Anion channels modulate store-operated calcium influx in human microglia

Recent work from this laboratory has demonstrated that purinergic-mediated depolarization of human microglia inhibited a store-operated pathway for entry of Ca2+. We have used Fura-2 spectrofluorometry to investigate the effects on store-operated Ca2+ influx induced by replacement of NaCl with Na-gluconate in extracellular solutions. Three separate procedures were used to activate store-operated channels. Platelet activating factor (PAF) was used to generate a sustained influx of Ca2+ in standard physiological saline solution (PSS). The magnitude of this response was depressed by 70% after replacement of PSS with low Cl- PSS. A second procedure used ATP, initially applied in Ca2+-free PSS solution to deplete intracellular stores. The subsequent perfusion of PSS solution containing Ca2+ resulted in a large and sustained entry of Ca2+, which was inhibited by 75% with low Cl- PSS. The SERCA inhibitor cyclopiazonic acid (CPA) was used to directly deplete stores in zero-Ca2+ PSS. Following the introduction of PSS containing Ca2+, a maintained stores-operated influx of Ca2+ was evident which was inhibited by 77% in the presence of the low Cl- PSS. Ca2+ influx was linearly reduced with cell depolarization in elevated K+ (7.5 to 35 mM) suggesting that changes in external Cl- were manifest as altered electrical driving force for Ca2+ entry. However, 50 mM external KCl effectively eliminated divalent entry which may indicate inactivation of this pathway with high magnitudes of depolarization. Patch clamp studies showed low Cl-PSS to cause depolarizing shifts in both holding currents and reversal potentials of currents activated with voltage ramps. The results demonstrate that Cl- channels play an important role in regulating store-operated entry of Ca2+ in human microglia.     

Dual role of tubulin-cytoskeleton in store-operated calcium entry in human platelets

Two mechanisms for store-operated Ca(2+) entry (SOCE) regulated by two independent Ca(2+) stores, the dense tubular system (DTS) and the acidic stores, have been described in platelets. We have previously suggested that coupling between the type II IP(3) receptor (IP(3)RII) and hTRPC1, involving reorganization of the actin microfilaments, play an important role in SOCE. However, the involvement of the tubulin microtubules, located beneath the plasma membrane, remains unclear. Here we show that the microtubule disrupting agent colchicine reduced Ca(2+) entry stimulated by low concentrations (0.1 U/mL) of thrombin, which activates SOCE mostly by depleting acidic Ca(2+)-store. Consistently, colchicine reduced SOCE activated by 2,5 di-(tertbutyl)-1,4-hydroquinone (TBHQ), which selectively depletes the acidic Ca(2+) stores. In contrast, colchicine enhanced SOCE mediated by depletion of the DTS, induced by high concentrations of thapsigargin (TG), which depletes both the acidic Ca(2+) stores and the DTS, the major releasable Ca(2+) store in platelets. These findings were confirmed by using Sr(2+) as a surrogate for Ca(2+) entry. Colchicine attenuated the coupling between IP(3)RII and hTRPC1 stimulated by thrombin while it enhanced that evoked by TG. Paclitaxel, which induces microtubular stabilization and polymerization, exerted the opposite effects on thrombin- and TG-evoked SOCE and coupling between IP(3)RII and hTRPC1 compared with colchicine. Neither colchicine nor paclitaxel altered the ability of platelets to extrude Ca(2+). These findings suggest that tubulin microtubules play a dual role in SOCE, acting as a barrier that prevents constitutive SOCE regulated by DTS, but also supporting SOCE mediated by the acidic Ca(2+) stores.     

Calcium-activated potassium channels in human platelets

The cationic fluorescent probe, DiSC3(5) was used to measure the membrane potential in human platelets. Hyperpolarization was induced by the addition of Ca2+ to the medium and also by the addition of the Ca2+ ionophore, A23187. In the absence of extracellular Ca2+ ([Ca2+]o) there was no response to A23187. The threshold concentration for [Ca2+]o was 20 microM and for A23187 was 12 nM. The increase polarity induced by [Ca2+]o was not affected by various K+ channel blockers. However, the effect of A23187 was inhibited by quinine and charybdotoxin, while apamin, tetraethylammonium, and the calmodulin inhibitors trifluoperazine and compound R24571 were ineffective. The resting membrane potential was -66 +/- 0.9 mV and was decreased by quinine. There are three conclusions from this study: (i) Ca2+-activated K+ channels exist in human platelets; (ii) they are the type that are apamin insensitive, charybdotoxin sensitive; and (iii) they may contribute to the resting membrane potential.     

Receptor-activated single channels in intact human platelets.

We report "cell-attached" patch clamp studies of intact human platelets which show receptor-activated single channels. Inclusion of ADP in the patch pipette, but not in the bath, resulted in the appearance of inward currents indicative of single channels tightly coupled to the ADP receptors. The channels had a slope conductance of 11 picosiemens at the resting potential. Removal of 1 mM Ca2+ or replacement of chloride by gluconate in the pipette filling solution had little effect on the slope conductance at the resting potential or on the estimated reversed potential. With isotonic BaCl2 in the pipette, ADP evoked single channel currents with a slope conductance of 10 picosiemens. Thus these channels appear to be permeable to monovalent and divalent cations and selective for cations over anions. Addition of 5 mM Ni2+ (which blocks ADP-evoked rapid calcium entry in fura-2-loaded platelets) to the pipette solution blocked ADP-evoked channel activity. These channels may therefore provide an important mechanism for ADP to activate human platelets within a small fraction of a second.     

TRPC channels as STIM1-regulated store-operated channels

Receptor-activated Ca(2+) influx is mediated largely by store-operated channels (SOCs). TRPC channels mediate a significant portion of the receptor-activated Ca(2+) influx. However, whether any of the TRPC channels function as a SOC remains controversial. Our understanding of the regulation of TRPC channels and their function as SOCs is being reshaped with the discovery of the role of STIM1 in the regulation of Ca(2+) influx channels. The findings that STIM1 is an ER resident Ca(2+) binding protein that regulates SOCs allow an expanded and molecular definition of SOCs. SOCs can be considered as channels that are regulated by STIM1 and require the clustering of STIM1 in response to depletion of the ER Ca(2+) stores and its translocation towards the plasma membrane. TRPC1 and other TRPC channels fulfill these criteria. STIM1 binds to TRPC1, TRPC2, TRPC4 and TRPC5 but not to TRPC3, TRPC6 and TRPC7, and STIM1 regulates TRPC1 channel activity. Structure-function analysis reveals that the C-terminus of STIM1 contains the binding and gating function of STIM1. The ERM domain of STIM1 binds to TRPC channels and a lysine-rich region participates in the gating of SOCs and TRPC1. Knock-down of STIM1 by siRNA and prevention of its translocation to the plasma membrane inhibit the activity of native SOCs and TRPC1. These findings support the conclusion that TRPC1 is a SOC. Similar studies with other TRPC channels demonstrate their regulation by STIM1 and indicate that all TRPC channels, except TRPC7, function as SOCs.     

Mitochondrial regulation of store-operated CRAC channels

In eukaryotic cells, one major route for Ca(2+) influx is through store-operated CRAC channels, which are activated following a fall in Ca(2+) content within the endoplasmic reticulum. Mitochondria are key regulators of this ubiquitous Ca(2+) influx pathway. Respiring mitochondria rapidly take up some of the Ca(2+) released from the stores, resulting in more extensive store depletion and thus robust activation of CRAC channels. As CRAC channels open, the ensuing rise in cytoplasmic Ca(2+) feeds back to inactivate the channels. By buffering some of the incoming Ca(2+) mitochondria reduce Ca(2+)-dependent inactivation of the CRAC channels, resulting in more prolonged Ca(2+) influx. However, mitochondria can release Ca(2+) close to the endoplasmic reticulum, accelerating store refilling and thus promoting deactivation of the CRAC channels. Mitochondria thus regulate all major transitions in CRAC channel gating, revealing remarkable versatility in how this organelle impacts upon Ca(2+) influx. Recent evidence suggests that mitochondria also control CRAC channels through mechanisms that are independent of their Ca(2+)-buffering actions and ability to generate ATP. Furthermore, pyruvic acid, a key intermediary metabolite and precursor substrate for the Krebs cycle, reduces the extent of Ca(2+)-dependent inactivation of CRAC channels. Hence mitochondrial metabolism impacts upon Ca(2+) influx through CRAC channels and thus on a range of key downstream cellular responses.     

Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a versatile regulator of TRP channels. We report that inclusion of a PIP2 analogue, PIP2 1,2-dioctanoyl, does not induce non-capacitative Ca2+ entry per se but enhanced Ca2+ entry stimulated either by thrombin or by selective depletion of the Ca2+ stores in platelets, the dense tubular system, using 10 nM TG, and the acidic stores, using 20 microM 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Reduction of PIP2 levels by blocking PIP2 resynthesis with Li+ or introducing a monoclonal anti-PIP2 antibody, or sequestering PIP2 using poly-lysine, attenuated Ca2+ entry induced by thrombin, TG and TBHQ, and reduced thrombin-evoked, but not TG- or TBHQ-induced, Ca2+ release from the stores. Incubation with the anti-hTRPC1 antibody did not alter the stimulation of Ca2+ entry by PIP2, whilst introduction of anti-hTRPC6 antibody directed towards the C-terminus of hTRPC6 reduced Ca2+ and Mn2+ entry induced by thrombin, TG or TBHQ, and abolished the stimulation of Ca2+ entry by PIP2. The anti-hTRPC6 antibody, but not the anti-hTRPC1 antibody or PIP2, reduced non-capacitative Ca2+ entry by the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol. In summary, hTRPC6 plays a role both in store-operated and in non-capacitative Ca2+ entry. PIP2 enhances store-operated Ca2+ entry in human platelets, most probably by stimulation of hTRPC6 channels.     

Dihydropyridine-sensitive and insensitive Ca2+ channels in human platelets

The Ca2+ channel blocker, nifedipine, a dihydropyridine derivative, inhibited the Ca2+ influx and release from internal stores caused by collagen or a low concentration of the thromboxane A2 (TXA2) analogue, 9,11-epithio-11,12-methano-TXA2 (STA2) (10 nM), but did not inhibit those caused by thrombin or a high concentration of STA2 (100 nM). These results indicate the presence of two distinct, dihydropyridine-sensitive and insensitive, Ca2+ channels dependent on the concentrations and classes of agonists in human platelets.     

Two thrombin-activated Ca2+ channels in human platelets.

The regulation of extracellular Ca2+ entry into fura-2-loaded human platelets was examined following stimulation with thrombin. In the presence of external Ca2+, stimulation of platelets with thrombin resulted in a rapid increase, followed by a plateau, in intracellular Ca2+ concentration ([Ca2+]i). Pretreatment with wortmannin, a specific inhibitor of myosin light chain kinase, suppressed only the plateau phase and had no effect on the initial rapid increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thrombin induced a transient and relatively small increase in [Ca2+]i caused by Ca2+ release from internal stores. When Ca2+ was added subsequently to the Ca(2+)-free medium within 10 min after thrombin activation, a marked increase in [Ca2+]i was seen, reflecting thrombin-stimulated external Ca2+ entry. With the Ca(2+)-free medium, wortmannin did not affect either the Ca2+ mobilization from the internal stores or the rapid external Ca2+ entry at early time points (within 5 s) after thrombin stimulation, whereas it significantly inhibited Ca2+ entry when Ca2+ was added later (at 3 min). Wortmannin inhibition of this late Ca2+ entry and that of 20-kDa myosin light chain phosphorylation after thrombin stimulation were dose- and preincubation time-dependent and correlated well with each other. These results suggest that two different channels are responsible for Ca2+ entry in human platelets at the early and late phases of thrombin stimulation and that the channel responsible for the late phase of Ca2+ entry may be activated by a mechanism involving myosin light chain kinase.     

SERCA2b and 3 play a regulatory role in store-operated calcium entry in human platelets

Two agonist-releasable Ca(2+)stores have been identified in human platelets differentiated by the distinct sensitivity of their SERCA isoforms to thapsigargin (TG) and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Here we have examined whether the SERCA isotypes might be involved in store-operated Ca(2+)entry (SOCE) activated by the physiological agonist thrombin in human platelets. Ca(2+)-influx evoked by thrombin (0.01 U/mL) reached a maximum after 3 min, which was consistent with the decrease in the Ca(2+)content in the stores; afterwards, the extent of SOCE decreased with no correlation with the accumulation of Ca(2+)in the stores. Inhibition of SERCA2b, by 10 nM TG, and SERCA3, with 20 microM TBHQ, individually or simultaneously, accelerated Ca(2+) store discharge and subsequently enhanced the extent of SOCE stimulated by thrombin. In addition, TG and TBHQ modified the time course of thrombin-evoked SOCE from a transient to a sustained increase in Ca(2+) influx, which reveals a negative role for SERCAs in the regulation of SOCE. This effect was consistent under conditions that inhibit Ca(2+) extrusion by PMCA or the Na(+)/Ca(2+) exchanger. Coimmunoprecipitation experiments revealed that thrombin stimulates direct interaction between SERCA2b and 3 with the hTRPC1 channel, an effect that was found to be independent of SERCA activity. In summary, our results suggest that SERCA2b and 3 modulate thrombin-stimulated SOCE probably by direct interaction with the hTRPC1 channel in human platelets.     

Bretylium opens mucosal amiloride-sensitive sodium channels

Addition of the quanternary ammonium compound, bretylium, to the outer surface of a frog skin leads to an increase in the potential difference and in the short circuit current across the skin. Bretylium does not have any effect when applied to the inside face of the frog skin. The effect of bretylium is dependent upon the presence of sodium ions in the outer medium; it is depressed when sodium is replaced by choline or potassium but not when lithium substitutes for sodium. The bretylium effect is blocked by the specific sodium channel blocker, amiloride. It is proposed that bretylium opens mucosal, amiloride-sensitive sodium channels.     

Bretylium opens mucosal amiloride-sensitive sodium channels

Addition of the quanternary ammonium compound, bretylium, to the outer surface of a frog skin leads to an increase in the potential difference and in the short circuit current across the skin. Bretylium does not have any effect when applied to the inside face of the frog skin. The effect of bretylium is dependent upon the presence of sodium ions in the outer medium; it is depressed when sodium is replaced by choline or potassium but not when lithium substitutes for sodium. The bretylium effect is blocked by the specific sodium channel blocker, amiloride. It is proposed that bretylium opens mucosal, amiloride-sensitive sodium channels.     

Functional consequences of activating store-operated CRAC channels

Store-operated CRAC channels, which are activated by the emptying of the endoplasmic reticulum Ca(2+) stores, are an important and widespread route for triggering rises in cytoplasmic Ca(2+). The cellular responses that are activated in response to Ca(2+) entry through CRAC channels are being dissected out, and recent evidence has established that CRAC channels can induce both short-term (safeguarding the Ca(2+) content of the endoplasmic reticulum, maintenance of cytoplasmic Ca(2+) oscillations, enzyme activation, secretion) and long-term (gene expression) changes in cells. CRAC channel activation is therefore capable of evoking a range of temporally distinct responses, highlighting the versatility of this ubiquitous Ca(2+) entry pathway.     

Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes

The depletion of intracellular Ca2+ stores triggers the opening of Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane of T lymphocytes. We have investigated the additional role of extracellular Ca2+ (Ca02+) in promoting CRAC channel activation in Jurkat leukemic T cells. Ca2+ stores were depleted with 1 microM thapsigargin in the nominal absence of Ca02+ with 12 mM EGTA or BAPTA in the recording pipette. Subsequent application of Ca02+ caused ICRAC to appear in two phases. The initial phase was complete within 1 s and reflects channels that were open in the absence of Ca02+. The second phase consisted of a severalfold exponential increase in current amplitude with a time constant of 5-10 s; we call this increase Ca(2+)-dependent potentiation, or CDP. The shape of the current-voltage relation and the inferred single-channel current amplitude are unchanged during CDP, indicating that CDP reflects an alteration in channel gating rather than permeation. The extent of CDP is modulated by voltage, increasing from approximately 50% at +50 mV to approximately 350% at -75 mV in the presence of 2 mM Ca02+. The voltage dependence of CDP also causes ICRAC to increase slowly during prolonged hyperpolarizations in the constant presence of Ca02+. CDP is not affected by exogenous intracellular Ca2+ buffers, and Ni2+, a CRAC channel blocker, can cause potentiation. Thus, the underlying Ca2+ binding site is not intracellular. Ba2+ has little or no ability to potentiate CRAC channels. These results demonstrate that the store-depletion signal by itself triggers only a small fraction of capacitative Ca2+ entry and establish Ca2+ as a potent cofactor in this process. CDP confers a previously unrecognized voltage dependence and slow time dependence on CRAC channel activation that may contribute to the dynamic behavior of ICRAC.     

Two types of store-operated channels in A431 cells

Activation of phospholipase C-coupled receptors leads to the release of Ca2+ from Ca2+ stores, and subsequent activation of store-operated cation (SOC) channels, promoting sustained Ca2+ influx. The most studied SOC channels are CRAC ("calcium-release activated calcium") channels exhibiting a very high selectivity for Ca2+. However, there are many SOC channels permeable for Ca2+ but having a lower selectivity. And while Ca2+ influx is important for many biological processes, little is known about the types of SOC channels and mechanisms of SOC channel activation. Previously, we described store-operated Imin channels in A431 cells. Here, by whole-cell recordings, we demonstrated that the store depletion activates two types of current in A431 cells--highly selective for divalent cations (presumably, ICRAC), and moderately selective (ISOC supported by Imin channels). These currents can be registered separately and have different developing time and amplitude. Coexisting of two different types of SOC channels in A431 cells seems to facilitate the control of intracellular Ca(2+)-dependent processes.     

Monitoring of the activation of receptor-operated calcium channels in human platelets

Activation of receptor-operated calcium channels has been monitored by measurements of the quenching of the fluorescence of intracellularly trapped fura-2 by Mn entering from the extracellular medium. Release of calcium from intracellular stores was followed simultaneously by measurements of the ratio of the fluorescence excited at 340 and 380 nm. Thrombin, ADP, platelet-activating-factor (PAF) and collagen, all produced both release of calcium from the intracellular stores and uptake of Mn from the extracellular medium. The uptake of Mn, but not the increase of (Ca2+)i, was blocked by nickel. These results suggest the existence of plasma membrane calcium channels which can be activated by the different agonists tested here. The activation of calcium channels was very fast and transient with ADP and PAF, fast and maintained with thrombin, and delayed with collagen.     

A role for the intracellular protease calpain in the activation of store-operated calcium entry in human platelets

Here, we report a novel role for the cysteine protease calpain in store-operated calcium entry. Several structurally and mechanistically unrelated inhibitors of calpain inhibited Ca2+ entry activated in human platelets by thapsigargin-evoked Ca2+ store depletion or the physiological agonist thrombin, whereas inhibitors of other cysteine proteases were without effect. The use of the cell-permeable fluorogenic calpain substrate 7-amino-4-chloromethylcoumarin, t-BOC-l-leucyl-l-methionine amide revealed rapid activation of calpain which was closely temporally correlated with Ca2+ store depletion even in the absence of a rise in cytosolic [Ca2+]. Calpain inhibition prevented the tyrosine phosphorylation of several proteins upon Ca2+ store depletion, suggesting that calpain may lie upstream of protein tyrosine phosphorylation that is known to be required for the activation of store-operated Ca2+ entry in human platelets. Earlier studies using calpain inhibitors may need reinterpretation in the light of this finding that calpain plays a role in the activation of physiological Ca2+ entry pathways.     

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.