom body of the cricket Gryllus bimaculatus. Current-clamp studies using amphotericin B perforated-patch recordings showed that freshly dispersed cricket Kenyon cells could produce repetitive Na⁺ spikes in response to prolonged depolarizing current injection. Bath application of ACh increased both the instantaneous frequency and the amplitudes of Na⁺ spikes. This excitatory action of ACh on Kenyon cells is attenuated by the pre-treatment of the cells with the muscarinic receptor antagonists, atropine and scopolamine, but not by the nicotinic receptor antagonist mecamylamine. Voltage-clamp studies further showed that bath application of ACh caused an increase in net inward currents that are sensitive to TTX, whereas outward currents were decreased by this treatment. These results indicate that in order to mediate CS, ACh may modulate the firing properties of Na⁺ spikes of Kenyon cells through muscarinic receptor activation, thus increasing Na conductance and decreasing K conductance.     

The alpha 1- and alpha 2-adrenoceptor and muscarinic responses of normal and axotomized bullfrog sympathetic ganglia

When neurones in bullfrog paravertebral sympathetic ganglia are studied by means of the sucrose-gap technique, muscarinic agonists produce a biphasic response (an initial hyperpolarization of ganglionic C cells followed by a depolarization of ganglionic B cells). Activation of ganglionic alpha 2-adrenoceptors promotes hyperpolarization. The present experiments with selective alpha 1- and alpha 2-adrenoceptor agonists and antagonists provided evidence for the existence of hitherto undescribed alpha 1-adrenoceptors, which are responsible for the production of depolarizing responses in these ganglia. Fifteen to twenty-five days after cutting postganglionic axons (axotomy), there was a nonselective depression of both alpha 1- and alpha 2-adrenoceptor mechanisms but little change in muscarinic responses. These results argue against the hypothesis that C cells assume all the properties of B cells after axotomy. Since the alpha-selective agonist phenylephrine failed to depolarize axotomized ganglia, it is unlikely that an alpha 1-adrenoceptor mechanism is prominent in axotomized neurones as it is in some immature adrenergic neurones. The data are consistent with the idea that axotomy selectively affects the properties of certain types of cation channels and raise questions as to the mechanisms involved in regulating the expression and maintenance of specific neurotransmitter responses on ganglionic neurones.     

Graded activation of CRAC channel by binding of different numbers of STIM1 to Orai1 subunits

The Ca²⁺ release-activated Ca²⁺ (CRAC) channel pore is formed by Orai1 and gated by STIM1 after intracellular Ca²⁺ store depletion. To resolve how many STIM1 molecules are required to open a CRAC channel, we fused different numbers of Orai1 subunits with functional two-tandem cytoplasmic domains of STIM1 (residues 336-485, designated as S domain). Whole-cell patch clamp recordings of these chimeric molecules revealed that CRAC current reached maximum at a stoichiometry of four Orai1 and eight S domains. Further experiments indicate that two-tandem S domains specifically interact with the C-terminus of one Orai1 subunit, and CRAC current can be gradually increased as more Orai1 subunits can interact with S domains or STIM1 proteins. Our data suggest that maximal opening of one CRAC channel requires eight STIM1 molecules, and support a model that the CRAC channel activation is not in an “all-or-none” fashion but undergoes a graded process via binding of different numbers of STIM1.     

A model of the muscarinic receptor-induced changes in K(+)-current and action potentials in the bullfrog atrial cell.

A model is formulated for characterizing the behavior of the acetylcholine (ACh)-sensitive K+ membrane channel (muscarinic channel) in bullfrog atrial myocytes. Parameters of the muscarinic current model are chosen in fit available data from the literature on bullfrog atrial myocytes (3, 4, 45). This model is subsequently incorporated into a large mathematical model of the bullfrog myocyte that is based on quantitative whole-cell voltage clamp data (40). Simulations are conducted on the active atrial cell model in bathing media containing ACh at different concentrations to explore the effect of this muscarinic channel on the electrical behavior of the myocyte. The model predicts a progressive shortening of the action potential with increasing [ACh], as well as an indirect influence of the muscarinic K+ current on the other membrane currents of the atrial cell. Interpretation of the simulation results provides suggestions for the probable mechanisms underlying the shortening of the action potential due to activity of the muscarinic channel. Specifically, the model predicts that with an increase in ACh concentration: (a) the outward muscarinic current, IK,ACh(t), increases in magnitude but shortens in duration; (b) the calcium current, ICa(t), may increase in magnitude, but when it does so it decreases in duration compared with the control conditions; (c) the intracellular Ca2+ concentration [Ca2+]i waveform during the action potential decreases in both magnitude and duration. Because the contractile activity of the cell is controlled by the [Ca2+]i waveform, the model predicts a decrease in contractile strength with an increase in ACh concentration in the bathing medium; i.e., a negative inotropic effect.     

Distinct properties of CRAC and MIC channels in RBL cells

In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca(2+) release-activated Ca(2+) (CRAC) channels open in response to passive Ca(2+) store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg(2+) exposes an outwardly rectifying current (Mg(2+)-inhibited cation [MIC]) that also admits monovalent cations when external divalent ions are removed. Here we demonstrate that CRAC and MIC currents are separable by ion selectivity and rectification properties: by kinetics of activation and susceptibility to run-down and by pharmacological sensitivity to external Mg(2+), spermine, and SKF-96365. Importantly, selective run-down of MIC current allowed CRAC and MIC current to be characterized under identical ionic conditions with low internal Mg(2+). Removal of internal Mg(2+) induced MIC current despite widely varying Ca(2+) and EGTA levels, suggesting that Ca(2+)-store depletion is not involved in activation of MIC channels. Increasing internal Mg(2+) from submicromolar to millimolar levels decreased MIC currents without affecting rectification but did not alter CRAC current rectification or amplitudes. External Mg(2+) and Cs(+) carried current through MIC but not CRAC channels. SKF-96365 blocked CRAC current reversibly but inhibited MIC current irreversibly. At micromolar concentrations, both spermine and extracellular Mg(2+) blocked monovalent MIC current reversibly but not monovalent CRAC current. The biophysical characteristics of MIC current match well with cloned and expressed TRPM7 channels. Previous results are reevaluated in terms of separate CRAC and MIC channels.     

Calumin, a Ca²⁺-binding protein on the endoplasmic reticulum, alters the ion permeability of Ca²⁺ release-activated Ca²⁺ (CRAC) channels

Store-operated channels (SOC) are Ca(2+)-permeable channels that are activated by IP(3)-receptor-mediated Ca(2+) depletion of the endoplasmic reticulum (ER). Recent studies identify a membrane pore subunits, Orai1 and a Ca(2+) sensor on ER, STIM1 as components of Ca(2+) release-activated Ca(2+) (CRAC) channels, which are well-characterized SOCs. On the other hand, proteins that act as modulators of SOC activity remain to be identified. Calumin is a Ca(2+)-binding protein that resides on the ER and functional experiments using calumin-null mice demonstrate that it is involved in SOC function, although its role is unknown. This study used electrophysiological analysis to explore whether calumin modulates CRAC channel activity. CRAC channel currents were absent in HEK293 cells co-expressing calumin with the CRAC channel components, Orai1 or STIM1. Meanwhile, HEK cells that co-expressed calumin with CRAC channels exhibited larger currents with slower inactivation than cells expressing CRAC channels alone. The current-voltage relationship showed an inwardly rectifying current, but a negative shift in the reversal potential of greater than 60mV was observed in HEK cells co-expressing calumin with CRAC channels. In addition, the permeability coefficient ratio of Ca(2+) over monovalent cations was much lower than that of cells expressing CRAC channels alone. Replacement of Na(+) with N-methyl-d-glucamine(+) in the external solution noticeably diminished the CRAC current in HEK cells co-expressing calumin and CRAC channels. In a Cs(+)-based external solution, CRAC current was not observed in either cell-type. In addition, Ca(2+) imaging analysis revealed that co-transfection of calumin reduced extracellular Ca(2+) influx via CRAC channels. Further, calumin was shown to be directly associated with CRAC channels. These results reveal a novel mechanism for the regulation of CRAC channels by calumin.     

Ca2+ modulation of Ca2+ release-activated Ca2+ channels is responsible for the inactivation of its monovalent cation current

The Ca(2+) release-activated Ca(2+) (CRAC) channel is the most well documented of the store-operated ion channels that are widely expressed and are involved in many important biological processes. However, the regulation of the CRAC channel by intracellular or extracellular messengers as well as its molecular identity is largely unknown. Specifically, in the absence of extracellular divalent cations it becomes permeable to monovalent cations with a larger conductance, however this monovalent cation current inactivates rapidly by an unknown mechanism. Here we found that Ca(2+) dissociation from a site on the extracellular side of the CRAC channel is responsible for the inactivation of its Na(+) current, and Ca(2+) occupancy of this site otherwise potentiates its Ca(2+) as well as Na(+) currents. This Ca(2+)-dependent potentiation is required for the normal functioning of CRAC channels.     

Permeation through store-operated CRAC channels in divalent-free solution: potential problems and implications for putative CRAC channel genes

CRAC channels are key calcium conduits in both physiological and pathological states. Understanding how these channels are controlled is important as this will not only provide insight into a novel signal transduction pathway coupling intracellular stores to the channels in the plasma membrane, but might also be of clinical relevance. Determining the molecular identity of the CRAC channels will certainly be a major step forward. Like all Ca(2+)-selective channels, CRAC channels lose their selectivity in divalent-free external solution to support large monovalent Na(+) currents. This approach has provided new insight into channel permeation and selectivity, and identifies some interesting differences between CRAC channels and voltage-operated calcium channels (VOCCs). Studies in divalent-free solution are a double-edged sword, however. Electrophysiologists need to be wary because some of the conditions used to study I(CRAC) in divalent-free external solution, notably omission of Mg(2+)/Mg-ATP from the recording pipette solution, activates an additional current permeating through Mg(2+)-nucleotide-regulated metal ion current (MagNuM; TRPM7) channels. This channel underlies the large single-channel events that have been attributed to CRAC channels in the past and which have been used to as a tool to identify store-operated channels in native cells and recombinant expression systems.Are we any closer to identifying the elusive CRAC channel gene(s)? TRPV6 seemed a very attractive candidate, but one of the main arguments supporting it was a single-channel conductance in divalent-free solution similar to that for CRAC reported under conditions where MagNuM is active. We now know that the conductance of TRPV6 is approximately 200-fold larger than that of CRAC in native tissue. Moreover, it is unclear if TRPV6 is store-operated. Further work on TRPV6, particularly whether its single-channel conductance is still high under conditions where it apparently forms multimers with endogenous store-operated channels, and whether it is activated by a variety of store depletion protocols, will be helpful in finally resolving this issue.     

Emergence of organized bursting in clusters of pancreatic beta-cells by channel sharing.

Pancreatic beta-cells in an intact Islet of Langerhans exhibit bursting electrical behavior. The Chay-Keizer model describes this using a calcium-activated potassium (K-Ca) channel, but cannot account for the irregular spiking of isolated beta-cells. Atwater I., L. Rosario, and E. Rojas, Cell Calcium. 4:451-461, proposed that the K-Ca channels, which are rarely open, are shared by several cells. This suggests that the chaotic behavior of isolated cells is stochastic. We have revised the Chay-Keizer model to incorporate voltage clamp data of Rorsman and Trube and extended it to include stochastic K-Ca channels. This model can describe the behavior of single cells, as well as that of clusters of cells tightly coupled by gap junctions. As the size of the clusters is increased, the electrical activity shows a transition from chaotic spiking to regular bursting. Although the model of coupling is over-simplified, the simulations lend support to the hypothesis that bursting is the result of channel sharing.     

CRACM1, CRACM2, and CRACM3 are store-operated Ca2+ channels with distinct functional properties

STIM1 in the endoplasmic reticulum and CRACM1 in the plasma membrane are essential molecular components for controlling the store-operated CRAC current. CRACM1 proteins multimerize and bind STIM1, and the combined overexpression of STIM1 and CRACM1 reconstitutes amplified CRAC currents. Mutations in CRACM1 determine the selectivity of CRAC currents, demonstrating that CRACM1 forms the CRAC channel's ion-selective pore, but the CRACM1 homologs CRACM2 and CRACM3 are less well characterized. Here, we show that both CRACM2 and CRACM3, when overexpressed in HEK293 cells stably expressing STIM1, potentiate I(CRAC) to current amplitudes 15-20 times larger than native I(CRAC). A nonconducting mutation of CRACM1 (E106Q) acts as a dominant negative for all three CRACM homologs, suggesting that they can form heteromultimeric channel complexes. All three CRACM homologs exhibit distinct properties in terms of selectivity for Ca(2+) and Na(+), differential pharmacological effects in response to 2-APB, and strikingly different feedback regulation by intracellular Ca(2+). Each of the CRAC channel proteins' specific functional features and the potential heteromerization provide for flexibility in shaping Ca(2+) signals, and their characteristic biophysical and pharmacological properties will aid in identifying CRAC-channel species in native cells that express them.     

All-or-none activation of CRAC channels by agonist elicits graded responses in populations of mast cells

In nonexcitable cells, receptor stimulation evokes Ca(2+) release from the endoplasmic reticulum stores followed by Ca(2+) influx through store-operated Ca(2+) channels in the plasma membrane. In mast cells, store-operated entry is mediated via Ca(2+) release-activated Ca(2+) (CRAC) channels. In this study, we find that stimulation of muscarinic receptors in cultured mast cells results in Ca(2+)-dependent activation of protein kinase Calpha and the mitogen activated protein kinases ERK1/2 and this is required for the subsequent stimulation of the enzymes Ca(2+)-dependent phospholipase A(2) and 5-lipoxygenase, generating the intracellular messenger arachidonic acid and the proinflammatory intercellular messenger leukotriene C(4). In cell population studies, ERK activation, arachidonic acid release, and leukotriene C(4) secretion were all graded with stimulus intensity. However, at a single cell level, Ca(2+) influx was related to agonist concentration in an essentially all-or-none manner. This paradox of all-or-none CRAC channel activation in single cells with graded responses in cell populations was resolved by the finding that increasing agonist concentration recruited more mast cells but each cell responded by generating all-or-none Ca(2+) influx. These findings were extended to acutely isolated rat peritoneal mast cells where muscarinic or P2Y receptor stimulation evoked all-or-none activation of Ca(2+)entry but graded responses in cell populations. Our results identify a novel way for grading responses to agonists in immune cells and highlight the importance of CRAC channels as a key pharmacological target to control mast cell activation.     

Permeation of monovalent cations through the non-capacitative arachidonate-regulated Ca2+ channels in HEK293 cells. Comparison with endogenous store-operated channels

In a manner similar to voltage-gated Ca(2+) channels and Ca(2+) release-activated Ca(2+) (CRAC) channels, the recently identified arachidonate-regulated Ca(2+) (ARC) channels display a large monovalent conductance upon removal of external divalent cations. Using whole-cell patch-clamp recording, we have characterized the properties of these monovalent currents in HEK293 cells stably transfected with the m3 muscarinic receptor and compared them with the corresponding currents through the endogenous store-operated Ca(2+) (SOC) channels in the same cells. Although the monovalent currents seen through these two channels displayed certain similarities, several marked differences were also apparent, including the magnitude of the monovalent current/Ca(2+) current ratio, the rate and nature of the spontaneous decline in the currents, and the effects of external monovalent cation substitutions and removal of internal Mg(2+). Moreover, monovalent ARC currents could be activated after the complete spontaneous inactivation of the corresponding SOC current in the same cell. We conclude that the non-capacitative ARC channels share, with voltage-gated Ca(2+) channels and store-operated Ca(2+) channels (e.g. SOC and CRAC the general property of monovalent ion permeation in the nominal absence of extracellular divalent ions. However, the clear differences between the properties of these currents through ARC and SOC channels in the same cell confirm that these represent distinct conductances.     

cGMP-gated conductance in retinal bipolar cells is suppressed by the photoreceptor transmitter.

Transmitter release from photoreceptors is decreased by light, resulting in a conductance increase in depolarizing bipolar cells. Addition of exogenous cGMP through a patch pipette to depolarizing bipolar cells from slices of dark-adapted tiger salamander retina resulted in an enhancement of the light response. This enhancement was blocked by GTP-gamma-S and dipyridamole, an inhibitor of phosphodiesterase. GTP-gamma-S and dipyridamole also blocked responses to exogenously applied 2-amino-4-phosphonobutyrate (APB), the glutamate agonist selective for this receptor. These data support the hypothesis that the postsynaptic receptor is linked via a G protein to a phosphodiesterase. The binding of glutamate or APB to the receptor suppresses a cGMP-activated current by increasing the rate of cyclic nucleotide hydrolysis.