Constitutively active calcium channels in plasma membrane of T-lymphocytes

Compensated influx and efflux of calcium ions maintain the constancy of Ca²⁺ concentration in cytoplasm of quiescent cells under variable external conditions. In cell plasma membrane there exist several types of Ca²⁺ channels with different properties, regulation mechanisms, and pharmacology. Using fluorescent Ca²⁺-sensitive probes, we have shown here that in T-lymphocytes under resting conditions, Ca²⁺ influx occurs through special constitutively active Ca²⁺ channels, permeable to Ni²⁺ and Mn²⁺. These channels differ from the receptor-activated SOC channels, from Ca²⁺ channels activated by arachidonic acid, and from calmidazolium-activated channels. Ca²⁺ influx rate in quiescent cells increases with a rise in temperature (Q₁₀ =1.9). The strong dependence of the constitutively active channel activity on temperature coincided with the plasma membrane Ca²⁺-ATPase dependence, indicating that intracellular enzymes regulate the channel activity. To identify the constitutively active channel, we analyzed the effects of L-type Ca²⁺ channels, SOC channels, Ca²⁺-independent phospholipase A2, and calmodulin inhibitors. Of all inhibitors listed only dihydropyridine blocker of L-type voltage-dependent Ca²⁺ channels, isradipin, at a concentration of 1.5 μM completely suppressed calcium influx. However, the channels did not exhibit sensitivity to changes in membrane potential. Our observations testify to the existence of a new nonselective Ca²⁺ channel in T-lymphocyte plasma membrane and characterize the new channels pharmacologically. The results obtained are important for understanding the regulation mechanisms of Ca²⁺ channels in plasma membrane of non-excitable cells.     

Dissecting out the Complex Ca2+-Mediated Phenylephrine-Induced Contractions of Mouse Aortic Segments

L-type Ca²⁺ channel (VGCC) mediated Ca²⁺ influx in vascular smooth muscle cells (VSMC) contributes to the functional properties of large arteries in arterial stiffening and central blood pressure regulation. How this influx relates to steady-state contractions elicited by α1-adrenoreceptor stimulation and how it is modulated by small variations in resting membrane potential (V_m) of VSMC is not clear yet. Here, we show that α1-adrenoreceptor stimulation of aortic segments of C57Bl6 mice with phenylephrine (PE) causes phasic and tonic contractions. By studying the relationship between Ca²⁺ mobilisation and isometric tension, it was found that the phasic contraction was due to intracellular Ca²⁺ release and the tonic contraction determined by Ca²⁺ influx. The latter component involves both Ca²⁺ influx via VGCC and via non-selective cation channels (NSCC). Influx via VGCC occurs only within the window voltage range of the channel. Modulation of this window Ca²⁺ influx by small variations of the VSMC V_m causes substantial effects on the contractile performance of aortic segments. The relative contribution of VGCC and NSCC to the contraction by α1-adrenoceptor stimulation could be manipulated by increasing intracellular Ca²⁺ release from non-contractile sarcoplasmic reticulum Ca²⁺ stores. Results of this study point to a complex interactions between α1-adrenoceptor-mediated VSMC contractile performance and Ca²⁺ release form contractile or non-contractile Ca²⁺ stores with concomitant Ca²⁺ influx. Given the importance of VGCC and their blockers in arterial stiffening and hypertension, they further point toward an additional role of NSCC (and NSCC blockers) herein.     

Passive Ca2+ Transport and Ca2+-Dependent K+ Transport in Plasmodium falciparum-Infected Red Cells

Previous reports have indicated that Plasmodium falciparum-infected red cells (pRBC) have an increased Ca²⁺ permeability. The magnitude of the increase is greater than that normally required to activate the Ca²⁺-dependent K⁺ channel (K _Ca channel) of the red cell membrane. However, there is evidence that this channel remains inactive in pRBC. To clarify this discrepancy, we have reassessed both the functional status of the K _Ca channel and the Ca²⁺ permeability properties of pRBC. For pRBC suspended in media containing Ca²⁺, K _Ca channel activation was elicited by treatment with the Ca²⁺ ionophore A23187. In the absence of ionophore the channel remained inactive. In contrast to previous claims, the unidirectional influx of Ca²⁺ into pRBC in which the Ca²⁺ pump was inhibited by vanadate was found to be within the normal range (30–55 μmol (10¹³ cells · hr)⁻¹), provided the cells were suspended in glucose-containing media. However, for pRBC in glucose-free media the Ca²⁺ influx increased to over 1 mmol (10¹³ cells · hr)⁻¹, almost an order of magnitude higher than that seen in uninfected erythrocytes under equivalent conditions. The pathway responsible for the enhanced influx of Ca²⁺ into glucose-deprived pRBC was expressed at approximately 30 hr post-invasion, and was inhibited by Ni²⁺. Possible roles for this pathway in pRBC are considered. Received: 12 May 1999/Revised: 8 July 1999     

SPCA2 Regulates Orai1 Trafficking and Store Independent Ca2+ Entry in a Model of Lactation

An unconventional interaction between SPCA2, an isoform of the Golgi secretory pathway Ca²⁺-ATPase, and the Ca²⁺ influx channel Orai1, has previously been shown to contribute to elevated Ca²⁺ influx in breast cancer derived cells. In order to investigate the physiological role of this interaction, we examined expression and localization of SPCA2 and Orai1 in mouse lactating mammary glands. We observed co-induction and co-immunoprecipitation of both proteins, and isoform-specific differences in the localization of SPCA1 and SPCA2. Three-dimensional cultures of normal mouse mammary epithelial cells were established using lactogenic hormones and basement membrane. The mammospheres displayed elevated Ca²⁺ influx by store independent mechanisms, consistent with upregulation of both SPCA2 and Orai1. Knockdown of either SPCA2 or Orai1 severely depleted Ca²⁺ influx and interfered with mammosphere differentiation. We show that SPCA2 is required for plasma membrane trafficking of Orai1 in mouse mammary epithelial cells and that this function can be replaced, at least in part, by a membrane-anchored C-terminal domain of SPCA2. These findings clearly show that SPCA2 and Orai1 function together to regulate Store-independent Ca²⁺ entry (SICE), which mediates the massive basolateral Ca²⁺ influx into mammary epithelia to support the large calcium transport requirements for milk secretion.     

Mechanosensitive Ca2+ permeant cation channels in human prostate tumor cells

The acquisition of cell motility plays a critical role in the spread of prostate cancer (PC), therefore, identifying a sensitive step that regulates PC cell migration should provide a promising target to block PC metastasis. Here, we report that a mechanosensitive Ca²⁺-permeable cation channel (MscCa) is expressed in the highly migratory/invasive human PC cell line, PC-3 and that inhibition of MscCa by Gd³⁺ or GsMTx-4 blocks PC-3 cell migration and associated elevations in [Ca²⁺]_i. Genetic suppression or overexpression of specific members of the canonical transient receptor potential Ca²⁺ channel family (TRPC1 and TRPC3) also inhibit PC-3 cell migration, but they do so by mechanisms other that altering MscCa activity. Although LNCaP cells are nonmigratory, they also express relatively large MscCa currents, indicating that MscCa expression alone cannot confer motility on PC cells. MscCa in both cell lines show similar conductance and ion selectivity and both are functionally coupled via Ca²⁺ influx to a small Ca²⁺-activated K⁺ channel. However, MscCa in PC-3 and LNCaP cell patches show markedly different gating dynamics—while PC-3 cells typically express a sustained, non-inactivating MscCa current, LNCaP cells express a mechanically-fragile, rapidly inactivating MscCa current. Moreover, mechanical forces applied to the patch, can induce an irreversible transition from the transient to the sustained MscCa gating mode. Given that cancer cells experience increasing compressive and shear forces within a growing tumor, a similar shift in channel gating in situ would have significant effects on Ca²⁺ signaling that may play a role in tumor progression.     

Mechanosensitive Ca2+ permeant cation channels in human prostate tumor cells

The acquisition of cell motility plays a critical role in the spread of prostate cancer (PC), therefore, identifying a sensitive step that regulates PC cell migration should provide a promising target to block PC metastasis. Here, we report that a mechanosensitive Ca²⁺-permeable cation channel (MscCa) is expressed in the highly migratory/invasive human PC cell line, PC-3 and that inhibition of MscCa by Gd³⁺ or GsMTx-4 blocks PC-3 cell migration and associated elevations in [Ca²⁺]_i. Genetic suppression or overexpression of specific members of the canonical transient receptor potential Ca²⁺ channel family (TRPC1 and TRPC3) also inhibit PC-3 cell migration, but they do so by mechanisms other that altering MscCa activity. Although LNCaP cells are nonmigratory, they also express relatively large MscCa currents, indicating that MscCa expression alone cannot confer motility on PC cells. MscCa in both cell lines show similar conductance and ion selectivity and both are functionally coupled via Ca²⁺ influx to a small Ca²⁺-activated K⁺ channel. However, MscCa in PC-3 and LNCaP cell patches show markedly different gating dynamics—while PC-3 cells typically express a sustained, non-inactivating MscCa current, LNCaP cells express a mechanically-fragile, rapidly inactivating MscCa current. Moreover, mechanical forces applied to the patch, can induce an irreversible transition from the transient to the sustained MscCa gating mode. Given that cancer cells experience increasing compressive and shear forces within a growing tumor, a similar shift in channel gating in situ would have significant effects on Ca²⁺ signaling that may play a role in tumor progression.     

Functional differentiation of a human ganglion cell derived neuroblastoma cell line SH-SY5Y induced by a phorbol ester (TPA)

When the human neuroblastoma cell line SH-SY5Y is exposed to 12-o-tetradecanoyl-phorbol-13-acetate (TPA) the cells grow long processes indicative of neural differentiation. Concomitantly there is an increase in the resting membrane potential from ?44 ± 2 mV found in untreated cells to ?63 ± 4 mV after induction. The TPA treated cells are depolarized when the external potassium concentration is increased to 46 mM and upon addition of veratridine. In contrast to the untreated cells depolarization in differentiated cells leads to an increase in the rate of Ca²⁺ influx. This increase in Ca²⁺ influx is blocked by the Ca²⁺ channel antagonist, verapamil, while the Na⁺ channel blocker tetrodotoxin only marginally inhibits the K⁺ depolarization-induced Ca²⁺ influx.The results suggest that the induction of morphological differentiation in this cell line is associated with the appearance of features of excitable cells.     

Characterization of store-operated Ca channels in pancreatic duct epithelia

Store-operated Ca²⁺ channels (SOCs) are activated by depletion of intracellular Ca²⁺ stores following agonist-mediated Ca²⁺ release. Previously we demonstrated that Ca²⁺ influx through SOCs elicits exocytosis efficiently in pancreatic duct epithelial cells (PDEC). Here we describe the biophysical, pharmacological, and molecular properties of the duct epithelial SOCs using Ca²⁺ imaging, whole-cell patch-clamp, and molecular biology. In PDEC, agonists of purinergic, muscarinic, and adrenergic receptors coupled to phospholipase C activated SOC-mediated Ca²⁺ influx as Ca²⁺ was released from intracellular stores. Direct measurement of [Ca²⁺] in the ER showed that SOCs greatly slowed depletion of the ER. Using IP₃ or thapsigargin in the patch pipette elicited inwardly rectifying SOC currents. The currents increased ∼8-fold after removal of extracellular divalent cations, suggesting competitive permeation between mono- and divalent cations. The current was completely blocked by high doses of La³⁺ and 2-aminoethoxydiphenyl borate (2-APB) but only partially depressed by SKF-96365. In polarized PDEC, SOCs were localized specifically to the basolateral membrane. RT-PCR screening revealed the expression of both STIM and Orai proteins for the formation of SOCs in PDEC. By expression of fluorescent STIM1 and Orai1 proteins in PDEC, we confirmed that colocalization of the two proteins increases after store depletion. In conclusion, basolateral Ca²⁺ entry through SOCs fills internal Ca²⁺ stores depleted by external stimuli and will facilitate cellular processes dependent on cytoplasmic Ca²⁺ such as salt and mucin secretion from the exocrine pancreatic ducts.     

Cch1p Mediates Ca2+ Influx to Protect Saccharomyces cerevisiae against Eugenol Toxicity

Eugenol has antifungal activity and is recognised as having therapeutic potential. However, little is known of the cellular basis of its antifungal activity and a better understanding of eugenol tolerance should lead to better exploitation of eugenol in antifungal therapies. The model yeast, Saccharomyces cerevisiae, expressing apoaequorin was used to show that eugenol induces cytosolic Ca²⁺ elevations. We investigated the eugenol Ca²⁺ signature in further detail and show that exponentially growing cells exhibit Ca²⁺ elevation resulting exclusively from the influx of Ca²⁺ across the plasma membrane whereas in stationary growth phase cells Ca²⁺ influx from intracellular and extracellular sources contribute to the eugenol-induced Ca²⁺ elevation. Ca²⁺ channel deletion yeast mutants were used to identify the pathways mediating Ca²⁺ influx; intracellular Ca²⁺ release was mediated by the vacuolar Ca²⁺ channel, Yvc1p, whereas the Ca²⁺ influx across the plasma membrane could be resolved into Cch1p-dependent and Cch1p-independent pathways. We show that the growth of yeast devoid the plasma membrane Ca²⁺ channel, Cch1p, was hypersensitive to eugenol and that this correlated with reduced Ca²⁺ elevations. Taken together, these results indicate that a cch1p-mediated Ca²⁺ influx is part of an intracellular signal which protects against eugenol toxicity. This study provides fresh insight into the mechanisms employed by fungi to tolerate eugenol toxicity which should lead to better exploitation of eugenol in antifungal therapies.     

Properties of AMPA Receptors Expressed in Rat Cerebellar Granule Cell Cultures: Ca2+ Influx Studies

Abstract: Cultured cerebellar granule cells become vulnerable to excitatory amino acids, especially to NMDA and kainate, by 9 days in vitro. In the same time, the sensitivity of cells to (RS)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), in terms of AMPA-induced toxicity or ⁴⁵Ca²⁺ uptake, was very low. The low AMPA responsiveness was due to receptor desensitization, because agents known to block desensitization, cyclothiazide and the lectins concanavalin A and wheat germ agglutinin, rendered granule cells vulnerable to AMPA and produced a pronounced stimulation of ⁴⁵Ca²⁺ accumulation. ⁴⁵Ca²⁺ influx was induced specifically by AMPA-receptor stimulation, because it was blocked virtually completely by 2,3-dihydroxy-6-nitro-7-sulfamoylbenzoquinoxaline (NBQX) and the benzodiazepine GYKI 52466 (selective non-NMDA receptor antagonists). Nevertheless, indirect routes activated by cellular responses to AMPA-receptor stimulation contributed significantly to the overall ⁴⁵Ca²⁺ influx. These included Ca²⁺ uptake through NMDA-receptor channels, voltage-sensitive Ca²⁺ channels, and via Na⁺/Ca²⁺ exchange. However, nearly one-fifth of the total ⁴⁵Ca²⁺ influx remained unaccounted for and this estimate was similar to ⁴⁵Ca²⁺ influx observed under Na⁺-free conditions. This observation suggested that a significant proportion of the Ca²⁺ flux passes through the AMPA-receptor channel proper, a view supported by Co²⁺ uptake into nearly all granule cells on exposure to AMPA in the presence of cyclothiazide. Results are discussed in light of the reported AMPA receptor-subunit composition of cerebellar granule cells in vitro.     

TRPP2 channels regulate apoptosis through the Ca2+ concentration in the endoplasmic reticulum

Ca²⁺ is an important signalling molecule that regulates multiple cellular processes, including apoptosis. Although Ca²⁺ influx through transient receptor potential (TRP) channels in the plasma membrane is known to trigger cell death, the function of intracellular TRP proteins in the regulation of Ca²⁺‐dependent signalling pathways and apoptosis has remained elusive. Here, we show that TRPP2, the ion channel mutated in autosomal dominant polycystic kidney disease (ADPKD), protects cells from apoptosis by lowering the Ca²⁺ concentration in the endoplasmic reticulum (ER). ER‐resident TRPP2 counteracts the activity of the sarcoendoplasmic Ca²⁺ ATPase by increasing the ER Ca²⁺ permeability. This results in diminished cytosolic and mitochondrial Ca²⁺ signals upon stimulation of inositol 1,4,5‐trisphosphate receptors and reduces Ca²⁺ release from the ER in response to apoptotic stimuli. Conversely, knockdown of TRPP2 in renal epithelial cells increases ER Ca²⁺ release and augments sensitivity to apoptosis. Our findings indicate an important function of ER‐resident TRPP2 in the modulation of intracellular Ca²⁺ signalling, and provide a molecular mechanism for the increased apoptosis rates in ADPKD upon loss of TRPP2 channel function.     

Divergent Ca2+/calmodulin feedback regulation of CaV1 and CaV2 voltage-gated calcium channels evolved in the common ancestor of Placozoa and Bilateria

Ca_V1 and Ca_V2 voltage-gated calcium channels evolved from an ancestral Ca_V1/2 channel via gene duplication somewhere near the stem animal lineage. The divergence of these channel types led to distinguishing functional properties that are conserved among vertebrates and bilaterian invertebrates and contribute to their unique cellular roles. One key difference pertains to their regulation by calmodulin (CaM), wherein bilaterian Ca_V1 channels are uniquely subject to pronounced, buffer-resistant Ca²⁺/CaM-dependent inactivation, permitting negative feedback regulation of calcium influx in response to local cytoplasmic Ca²⁺ rises. Early diverging, nonbilaterian invertebrates also possess Ca_V1 and Ca_V2 channels, but it is unclear whether they share these conserved functional features. The most divergent animals to possess both Ca_V1 and Ca_V2 channels are placozoans such as Trichoplax adhaerens, which separated from other animals over 600 million years ago shortly after their emergence. Hence, placozoans can provide important insights into the early evolution of Ca_V1 and Ca_V2 channels. Here, we build upon previous characterization of Trichoplax Ca_V channels by determining the cellular expression and ion-conducting properties of the Ca_V1 channel orthologue, TCa_V1. We show that TCa_V1 is expressed in neuroendocrine-like gland cells and contractile dorsal epithelial cells. In vitro, this channel conducts dihydropyridine-insensitive, high-voltage–activated Ca²⁺ currents with kinetics resembling those of rat Ca_V1.2 but with left-shifted voltage sensitivity for activation and inactivation. Interestingly, TCa_V1, but not TCa_V2, exhibits buffer-resistant Ca²⁺/CaM-dependent inactivation, indicating that this functional divergence evolved prior to the emergence of bilaterian animals and may have contributed to their unique adaptation for cytoplasmic Ca²⁺ signaling within various cellular contexts.     

Inhibition of mechanical strain-induced fetal rat lung cell proliferation by gadolinium,a stretch-activated channel blocker

Normal growth of the fetal lung is dependent upon fetal breathing movements. We have previously demonsrated that mechanical strain, simulating fetal breathing movements, stimulated DNA synthesis and cell division by reaggregated alveolar-like structures of fetal rat lung cells. Herein, we report that both intracellular and extracellular calcium modulate strain-induced proliferative activity. Strain-induced cell proliferation was inhibited by BAPTA/AM, an intracellular calcium chelator. The intracellular calcium modulators, cyclopiazonic acid and 2,5-di-(tert-butyl)-1, 4-benzohydroquinone, increased DNA synthesis of unstrained cultures and partially reduced strain-induced cell growth activity. A similar effect was noted with the calcium ionophore A23187. Extracellular Ca²⁺ increased DNA synthesis in unstrained cultures in a concentration-dependent fashion. The stimulatory effect of strain on DNA synthesis was also dependent on the calcium concentration in the medium. Furthermore, strain-enhanced DNA synthesis was inhibited by the presence of a divalent ion chelator, EGTA, in the medium. Mechanical strain increased ⁴⁵Ca²⁺ influx within 1 min after the onset strain. This rapid entry of calcium was not affected by calcium channel blockers, such as verapamil or Ni²⁺. Calcium channel blockers verapamil, nifedipine, Ni²⁺, Co²⁺, or La³⁺ also did not inhibit strain-induced cell growth activity. In contrast, gadolinium, a stretch-activated channel blocker, inhibited strain-induced ⁴⁵Ca²⁺ influx and suppressed strain-enhanced DNA synthesis. We conclude that the entry of calcium into cells through stretch-activated ion channels plays a critical role in strain-induced fetal lung cell proliferation. © 1994 Wiley-Liss, Inc.     

The steroid hormone 20-hydroxyecdysone upregulates calcium release-activated calcium channel modulator 1 expression to induce apoptosis in the midgut of Helicoverpa armigera

Animal steroid hormones stimulate extracellular Ca²⁺ influx into cells; however, the mechanism remains unclear. In this study, we determined that the Ca²⁺ influx induced by steroid hormone 20-hydroxyecdysone (20E) is mediated by the calcium release-activated calcium channel modulator 1 (CRACM1/Orai1). The Orai1 mRNA is highly expressed during midgut programmed cell death in the lepidopteran insect Helicoverpa armigera. 20E upregulated the expression of Orai1 in H. armigera larvae and in an epidermal cell line (HaEpi). Knockdown of Orai1 in HaEpi cells blocked 20E-induced Ca²⁺ influx, and the inhibitor of inositol 1, 4, 5-trisphosphate receptor (IP₃R) Xestospongin (XeC) blocked 20E-induced Ca²⁺ influx, suggesting that 20E, via Orai1, induces stored-operated Ca²⁺ influx. Orai1 interacts with stromal interaction molecule 1(Stim1) to exert its function in 20E-induced Ca²⁺ influx. 20E promotes Orai1 aggregation through G-protein-coupled receptors, phospholipase C gamma 1, and Stim1. Knockdown of Orai1 in the HaEpi cell line repressed apoptosis and maintained autophagy under 20E regulation. Knockdown of Orai1 in larvae delayed pupation, repressed midgut apoptosis, maintained the midgut in an autophagic state, and repressed 20E-pathway gene expression. These results revealed that steroid hormone 20E, via Orai1, induces Ca²⁺ influx to promote the transition of midgut from autophagy to apoptosis.     

Trans to Cis proton concentration gradients accelerate ionophore A23187-mediated net fluxes of Ca2+ across the human red cell membrane

Ionophore A23187-mediated net influx of Ca²⁺ in ATP-depleted human red cells was studied as a function of the pH and the proton concentration gradient across the membranes. Utilizing the Ca²⁺-induced increase in K⁺ conductance of the cell membranes, various CCCP-mediated proton gradients were raised across the membranes of cells suspended in unbuffered salt solutions with different K⁺ concentrations. In ionophore-mediated equilibrium the concentration ratios of ionized Ca between ATP-depleted, DIDS-treated cells and their suspension medium were equal to the concentration ratios of protons raised to the second power. With no proton concentration gradient across the membranes the net influxes of Ca²⁺ as a function of pH resembled a titration curve of a weak acid, with half maximal net influx at pH 7.3, at 100 μM extracellular Ca²⁺. With cellular pH fixed at various values, the net influx of Ca²⁺ was determined as a function of the proton concentration gradient. A linear relationship between the logarithm of net influx and the difference between extracellular and cellular pH was found at all cellular pH values tested, but the proton concentration gradient acceleration was a function of the cellular pH. Accelerations between 10- and 40- times per unit ΔpH were found and net effluxes were correspondingly decreased. The results are discussed in relation to present models of the mechanism of ionophore A23187-mediated Ca²⁺ transport. The importance of the proton concentration gradient dependency is discussed in relation to the induced oscillations in K⁺-conductance of human red cell membranes previously reported (Vestergaard-Bogind and Bennekou (1982) Biochim. Biophys. Acta 688, 37ndash;44).     

A non-invasive vibrating calcium-selective electrode measures acetylcholine-induced calcium flux across the sarcolemma of a smooth muscle

To determine possible sources of Ca²⁺ during excitation-contraction coupling in smooth muscle, a vibrating Ca²⁺-selective electrode was used to measure Ca²⁺ flux during the process of contraction. The smooth muscle model was the longitudinal muscle of the body wall of a sea cucumberSclerodactyla briareus. Because acetylcholine caused slow contractions of the muscle that were inhibited by Ca²⁺ channel blockers diltiazem and verapamil in earlier mechanical studies, we chose a vibrating Ca²⁺-selective electrode as our method to test the hypothesis that acetylcholine may be stimulating Ca²⁺ influx across the sarcolemma, providing a Ca²⁺ source during excitation-contraction coupling. Acetylcholine treatment stimulated a net Ca²⁺ efflux that was both dose and time dependent. We then tested two L-type Ca²⁺ channel blockers, diltiazem and verapamil, and two non-specific Ca²⁺ blockers, cobalt (Co²⁺) and lanthanum (La³⁺) on acetylcholine-induced Ca²⁺ flux. All four Ca²⁺ blockers tested potently inhibited Ca²⁺ efflux induced by physiological doses of acetylcholine. We propose that the acetylcholine-induced Ca²⁺ efflux was the result of, first, Ca²⁺ influx through voltage-sensitive L-type Ca²⁺ channels, then the rapid extrusion of Ca²⁺ by an outwardly directed carrier such as the Na–Ca exchanger as suggested by Li⁺ substitution experiments. The vibrating Ca²⁺ electrode has provided new insights on the active and complex role the sarcolemma plays in Ca²⁺ homeostasis and regulating Ca²⁺ redistribution during excitation-contraction coupling.Abbreviations ACh acetylcholine - E-C coupling excitation-contraction coupling - LMBW longitudinal muscle of the body wall     

Chemico-genetic identification of drebrin as a regulator of calcium responses

Store-operated calcium channels are plasma membrane Ca²⁺ channels that are activated by depletion of intracellular Ca²⁺ stores, resulting in an increase in intracellular Ca²⁺ concentration, which is maintained for prolonged periods in some cell types. Increases in intracellular Ca²⁺ concentration serve as signals that activate a number of cellular processes, however, little is known about the regulation of these channels. We have characterized the immuno-suppressant compound BTP, which blocks store-operated channel mediated calcium influx into cells. Using an affinity purification scheme to identify potential targets of BTP, we identified the actin reorganizing protein, drebrin, and demonstrated that loss of drebrin protein expression prevents store-operated channel mediated Ca²⁺ entry, similar to BTP treatment. BTP also blocks actin rearrangements induced by drebrin. While actin cytoskeletal reorganization has been implicated in store-operated calcium channel regulation, little is known about actin-binding proteins that are involved in this process, or how actin regulates channel function. The identification of drebrin as a mediator of this process should provide new insight into the interaction between actin rearrangement and store-operated channel mediated calcium influx.     

The role of nitric oxide and l-type calcium channel blocker in the contractility of rabbit ileum in vitro

Nitric oxide (NO) and calcium channel blockers are two agents that can affect gastrointestinal motility. The goal of this work was to study the rabbit intestinal smooth muscle contraction response to (1) sodium nitroprusside (SNP), the NO donor, and its potential mechanism of action, and (2) nifedipine, the l-type Ca²⁺ channel blocker; to clarify the degree of participation by extra- and intracellular Ca²⁺ in smooth muscle contraction. We used standard isometric tension and intracellular micro-electrode recordings. To record the activity of the longitudinal smooth muscle of the ileum, segments of 1.5?cm length of the ileum were suspended vertically in organ baths of Krebs solution. The mechanical activity of the isolated ileal longitudinal muscle was recorded. Different substances were added, and the changes produced on spontaneous contraction were recorded. We found that SNP produced significant decrease, while nitric oxide synthase inhibitor produced significant increase in the amplitude of spontaneous contractions. Both apamin, the Ca²⁺-dependent K⁺ channel blocker, and methylene blue, the inhibitor of soluble guanylate cyclase, alone, partially decreased relaxation induced by SNP. Addition of both methylene blue and apamine together abolished the inhibitory effect produced by SNP on spontaneous contractions. Nifedipine produced significant decrease in the amplitude of spontaneous contractions. In conclusion, in longitudinal muscle of rabbit ileum, calcium channels blocker are potent inhibitors of spontaneous activity. However, both extracellular and intracellular Ca²⁺ participates in the spontaneous contractions. NO also has inhibitory effect on spontaneous activity, and this effect is mediated by cGMP generation system and Ca²⁺-dependent K⁺ channels.     

Divalent cation influx and calcium homeostasis in germinal vesicle mouse oocytes

Prior to maturation, mouse oocytes are arrested at the germinal vesicle (GV) stage during which they experience constitutive calcium (Ca²⁺) influx and spontaneous Ca²⁺ oscillations. The oscillations cease during maturation but Ca²⁺ influx continues, as the oocytes’ internal stores attain maximal content at the culmination of maturation, the metaphase II stage. The identity of the channel(s) that underlie this Ca²⁺ influx has not been completely determined. GV and matured oocytes are known to express three Ca²⁺ channels, Ca_V3.2, TRPV3 and TRPM7, but females null for each of these channels are fertile and their oocytes display minor modifications in Ca²⁺ homeostasis, suggesting a complex regulation of Ca²⁺ influx. To define the contribution of these channels at the GV stage, we used different divalent cations, pharmacological inhibitors and genetic models. We found that the three channels are active at this stage. Ca_V3.2 and TRPM7 channels contributed the majority of Ca²⁺ influx, as inhibitors and oocytes from homologous knockout (KO) lines showed severely reduced Ca²⁺ entry. Sr²⁺ influx was promoted by Ca_V3.2 channels, as Sr²⁺ oscillations were negligible in Ca_V3.2-KO oocytes but robust in control and Trpv3-KO GV oocytes. Mn²⁺ entry relied on expression of Ca_V3.2 and TRPM7 channels, but Ni²⁺ entry depended on the latter. Ca_V3.2 and TRPV3 channels combined to fill the Ca²⁺ stores, although Ca_V3.2 was the most impactful. Studies with pharmacological inhibitors effectively blocked the influx of divalent cations, but displayed off-target effects, and occasionally agonist-like properties. In conclusion, GV oocytes express channels mediating Ca²⁺ and other divalent cation influx that are pivotal for fertilization and early development. These channels may serve as targets for intervention to improve the success of assisted reproductive technologies.     

Mechanisms underlying odorant-induced and spontaneous calcium signals in olfactory receptor neurons of spiny lobsters, Panulirus argus

We determined if a newly developed antennule slice preparation allows studying chemosensory properties of spiny lobster olfactory receptor neurons under in situ conditions with Ca²⁺ imaging. We show that chemical stimuli reach the dendrites of olfactory receptor neurons but not their somata, and that odorant-induced Ca²⁺ signals in the somata are sufficiently stable over time to allow stimulation with a substantial number of odorants. Pharmacological manipulations served to elucidate the source of odorant-induced Ca²⁺ transients and spontaneous Ca²⁺ oscillations in the somata of olfactory receptor neurons. Both Ca²⁺ signals are primarily mediated by an influx of extracellular Ca²⁺ through voltage-activated Ca²⁺ channels that can be blocked by CoCl₂ and the L-type Ca²⁺ channel blocker verapamil. Intracellular Ca²⁺ stores contribute little to odorant-induced Ca²⁺ transients and spontaneous Ca²⁺ oscillations. The odorant-induced Ca²⁺ transients as well as the spontaneous Ca²⁺ oscillations depend on action potentials mediated by Na⁺ channels that are largely TTX-insensitive but blocked by the local anesthetics tetracaine and lidocaine. Collectively, these results corroborate the conclusion that odorant-induced Ca²⁺ transients and spontaneous Ca²⁺ oscillations in the somata of olfactory receptor neurons closely reflect action potential activity associated with odorant-induced phasic-tonic responses and spontaneous bursting, respectively. Therefore, both types of Ca²⁺ signals represent experimentally accessible proxies of spiking.