I ARC, a Novel Arachidonate-regulated, Noncapacitative Ca2+ Entry Channel

Along with the inositol trisphosphate-induced release of stored Ca2+, a receptor-enhanced entry of Ca2+ is a critical component of intracellular Ca2+ signals generated by agonists acting at receptors coupled to the activation of phospholipase C. Although the simple emptying of the intracellular Ca2+ stores is known to be capable of activating Ca2+ entry via the so-called “capacitative” mechanism, recent evidence suggests that Ca2+ entry at physiological agonist concentrations, where oscillatory Ca2+ signals are typically observed, does not conform to such a model. Instead, a noncapacitative Ca2+entry pathway regulated by arachidonic acid appears to be responsible for Ca2+ entry under these conditions. Using whole-cell patch clamp techniques we demonstrate that low concentrations of arachidonic acid activate a Ca2+-selective current that is superficially similar to the store-operated current I CRAC, but which also demonstrates certain distinct features. We have named this novel currentI ARC (forarachidonate-regulated calcium current). Importantly, I ARC can be readily activated in cells whose Ca2+ stores have been maximally depleted. I ARC represents a novel Ca2+ entry pathway that is entirely separate from those activated by store depletion and is specifically activated at physiological levels of stimulation. Along with the inositol trisphosphate-induced release of stored Ca2+, a receptor-enhanced entry of Ca2+ is a critical component of intracellular Ca2+ signals generated by agonists acting at receptors coupled to the activation of phospholipase C. Although the simple emptying of the intracellular Ca2+ stores is known to be capable of activating Ca2+ entry via the so-called “capacitative” mechanism, recent evidence suggests that Ca2+ entry at physiological agonist concentrations, where oscillatory Ca2+ signals are typically observed, does not conform to such a model. Instead, a noncapacitative Ca2+entry pathway regulated by arachidonic acid appears to be responsible for Ca2+ entry under these conditions. Using whole-cell patch clamp techniques we demonstrate that low concentrations of arachidonic acid activate a Ca2+-selective current that is superficially similar to the store-operated current I CRAC, but which also demonstrates certain distinct features. We have named this novel currentI ARC (forarachidonate-regulated calcium current). Importantly, I ARC can be readily activated in cells whose Ca2+ stores have been maximally depleted. I ARC represents a novel Ca2+ entry pathway that is entirely separate from those activated by store depletion and is specifically activated at physiological levels of stimulation. store-operated channel (s) cytosolic free calcium ion concentration farad Ca2+ release-activated Ca2+ current store-operated current arachidonate-regulated calcium current sarcoplasmic-endoplasmic reticulum calcium pump N-methyl-d-glucamine transient receptor potential The prevailing model of receptor-activated Ca2+ entry in non-excitable cells is the so-called “capacitative” or store-operated model in which the emptying of the agonist-sensitive stores alone is both necessary and sufficient to activate Ca2+ entry (1. Putney J. W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2115) Google Scholar, 2. Putney J. W. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1264) Google Scholar). This capacitative entry, which occurs via store-operated channels (SOCs), 1 underlies both the sustained elevations in Ca2+i observed following stimulation with high agonist concentrations and the subsequent refilling of intracellular agonist-sensitive Ca2+ stores on the termination of such signals (1. Putney J. W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2115) Google Scholar, 2. Putney J. W. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1264) Google Scholar). However, physiological concentrations of appropriate agonists often result in oscillatory Ca2+i signals where the internal Ca2+ stores empty only partially and/or transiently making a capacitative mechanism for the control of Ca2+ entry unlikely (3. Shuttleworth T. J. Cell Calcium. 1999; 25: 237-246Crossref PubMed Scopus (94) Google Scholar). Furthermore, many of the specific characteristics of the Ca2+ entry seen during such signals, as well as the properties of those capacitative channels characterized to date, are inconsistent with such a role (3. Shuttleworth T. J. Cell Calcium. 1999; 25: 237-246Crossref PubMed Scopus (94) Google Scholar). Recently, we have described a novel mechanism of agonist-activated Ca2+ entry that is independent of store depletion (i. e. noncapacitative) (4. Shuttleworth T. J. Thompson J. L. Biochem. J. 1996; 316: 819-824Crossref PubMed Scopus (50) Google Scholar) and which appears to be specifically responsible for the entry of Ca2+ seen following stimulation with low agonist concentrations, such as those resulting in Ca2+oscillations (5. Shuttleworth T. J. J. Biol. Chem. 1996; 271: 21720-21725Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 6. Shuttleworth T. J. Thompson J. L. J. Biol. Chem. 1998; 273: 32636-32643Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The signal responsible for the activation of the entry of Ca2+ under these circumstances is a receptor-mediated generation of arachidonic acid (5. Shuttleworth T. J. J. Biol. Chem. 1996; 271: 21720-21725Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 6. Shuttleworth T. J. Thompson J. L. J. Biol. Chem. 1998; 273: 32636-32643Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Three key pieces of evidence support this contention: 1) arachidonic acid is generated at the same agonist concentrations that are known to produce Ca2+i oscillations in the same cells, 2) addition of low concentrations of exogenous arachidonic acid induces an entry of Ca2+ that is entirely independent of store depletion, and 3) inhibition of the agonist-induced generation of arachidonic acid specifically and rapidly blocks the Ca2+entry associated with Ca2+i oscillations, yet it is without effect on capacitative Ca2+ entry (5. Shuttleworth T. J. J. Biol. Chem. 1996; 271: 21720-21725Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 6. Shuttleworth T. J. Thompson J. L. J. Biol. Chem. 1998; 273: 32636-32643Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Recent evidence suggests that this Ca2+ entry pathway, which is activated by arachidonic acid itself not a metabolite (6. Shuttleworth T. J. Thompson J. L. J. Biol. Chem. 1998; 273: 32636-32643Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), is entirely distinct from the capacitative or store-operated pathway (7. Shuttleworth T. J. Thompson J. L. J. Biol. Chem. 1999; 274: 31174-31178Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Using whole-cell patch clamp techniques we have now characterized the current associated with this novel arachidonic acid-regulated Ca2+ entry pathway and demonstrate that it possesses certain unique properties that clearly distinguish it fromI CRAC, the archetypal store-operated Ca2+ channel current (8. Hoth M. Penner R. Nature. 1992; 355: 353-356Crossref PubMed Scopus (1495) Google Scholar, 9. Zweifach A. Lewis R. S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6259-6299Crossref Scopus (697) Google Scholar, 10. Hoth M. Penner R. J. Physiol. (Lond. ). 1993; 465: 359-386Crossref Scopus (662) Google Scholar, 11. McDonald T. V. Premack B. A. Gardner P. J. Biol. Chem. 1993; 268: 3889-3896Abstract Full Text PDF PubMed Google Scholar). We have named this novel current I ARC, for a rachidonate- r egulated c alcium current. HEK293 cells stably transfected with the human m3 muscarinic receptor were a generous gift from Dr. Craig Logsdon (University of Michigan). The cells were cultured under standard conditions in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and antibiotics at 37 °C in a humidified atmosphere of 95% air and 5% CO2 as reported previously (6. Shuttleworth T. J. Thompson J. L. J. Biol. Chem. 1998; 273: 32636-32643Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Cells were plated onto glass coverslips that formed the bottom of a perfusion chamber (Warner) immediately before experimentation. Whole-cell currents were recorded with an Axopatch-1C patch clamp amplifier (Axon Instruments) using standard patch clamp techniques at room temperature (20–22 °C). Patch pipettes (Garner, GC150-F) were fire-polished to a resistance of 3–6 megaOhms. Whole-cell currents were recorded using 250-ms voltage steps from a holding potential of 0 to −80 mV delivered every 2 s. Alternatively, current-voltage relationships were recorded using 150-ms voltage ramps from −100 to +30 mV. Ramps were terminated at +30 mV to avoid activation of a depolarization-activated Cl−current. Currents were sampled at 20 kHz during the voltage steps and 5. 5 kHz during voltage ramps and digitally filtered off-line at 1 KHz. Initial traces obtained on going whole-cell (i. e. before activation of I ARC orI SOC) were averaged and used for leak subtraction. Internal solutions contained (in mm): Cs+ acetate, 140; NaCl, 10; MgCl2, 1. 22; CaCl2, 1. 89; EGTA, 5; HEPES, 10 (pH 7. 2). The free Ca2+ concentration of this solution was calculated to be 100 nm as computed with Maxchelator (12. ref data missingGoogle Scholar). CaCl2was omitted for the experiments involving Ca2+-free internal solutions. Extracellular solutions contained (in mm): NaCl, 140; MgCl2, 1. 2; CaCl2, 10; CsCl, 5; d-glucose, 10; HEPES, 10 (pH 7. 4) unless otherwise specified. Both MgCl2 and CaCl2 were omitted from this solution for the experiments involving divalent-free external solutions and the osmolarity (320 mosmol/liter) was maintained with additional d-glucose. Fast inactivation/activation was examined by comparing peak and steady-state currents during hyperpolarizing pulses to −80 mV. Examination of the capacitative currents during pulses to −80 mV indicated a mean time constant for the capacitative transient of 240 ± 7. 6 μs (n = 159). To minimize contributions from these transients, peak currents (1 ms average) were determined at a point 3 ms after the start of the hyperpolarizing pulse. Steady-state currents were similarly determined at a point 200 ms later. Arachidonic acid was purchased from BioMol and all other chemicals from Sigma. To observe potential Ca2+ entry currents, hyperpolarizing voltage pulses were applied from a holding potential of 0 to −80 mV. Under these conditions, basal currents were usually small and variable (typically ranging between +0. 2 and −0. 3 pA/pF). 2Occasionally, a current was observed to spontaneously activate in the whole-cell mode. This current was much larger than either the endogenous store-operated current or the arachidonate-activated current (∼1. 8 pA/pF at −80 mV compared with ∼0. 6 pA/pF), and demonstrated a linear I/V curve with a reversal potential close to zero. Based on this evidence it would appear that this spontaneously activating current was nonselective for cations. Addition of arachidonic acid (8 μm) to the bath resulted in the activation of a small inward current that averaged 0. 56 ± 0. 05 pA/pF at −80 mV (n = 10) (Fig. 1 A). Taking all experiments together, activation was seen in 76% of cells (n = 120). Current-voltage relationships of the arachidonic acid-activated current were obtained by applying voltage ramps after subtracting leak currents. This current shows strong inward rectification and a reversal potential more positive than +30 mV (Fig. 1 B). The arachidonate-induced current was fully and reversibly inhibited by 50 μm La3+, a potent blocker of Ca2+influx (Fig. 1, A and B). Addition of 50 μm Cd2+, another blocker of Ca2+influx, produced a similar, although somewhat less complete, inhibition (approximately 85%; data not shown). Plots of the mean amplitude of the arachidonate-activated currents against membrane potential demonstrate that the magnitude of the current is reasonably consistent between cells (Fig. 1 C). Complete substitution of extracellular Na+ with NMDG+ had no significant effect on the magnitude of the inward current measured at −80 mV (0. 57 ± 0. 05 pA/pF in Na+ solutions versus0. 49 ± 0. 03 pA/pF in NMDG+ solutions, n = 4, p +30 mV), and potent inhibition by La3+ and Cd2+ are all consistent with the arachidonate-regulated current being a Ca2+ current (=I ARC). As such, these properties are very similar to those demonstrated by the only store-operated current that has been extensively characterized to date, namely the calcium release-activated current (I CRAC) originally described in mast cells, T lymphocytes, and RBL cells (8. Hoth M. Penner R. Nature. 1992; 355: 353-356Crossref PubMed Scopus (1495) Google Scholar, 9. Zweifach A. Lewis R. S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6259-6299Crossref Scopus (697) Google Scholar, 10. Hoth M. Penner R. J. Physiol. (Lond. ). 1993; 465: 359-386Crossref Scopus (662) Google Scholar, 11. McDonald T. V. Premack B. A. Gardner P. J. Biol. Chem. 1993; 268: 3889-3896Abstract Full Text PDF PubMed Google Scholar). However, the clear and critical distinction between these two currents is that, unlike I CRAC, activation ofI ARC is entirely independent of store depletion. Another feature of store-operated Ca2+ currents such asI CRAC is that, on removal of extracellular divalent cations, they become highly permeable to monovalent ions (10. Hoth M. Penner R. J. Physiol. (Lond. ). 1993; 465: 359-386Crossref Scopus (662) Google Scholar, 11. McDonald T. V. Premack B. A. Gardner P. J. Biol. Chem. 1993; 268: 3889-3896Abstract Full Text PDF PubMed Google Scholar, 13. Lepple-Wienhues A. Cahalan M. D. Biophys. J. 1996; 7: 787-794Abstract Full Text PDF Scopus (119) Google Scholar, 14. Kerschbaum H. H. Cahalan M. D. J. Gen. Physiol. 1998; 111: 521-537Crossref PubMed Scopus (85) Google Scholar, 15. Kerschbaum H. H. Cahalan M. D. Science. 1999; 283: 836-839Crossref PubMed Scopus (129) Google Scholar). This is also seen in voltage-activated Ca2+currents (16. Hess P. Tsien R. W. Nature. 1984; 309: 453-456Crossref PubMed Scopus (580) Google Scholar, 17. Almers W. J. Physiol. (Lond. ). 1984; Scopus Google Scholar). We examined the same was by I divalent-free extracellular addition of μm arachidonic acid a inward current that was inhibited by La3+ μm) (Fig. 1 This activation of a monovalent current was rapidly on of extracellular divalent that it is not a simple leak current. The magnitude of the monovalent current was larger than the Ca2+ current measured in the presence of extracellular divalent ions ± = 0. 56 ± 0. 05 pA/pF). the Ca2+ current in the monovalent current observed in divalent-free solutions shows inward rectification (Fig. 1 However, with internal the monovalent current observed in divalent-free extracellular solution 0 and an current is observed at positive be that these clearly distinguish this monovalent current from the nonselective current observed on going whole-cell The current that is seen with solutions in the of extracellular divalent is not seen with NMDG+ as the internal (Fig. 1 indicating that it Na+ and/or Cs+ the ARC I CRAC has also been demonstrated to a during hyperpolarizing voltage pulses that with a time constant of ms A. Lewis R. S. J. Gen. Physiol. PubMed Scopus Google Scholar, J. Biol. 1999; PubMed Scopus (94) Google Scholar). This is reported to be and to a by Ca2+ the of the CRAC channels A. Lewis R. S. J. Gen. Physiol. PubMed Scopus Google Scholar, J. Biol. 1999; PubMed Scopus (94) Google Scholar). To this ARC, peak and steady-state arachidonate-activated currents were recorded during hyperpolarizing pulses to −80 mV. these extracellular from 10 to 20 to This in extracellular Ca2+ not produce significant in the measured steady-state arachidonate-activated current ± pA/pF at −80 = of the measured peak and steady-state currents that cells either no significant in the measured current or a A). the cells, a significant (i. e. was The mean result from all cells a ± in current indicating that, in with I CRAC, I not consistent at under the conditions used in these As the endogenous store-operated Ca2+ channels of HEK293 cells have only been described C. J. Physiol. 1998; PubMed Scopus Google Scholar), it was to that this of significant was a unique feature ofI ARC and not an of the endogenous store-operated Ca2+ channels of the We examined the store-operated currents (I SOC) in the stores were by using a Ca2+-free Under these conditions, the stores become to the of the responsible for to The magnitude of the resulting currents SOC) measured at −80 mV with an external Ca2+ concentration of 20 was ± pA/pF (n = currents inward rectification and a reversal potential more positive than +30 mV (Fig. 2 B). of extracellular divalent resulted in the of current by monovalent ions shown). As such, the demonstrated properties that were consistent with those previously reported CRAC (8. Hoth M. Penner R. Nature. 1992; 355: 353-356Crossref PubMed Scopus (1495) Google Scholar, 9. Zweifach A. Lewis R. S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6259-6299Crossref Scopus (697) Google Scholar) and with the of I currents in the HEK293 cells C. J. Physiol. 1998; PubMed Scopus Google Scholar). Examination of the in the magnitude of the I during pulses to −80 mV using the described a highly consistent (Fig. 2 to ± with a time constant of ± ms (n = are similar to those reported for I T and RBL cells A. Lewis R. S. J. Gen. Physiol. PubMed Scopus Google Scholar, J. Biol. 1999; PubMed Scopus (94) Google Scholar). As such, this clearly from the observed for I ARC described (Fig. 2 A). This not be by the of Ca2+ in the intracellular solution used in the I experiments as data were obtained I was activated by extracellular addition of the pump (1 μm) using the standard solution 100 nm Ca2+. The data obtained ARC and I the of entirely distinct additional feature of store-operated Ca2+ entry in a of is to in extracellular has been to capacitative Ca2+entry in cells S. J. Physiol. PubMed Google Scholar, J. Physiol. Google Scholar, Biochem. Biophys. 1996; PubMed Scopus Google Scholar, C. 1996; PubMed Scopus Google Scholar), the cells (6. Shuttleworth T. J. Thompson J. L. J. Biol. Chem. 1998; 273: 32636-32643Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), and to I CRAC in RBL cells A. J. Biol. PubMed Scopus Google Scholar). we have previously reported that the noncapacitative entry of Ca2+ activated by arachidonic acid is to in extracellular to (6. Shuttleworth T. J. Thompson J. L. J. Biol. Chem. 1998; 273: 32636-32643Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). To that this to was demonstrated by the endogenous store-operated current (I SOC) of cells, we examined the currents in cells in which the intracellular Ca2+ stores were by using a Ca2+-free Under these conditions, from to produced a and inhibition ± = of the store-operated current that was on of the to ± of A and C). of the extracellular from to in cells to arachidonic acid (8 μm) had only a small and effect on the ARC (Fig. 3 to a of ± (n = 200 (Fig. 3 C). The data that the arachidonate-regulated current is entirely distinct from that activated by store depletion. This is consistent with where the of capacitative and arachidonate-activated Ca2+ entry to a transfected were used to between the two Ca2+ entry (7. Shuttleworth T. J. Thompson J. L. J. Biol. Chem. 1999; 274: 31174-31178Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). capacitative and noncapacitative Ca2+ entry occurs two entirely distinct membrane it that the two currents be To we the effect of addition of arachidonic acid to cells in which the endogenous store-operated current (I SOC) had been activated by using a Ca2+-free As such resulted in the activation of an inward current with a magnitude of pA/pF at −80 mV and which all the characteristics of a store-operated current. The subsequent addition of μm arachidonic acid to such cells resulted in a in the magnitude of the inward current measured at −80 mV to a of pA/pF A). This is consistent with the two currents (I being To be certain that intracellular Ca2+ stores had been maximally fully activating the endogenous store-operated Ca2+entry additional experiments were on cells that had been in (1 μm) for this activation of capacitative subsequent addition of μm arachidonic acid an additional inward current the activation ofI ARC (Fig. B). The magnitude of the additional arachidonate-activated current ± pA/pF at −80 was to that previously seen ARC in the of Furthermore, subsequent addition of La3+ μm) to the and cells resulted in the overall current to a than that observed before addition of arachidonic acid that La3+ an additional Ca2+ current activated by the I the two currents, I ARC, are We that low concentrations of exogenous arachidonic acid activate a novel Ca2+ current, I ARC, and that this current for the arachidonic noncapacitative Ca2+ entry observed in and cells (5. Shuttleworth T. J. J. Biol. Chem. 1996; 271: 21720-21725Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 6. Shuttleworth T. J. Thompson J. L. J. Biol. Chem. 1998; 273: 32636-32643Abstract Full Text Full Text PDF PubMed Scopus (78) Google ARC properties with the archetypal store-operated current I the presence of extracellular Ca2+ both are small magnitude currents pA/pF at −80 mV), with a high for Ca2+, and inward removal of extracellular divalent both ARC channels and CRAC channels a similar of permeable to monovalent these are between I CRAC and I ARC shows the the to extracellular that is ofI the activation ofI ARC is observed after depletion of the intracellular Ca2+ noncapacitative data (7. Shuttleworth T. J. Thompson J. L. J. Biol. Chem. 1999; 274: 31174-31178Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) that capacitative and the arachidonate-regulated noncapacitative Ca2+ entry occurs two entirely distinct membrane this we used the reported of a transfected to stimulation specifically by capacitative We that, Ca2+entry produced a in the in the transfected cells, the entry activated by arachidonic acid to clearly between the two Ca2+ entry (7. Shuttleworth T. J. Thompson J. L. J. Biol. Chem. 1999; 274: 31174-31178Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). recent much has been to the of the Ca2+ entry of non-excitable has be to the so-called receptor from that a channel in phospholipase Ca2+ entry and that be activated as a result of an inositol trisphosphate-induced emptying of intracellular Ca2+ stores Cell Calcium. PubMed Scopus Google Scholar). Although the characteristics of the endogenous channels of are very from I ARC from store-operated channels such asI a recent has that these channels with the similar are activated by certain arachidonic acid S. P. Nature. 1999; PubMed Scopus Google Scholar). of of the has been to to the of a of channel M. M. M. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). to noncapacitative entry only two of these and have that are specifically activated by agonists and not by store depletion, with activation of being independent of store depletion M. M. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). A recent has that both and can be activated by M. C. Nature. 1999; PubMed Scopus Google Scholar), and it was that these of a of However, of either or nonselective currents, with reversal close to 0 mV and with M. M. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, M. C. Nature. 1999; PubMed Scopus Google Scholar). of these are inconsistent with the properties we have described Based on it unlikely that of the known or the channel I As to the physiological role of I ARC, it has previously been that the receptor-activated entry of Ca2+ during oscillations is entirely on the generation of arachidonic acid (5. Shuttleworth T. J. J. Biol. Chem. 1996; 271: 21720-21725Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 6. Shuttleworth T. J. Thompson J. L. J. Biol. Chem. 1998; 273: 32636-32643Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The of this in two very cells from the and in HEK293 with recent of an arachidonate-regulated entry of Ca2+ in and in cells S. C. Cell Calcium. PubMed Scopus Google Scholar, J. Physiol. (Lond. ). 1999; Scopus Google Scholar), that this is a with noncapacitative the critical role of this arachidonate-regulated entry is not associated with store but with the control of (4. Shuttleworth T. J. Thompson J. L. Biochem. J. 1996; 316: 819-824Crossref PubMed Scopus (50) Google Scholar). as previously (3. Shuttleworth T. J. Cell Calcium. 1999; 25: 237-246Crossref PubMed Scopus (94) Google Scholar), the transient and/or of store depletion during oscillatory Ca2+ signals that the for store refilling in cells is during such to that arachidonate-regulated Ca2+ entry during the oscillatory Ca2+ of levels of stimulation. that cells are known to be to Ca2+ signals in the and that has been to activate specific as well as in a it is clear that I ARC represents a Ca2+ entry pathway of critical physiological We Dr. Craig Logsdon (University of for with the HEK293 cells stably transfected with the human m3 muscarinic for on an of the and Thompson and for