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Betel nut consumption has significant implications for the public health globally, as the wide-spread habit of Areca chewing throughout Asia and the Pacific is associated with a high prevalence of oral carcinoma and other diseases. Despite a clear causal association of betel nut chewing and oral mucosal diseases, the biological mechanisms that link Areca nut-contained molecules, inflammation and cancer remain underexplored. In this study we show that the whole Areca nut extract (ANE) is capable of mobilizing Ca2+ in various immune cell lines. Interestingly, none of the four major alkaloids or a range of other known constituents of Areca nut were able to induce such Ca2+ signals, suggesting that the active components might represent novel or so far unappreciated chemical structures. The separation of ANE into aqueous and organic fractions has further revealed that the calcium-mobilizing molecules are exclusively present in the aqueous extract. In addition, we found that these calcium signals are associated with the activation of several immune cell lines as shown by the release of pro-inflammatory cytokines and increased cell proliferation. These results indicate that calcium-mobilizing molecules present in the aqueous fraction of the Areca nut may critically contribute to the inflammatory disorders affecting betel nut chewers.

Intracellular levels of the divalent cations Ca2+ and Mg2+ are important regulators of cell cycle and proliferation. However, the precise mechanisms by which they are regulated in cancer remain incompletely understood. The channel kinases TRPM6 and TRPM7 are gatekeepers of human Ca2+/Mg2+ metabolism. Here, we investigated the human neuroblastoma cell line SHEP-21N in which the MYCN oncogene (encoding N-Myc) can be reversibly expressed under control of an inducible repressor. We report that N-Myc expression increases cell growth and up-regulates both TRPM6 and TRPM7 expression. Membrane current analyses reveal that endogenous TRPM6/TRPM7 currents exhibit reduced Mg·ATP suppression, increased Mg2+ sensitivity, and diminished sensitivity to 2-APB inhibition. These properties are consistent with N-Myc-induced increase of heteromeric TRPM7/TRPM6 channels promoting Ca2+ and Mg2+ uptake. Genetic suppression of TRPM6/TRPM7 through siRNA inhibits cell proliferation, suggesting that N-Myc can promote neuroblastoma cell proliferation through up-regulation of divalent cation-transporting channels.

TRPM7 is a member of the Transient-Receptor-Potential Melastatin ion channel family. TRPM7 is a unique fusion protein of an ion channel and an α-kinase. Although mammalian TRPM7 is well characterized biophysically and its pivotal role in cancer, ischemia and cardiovascular disease is becoming increasingly evident, the study of TRPM7 in mouse models has been hampered by embryonic lethality of transgenic ablations. In zebrafish, functional loss of TRPM7 (drTRPM7) manifests itself in an array of non-lethal physiological malfunctions. Here, we investigate the regulation of wild type drTRPM7 and multiple C-terminal truncation mutants. We find that the biophysical properties of drTRPM7 are very similar to mammalian TRPM7. However, pharmacological profiling reveals that drTRPM7 is facilitated rather than inhibited by 2-APB, and that the TRPM7 inhibitor waixenicin A has no effect. This is reminiscent of the pharmacological profile of human TRPM6, the sister channel kinase of TRPM7. Furthermore, using truncation mutations, we show that the coiled-coil domain of drTRPM7 is involved in the channel’s regulation by magnesium (Mg) and Mg·adenosine triphosphate (Mg·ATP). We propose that drTRPM7 has two protein domains that regulate inhibition by intracellular magnesium and nucleotides, and one domain that is concerned with sensing magnesium only.

The Ca2+-permeable channel TRPM2 stimulates monocytes to produce chemokines in response to reactive oxygen species, and channel inhibition attenuates inflammatory disease in mice. TRPM2 could be a new target for treating inflammatory diseases. Reactive oxygen species (ROS) induce chemokines responsible for the recruitment of inflammatory cells to sites of injury or infection. Here we show that the plasma membrane Ca2+-permeable channel TRPM2 controls ROS-induced chemokine production in monocytes. In human U937 monocytes, hydrogen peroxide (H2O2) evokes Ca2+ influx through TRPM2 to activate Ca2+-dependent tyrosine kinase Pyk2 and amplify Erk signaling via Ras GTPase. This elicits nuclear translocation of nuclear factor-κB essential for the production of the chemokine interleukin-8 (CXCL8). In monocytes from Trpm2-deficient mice, H2O2-induced Ca2+ influx and production of the macrophage inflammatory protein-2 (CXCL2), the mouse CXCL8 functional homolog, were impaired. In the dextran sulfate sodium-induced colitis inflammation model, CXCL2 expression, neutrophil infiltration and ulceration were attenuated by Trpm2 disruption. Thus, TRPM2 Ca2+ influx controls the ROS-induced signaling cascade responsible for chemokine production, which aggravates inflammation. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases.

CNNM2 is associated with the regulation of serum Mg concentration, and when mutated, with severe familial hypomagnesemia. The function and cellular localization of CNNM2 and its isomorphs (Iso) remain controversial. The objective of this work was to examine the following: (1) the transcription-responsiveness of CNNM2 to Mg starvation, (2) the cellular localization of Iso1 and Iso2, (3) the ability of Iso1 and Iso2 to transport Mg(2+), and (4) the complex-forming ability and spectra of potential interactors of Iso1 and Iso2. The five main findings are as follows. (1) Mg-starvation induces CNNM2 overexpression that is markedly higher in JVM-13 cells (lymphoblasts) compared with Jurkat cells (T-lymphocytes). (2) Iso1 and Iso2 localize throughout various subcellular compartments in transgenic HEK293 cells overexpressing Iso1 or Iso2. (3) Iso1 and Iso2 do not transport Mg(2+) in an electrogenic or electroneutral mode in transgenic HEK293 cells overexpressing Iso1 or Iso2. (4) Both Iso1 and Iso2 form complexes of a higher molecular order. (5) The spectrum of potential interactors of Iso1 is ten times smaller than that of Iso2. We conclude that sensitivity of CNNM2 expression to extracellular Mg(2+) depletion depends on cell type. Iso1 and Iso2 exhibit a dispersed pattern of cellular distribution; thus, they are not exclusively integral to the cytoplasmic membrane. Iso1 and Iso2 are not Mg(2+) transporters per se. Both isomorphs form protein complexes, and divergent spectra of potential interactors of Iso1 and Iso2 indicate that each isomorph has a distinctive function. CNNM2 is therefore the first ever identified Mg(2+) homeostatic factor without being a Mg(2+) transporter per se.

Small-conductance, Ca2+ activated K+ channels (SK channels) are expressed at high levels in brain regions responsible for learning and memory. In the current study we characterized the contribution of SK2 channels to synaptic plasticity and to different phases of hippocampal memory formation. Selective SK2 antisense-treatment facilitated basal synaptic transmission and theta-burst induced LTP in hippocampal brain slices. Using the selective SK2 antagonist Lei-Dab7 or SK2 antisense probes, we found that hippocampal SK2 channels are critical during two different time windows: 1) blockade of SK2 channels before the training impaired fear memory, whereas, 2) blockade of SK2 channels immediately after the training enhanced contextual fear memory. We provided the evidence that the post-training cleavage of the SK2 channels was responsible for the observed bidirectional effect of SK2 channel blockade on memory consolidation. Thus, Lei-Dab7-injection before training impaired the C-terminal cleavage of SK2 channels, while Lei-Dab7 given immediately after training facilitated the C-terminal cleavage. Application of the synthetic peptide comprising a leucine-zipper domain of the C-terminal fragment to Jurkat cells impaired SK2 channel-mediated currents, indicating that the endogenously cleaved fragment might exert its effects on memory formation by blocking SK2 channel-mediated currents. Our present findings suggest that SK2 channel proteins contribute to synaptic plasticity and memory not only as ion channels but also by additionally generating a SK2 C-terminal fragment, involved in both processes. The modulation of fear memory by down-regulating SK2 C-terminal cleavage might have applicability in the treatment of anxiety disorders in which fear conditioning is enhanced.

Capacitative Ca(2+) entry (CCE) activated by release/depletion of Ca(2+) from internal stores represents a major Ca(2+) influx mechanism in lymphocytes and other nonexcitable cells. Despite the importance of CCE in antigen-mediated lymphocyte activation, molecular components constituting this mechanism remain elusive. Here we demonstrate that genetic disruption of transient receptor potential (TRP)1 significantly attenuates both Ca(2+) release-activated Ca(2+) currents and inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from endoplasmic reticulum (ER) in DT40 B cells. As a consequence, B cell antigen receptor-mediated Ca(2+) oscillations and NF-AT activation are reduced in TRP1-deficient cells. Thus, our results suggest that CCE channels, whose formation involves TRP1 as an important component, modulate IP(3) receptor function, thereby enhancing functional coupling between the ER and plasma membrane in transduction of intracellular Ca(2+) signaling in B lymphocytes.

Transient receptor potential melastatin 7 (TRPM7) is a divalent-selective cation channel fused to an atypical α-kinase. TRPM7 is a key regulator of cell growth and proliferation, processes accompanied by mandatory cell volume changes. Osmolarity-induced cell volume alterations regulate TRPM7 through molecular crowding of solutes that affect channel activity, including magnesium (Mg(2+)), Mg-nucleotides and a further unidentified factor. Here, we assess whether chloride and related halides can act as negative feedback regulators of TRPM7. We find that chloride and bromide inhibit heterologously expressed TRPM7 in synergy with intracellular Mg(2+) ([Mg(2+)]i) and this is facilitated through the ATP-binding site of the channel's kinase domain. The synergistic block of TRPM7 by chloride and Mg(2+) is not reversed during divalent-free or acidic conditions, indicating a change in protein conformation that leads to channel inactivation. Iodide has the strongest inhibitory effect on TRPM7 at physiological [Mg(2+)]i. Iodide also inhibits endogenous TRPM7-like currents as assessed in MCF-7 breast cancer cells, where upregulation of SLC5A5 sodium-iodide symporter enhances iodide uptake and inhibits cell proliferation. These results indicate that chloride could be an important factor in modulating TRPM7 during osmotic stress and implicate TRPM7 as a possible molecular mechanism contributing to the anti-proliferative characteristics of intracellular iodide accumulation in cancer cells.

Store-operated Ca2+ entry is controlled by the interaction of stromal interaction molecules (STIMs) acting as endoplasmic reticulum ER Ca2+ sensors with calcium release–activated calcium (CRAC) channels (CRACM1/2/3 or Orai1/2/3) in the plasma membrane. Here, we report structural requirements of STIM1-mediated activation of CRACM1 and CRACM3 using truncations, point mutations, and CRACM1/CRACM3 chimeras. In accordance with previous studies, truncating the N-terminal region of CRACM1 or CRACM3 revealed a 20–amino acid stretch close to the plasma membrane important for channel gating. Exchanging the N-terminal region of CRACM3 with that of CRACM1 (CRACM3-N(M1)) results in accelerated kinetics and enhanced current amplitudes. Conversely, transplanting the N-terminal region of CRACM3 into CRACM1 (CRACM1-N(M3)) leads to severely reduced store-operated currents. Highly conserved amino acids (K85 in CRACM1 and K60 in CRACM3) in the N-terminal region close to the first transmembrane domain are crucial for STIM1-dependent gating of CRAC channels. Single-point mutations of this residue (K85E and K60E) eliminate store-operated currents induced by inositol 1,4,5-trisphosphate and reduce store-independent gating by 2-aminoethoxydiphenyl borate. However, short fragments of these mutant channels are still able to communicate with the CRAC-activating domain of STIM1. Collectively, these findings identify a single amino acid in the N terminus of CRAC channels as a critical element for store-operated gating of CRAC channels.

TRPM2 is a Ca 2+ -permeable cation channel that is specifically activated by adenosine diphosphoribose (ADPR). Channel activation in the plasma membrane leads to Ca 2+ influx and has been linked to apoptotic mechanisms. The primary agonist, ADPR, is produced both extra- and intracellularly and causes increases in intracellular calcium concentration ([Ca 2+ ] i ), but the mechanisms involved are not understood. Using short interfering RNA and a knockout mouse, we report that TRPM2, in addition to its role as a plasma membrane channel, also functions as a Ca 2+ -release channel activated by intracellular ADPR in a lysosomal compartment. We show that both functions of TRPM2 are critically linked to hydrogen peroxide–induced β cell death. Additionally, extracellular ADPR production by the ectoenzyme CD38 from its substrates NAD + (nicotinamide adenine dinucleotide) or cADPR causes IP 3 -dependent Ca 2+ release via P2Y and adenosine receptors. Thus, ADPR and TRPM2 represent multimodal signaling elements regulating Ca 2+ mobilization in β cells through membrane depolarization, Ca 2+ influx, and release of Ca 2+ from intracellular stores.

Depletion of intracellular calcium stores activates store-operated calcium entry across the plasma membrane in many cells. STIM1, the putative calcium sensor in the endoplasmic reticulum, and the calcium release-activated calcium (CRAC) modulator CRACM1 (also known as Orai1) in the plasma membrane have recently been shown to be essential for controlling the store-operated CRAC current (I(CRAC)). However, individual overexpression of either protein fails to significantly amplify I(CRAC). Here, we show that STIM1 and CRACM1 interact functionally. Overexpression of both proteins greatly potentiates I(CRAC), suggesting that STIM1 and CRACM1 mutually limit store-operated currents and that CRACM1 may be the long-sought CRAC channel.

SummaryReceptor-mediated Ca2+ release from the endoplasmic reticulum (ER) is often followed by Ca2+ entry through Ca2+-release-activated Ca2+ (CRAC) channels in the plasma membrane [1–5]. RNAi screens have identified STIM1 as the putative ER Ca2+ sensor [6–8] and CRACM1 (Orai1; [9–11]) as the putative store-operated Ca2+ channel. Overexpression of both proteins is required to reconstitute CRAC currents (ICRAC; [11–14]). We show here that CRACM1 forms multimeric assemblies that bind STIM1 and that acidic residues in the transmembrane (TM) and extracellular domains of CRACM1 contribute to the ionic selectivity of the CRAC-channel pore. Replacement of the conserved glutamate in position 106 of the first TM domain of CRACM1 with glutamine (E106Q) acts as a dominant-negative protein, and substitution with aspartate (E106D) enhances Na+, Ba2+, and Sr2+ permeation relative to Ca2+. Mutating E190Q in TM3 also affects channel selectivity, suggesting that glutamate residues in both TM1 and TM3 face the lumen of the pore. Furthermore, mutating a putative Ca2+ binding site in the first extracellular loop of CRACM1 (D110/112A) enhances monovalent cation permeation, suggesting that these residues too contribute to the coordination of Ca2+ ions to the pore. Our data provide unequivocal evidence that CRACM1 multimers form the Ca2+-selective CRAC-channel pore.

TRPM7 is a ubiquitously expressed and constitutively active divalent cation-selective ion channel, whose basal activity is regulated by intracellular levels of Mg 2+ and Mg·ATP. We have investigated receptor-mediated mechanisms that may actively regulate TRPM7 activity. We here report that TRPM7 currents are suppressed by intracellular GTPγS, suggesting the involvement of heterotrimeric G proteins. TRPM7 currents are also inhibited by stimulating endogenous muscarinic receptors, which is mediated by G i because the inhibitory effect is blunted by pertussis toxin. Conversely, stimulation of endogenous G s -coupled β-adrenergic receptors potentiates TRPM7 currents, whereas G q -coupled thrombin receptors have little effect. Consistent with the involvement of G s /G i in controlling adenylyl cyclase activity, elevations of intracellular cAMP levels enhance TRPM7 activity and prevent receptor-mediated modulation of TRPM7 activity by muscarinic and adrenergic agonists. This cAMP-dependent effect requires the functional integrity of both protein kinase A (PKA) and the endogenous kinase domain of TRPM7 because cAMP-mediated effects are abolished when treating cells with the PKA inhibitors H89 or KT5720 as well as in cells expressing phosphotransferase-deficient TRPM7 constructs. These mutant channels are also much less susceptible to GTPγS-mediated inhibition, suggesting that the main regulatory effect occurs through G i - and G s -mediated changes in cAMP. Taken together, our results demonstrate that TRPM7 activity is up- and down-regulated through its endogenous kinase in a cAMP- and PKA-dependent manner.

1. We studied the immediate and short-term effects of UV light in the near-visible range at the cellular and membrane level using the whole-cell patch-clamp technique in combination with digital fluorescence imaging. 2. Illumination with monochromatic UVA light (340-380 nm) induced a sustained non-saturable increase in membrane conductance dependent on wavelength and light intensity in several different mammalian cell types including RBL, mast, HEK, PC12 and 3T3 cells. 3. The current was non-selective for cations and permeable to Ca2+, but was inhibited by trivalent cations and was not due to the activation of an endogenous ion channel. We termed this novel current ILiNC for light-induced non-selective cation current. 4. A similar current was evoked by chemical peroxidants such as hydrogen peroxide and tertbutylhydroperoxide, but not by cytosolic oxidized glutathione. 5. The free-radical scavengers tocopherol (vitamin E) and ascorbic acid (vitamin C) significantly reduced the UV light effect. 6. The generation of the current was membrane delimited since it could be induced by the same UVA treatment in cell-free membrane patches showing a similar wavelength dependence. 7. These results suggest that ILiNC is activated by UVA light-induced generation of free radicals acting through lipid or protein peroxidation, and may represent a ubiquitous mechanism by which Na+ and Ca2+ can enter cells after phototoxic or free radical-induced membrane damage.

Background Airway inflammation and asthma have been linked to oxidative stress and the melastatin-related transient receptor potential cation channel, member 2 (TRPM2), which can be activated by reactive oxygen species (ROS), has emerged as a potential therapeutic target for inflammatory diseases. Objective Using TRPM2 deficient (TRPM2-/-) mice, we investigated whether the TRPM2 ion channel, which mediates calcium (Ca2+) influx and lysosomal Ca2+ release, plays a role in the pathophysiology of severe allergic asthma in mouse. Methods Severe allergic asthma was initiated in wild type (WT) and TRPM2-/- mice by repeated sensitization with ovalbumin (OVA)/aluminum hydroxide on Days 0, 7 and 14, followed by intranasal challenge on Days 21, 22 and 23. Mice were investigated for the presence of airway responsiveness, airway inflammation, production of allergen-specific antibodies, cytokine response and lung pathology. Results The absence of TRPM2 channels has no obvious effect on major etiologic markers of severe allergic asthma in this mouse model. Neither airway resistance nor mucus production are affected in TRPM2-/- mice. TRPM2 channel ablation also does not alter airway inflammation or immunocyte infiltration and does not affect antibody response or cytokine levels. Conclusions TRPM2 is not required for airway inflammation in OVA-induced severe allergic asthma in mice. Accordingly, TRPM2 might not be a suitable therapeutic target for airway inflammation caused by allergens in humans.

1. Whole-cell patch-clamp recording were employed to study facilitation of Ca2+ currents and excessive Ca2+ tail currents evoked by strong and long-lasting conditioning depolarizations in skeletal myoballs cultured from newborn rats. 2. Paired-pulse facilitation and excessive tail currents showed the same voltage dependence, becoming prominent at conditioning potentials above +30 mV. 3. Recruitment of excessive tail currents and facilitation occurred with the same time dependence (time constant (tau), approximately 200 ms to approximately 1 s), accelerating with the depolarization strength of conditioning pulses. 4. Reversal of Ca2+ current facilitation during the repolarization period between conditioning and test pulses was time- and voltage dependent. The time window of recruitment of facilitated Ca2+ currents narrowed considerably at more negative repolarization potentials (tau: approximately 10 ms at -100 mV, but approximately 1.5 at 0 mV). 5. Neither omission of internal ATP nor perfusion of the cells with the peptide inhibitor of protein kinase A (PKI) had significant effects on Ca2+ current facilitation, although internal perfusion with ATP gamma S slowly suppressed the facilitation currents by about 30%. External application of either ryanodine or caffeine under control conditions selectively and significantly suppressed the facilitated Ca2+ currents by about 30-40%. 6. We propose that facilitation of Ca2+ currents and excessive tail currents are consequences of a common mechanism linked to ryanodine receptors.

1. Whole-cell patch clamp recordings of membrane currents and fura-2 measurements of free intracellular calcium concentration ([Ca2+]i) were used to study the biophysical properties of a calcium current activated by depletion of intracellular calcium stores in rat peritoneal mast cells. 2. Calcium influx through an inward calcium release-activated calcium current (ICRAC) was induced by three independent mechanisms that result in store depletion: intracellular infusion of inositol 1,4,5-trisphosphate (InsP3) or extracellular application of ionomycin (active depletion), and intracellular infusion of calcium chelators (ethylene glycol bis-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)) to prevent reuptake of leaked-out calcium into the stores (passive depletion). 3. The activation of ICRAC induced by active store depletion has a short delay (4-14 s) following intracellular infusion of InsP3 or extracellular application of ionomycin. It has a monoexponential time course with a time constant of 20-30 s and, depending on the complementary Ca2+ buffer, a mean normalized amplitude (at 0 mV) of 0.6 pA pF-1 (with EGTA) and 1.1 pA pF-1 (with BAPTA). 4. After full activation of ICRAC by InsP3 in the presence of EGTA (10 mM), hyperpolarizing pulses to -100 mV induced an instantaneous inward current that decayed by 64% within 50 ms. This inactivation is probably mediated by [Ca2+]i, since the decrease of inward current in the presence of the fast Ca2+ buffer BAPTA (10 mM) was only 30%. 5. The amplitude of ICRAC was dependent on the extracellular Ca2+ concentration with an apparent dissociation constant (KD) of 3.3 mM. Inward currents were nonsaturating up to -200 mV. 6. The selectivity of ICRAC for Ca2+ was assessed by using fura-2 as the dominant intracellular buffer (at a concentration of 2 mM) and relating the absolute changes in the calcium-sensitive fluorescence (390 nm excitation) with the calcium current integral. This relationship was almost identical to the one determined for Ca2+ influx through voltage-activated calcium currents in chromaffin cells, suggesting a similar selectivity. Replacing Na+ and K+ by N-methyl-D-glucamine (with Ca2+ ions as exclusive charge carriers) reduced the amplitude of ICRAC by only 9% further suggesting a high specificity for Ca2+ ions. 7. The current amplitude was not greatly affected by variations of external Mg2+ in the range of 0-12 mM. Even at 12 mM Mg2+ the current amplitude was reduced by only 23%. 8. ICRAC was dose-dependently inhibited by Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)

In rat peritoneal mast cells, the activation of GTP-binding proteins (G proteins) by guanosine 5'-[gamma-thio]triphosphate GTP[gamma S] has been found to induce a transient rise in intracellular calcium as well as degranulation. A G protein that couples to phospholipase C (Gp) is thought to mediate the calcium response, whereas degranulation is mediated by a different G protein, termed Ge. In an attempt to activate mast-cell G proteins more selectively, the GTP analogues guanosine 5'-[alpha-thio]triphosphate (GTP[alpha S]) and guanosine 5'-[beta-thio]triphosphate (GTP[beta S]) (RP and SP diastereomers) were introduced into mast cells by means of patch pipettes. Degranulation and free intracellular calcium were monitored by cell capacitance and fura-2 measurements, respectively. It was found that RP-GTP[alpha S], like GTP[gamma S], induced both calcium release and exocytosis. In contrast, RP-GTP[beta S] induced repetitive calcium spikes that were not regularly accompanied by exocytosis. These results suggest that RP-GTP[beta S] selectively activates calcium signaling in mast cells. The RP-GTP[beta S]-induced oscillations were independent of extracellular calcium. They were absent in the presence of heparin or high concentrations of inositol 1,4,5-trisphosphate and modulated by compound 48/80, suggesting the involvement of the inositol phospholipid signaling pathway. Latency of appearance and spiking frequency were markedly modulated by varying the intracellular ATP concentration. The differential activation of intracellular calcium signaling and exocytosis by GTP[beta S] confirms the presence of independent signal-transduction pathways for the two cell responses. RP-GTP[beta S] may prove helpful in the biochemical and molecular characterization of Gp, the as-yet-unidentified G protein that couples receptors to intracellular calcium release.

Combined patch-clamp and fura-2 measurements were performed to study the calcium release properties of Chinese hamster ovary (CHO) cells transfected with the rabbit skeletal muscle ryanodine receptor cDNA carried by an expression vector. Both caffeine (1–50 mM) and ryanodine (100 μM) induced release of calcium from intracellular stores of transformed CHO cells but not from control (non-transfected) CHO cells. The calcium responses to caffeine and ryanodine closely resembled those commonly observed in skeletal muscle. Repetitive applications of caffeine produced characteristic all-or-none rises in intracellular calcium. Inositol 1,4,5-trisphosphate (IP3) neither activated the ryanodine receptor channel nor interfered with the caffeine-elicited calcium release. These results indicate that functional calcium release channels are formed by expression of the ryanodine receptor cDNA.

1. Stimulation of mast cells by externally applied secretagogues activated a slowly developing membrane current. With high external and low internal chloride (Cl-) concentrations, the current reversed at about -40 mV, but when external Cl- was made equal to internal Cl-, the reversal potential shifted to about 0 mV, demonstrating that the current carrier was Cl-. 2. In addition to external agonists, internally applied cyclic AMP and high concentrations of intracellular calcium [Ca2+]i could also activate the Cl- current. However, elevated [Ca2+]i produced only slow and incomplete activation. This suggests that the Cl- current is not directly Ca2+ activated. Also, activation of Cl- current by external agonists and by cyclic AMP was unimpaired when [Ca2+]i was clamped to low levels with internal ethylene glycol bis-N,N,N',N'-tetraacetic acid (EGTA), indicating that elevated [Ca2+]i is not necessary for activation of the Cl- current. Although activation by cyclic AMP was faster than that produced by elevated [Ca2+]i, it still required tens of seconds; thus the effect of cyclic AMP was also likely to be indirect. 3. Internal guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) could also activate the Cl- current, suggesting the involvement of a G protein in the control of the current. 4. The variance associated with the Cl- current was small, and noise analysis gave a lower limit of about 1-2 pS for the single-channel conductance. The Cl- current was reduced by 4,4'-diisothiocyano-2,2'-stilbenedisulphonate (DIDS), and during DIDS blockade, the variance of the current increased. This suggests that DIDS enters and blocks the open channel. 5. Activation of the Cl- current would make the membrane potential negative following stimulation of a mast cell, thus providing a driving force for entry of external calcium via the stimulation-induced influx pathways described in the preceding paper (Matthews, Neher & Penner, 1989).