Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells

Although the crucial role of Ca(2+) influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca(2+) channels in normal human T lymphocytes. The use of Na(+) as the permeant ion in divalent-free solution permitted Ca(2+) release-activated Ca(2+) (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca(2+) store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca(2+) in the micromolar range, selective Ca(2+) permeation in the millimolar range, and inactivation that depended upon intracellular Mg(2+) ions. The number of CRAC channels per cell increased greatly from approximately 15 in resting T cells to approximately 140 in activated T cells. Treatment with the phorbol ester PMA also increased CRAC channel expression to approximately 60 channels per cell, whereas the immunosuppressive drug cyclosporin A (1 microM) suppressed the PHA-induced increase in functional channel expression. Capacitative Ca(2+) influx induced by thapsigargin was also significantly enhanced in activated T cells. We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca(2+) influx in human resting T cells, and that the expression of CRAC channels increases approximately 10-fold during activation, resulting in enhanced Ca(2+) signaling.     

Hypoxia leads to Na,K-ATPase downregulation via Ca(2+) release-activated Ca(2+) channels and AMPK activation

To maintain cellular ATP levels, hypoxia leads to Na,K-ATPase inhibition in a process dependent on reactive oxygen species (ROS) and the activation of AMP-activated kinase α1 (AMPK-α1). We report here that during hypoxia AMPK activation does not require the liver kinase B1 (LKB1) but requires the release of Ca(2+) from the endoplasmic reticulum (ER) and redistribution of STIM1 to ER-plasma membrane junctions, leading to calcium entry via Ca(2+) release-activated Ca(2+) (CRAC) channels. This increase in intracellular Ca(2+) induces Ca(2+)/calmodulin-dependent kinase kinase β (CaMKKβ)-mediated AMPK activation and Na,K-ATPase downregulation. Also, in cells unable to generate mitochondrial ROS, hypoxia failed to increase intracellular Ca(2+) concentration while a STIM1 mutant rescued the AMPK activation, suggesting that ROS act upstream of Ca(2+) signaling. Furthermore, inhibition of CRAC channel function in rat lungs prevented the impairment of alveolar fluid reabsorption caused by hypoxia. These data suggest that during hypoxia, calcium entry via CRAC channels leads to AMPK activation, Na,K-ATPase downregulation, and alveolar epithelial dysfunction.     

The third transmembrane segment of orai1 protein modulates Ca2+ release-activated Ca2+ (CRAC) channel gating and permeation properties

Orai1, the pore subunit of Ca(2+) release-activated Ca(2+) channels, has four transmembrane segments (TMs). The first segment, TMI, lines the pore and plays an important role in channel activation and ion permeation. TMIII, on the other hand, does not line the pore but still regulates channel gating and permeation properties. To understand the role of TMIII, we have mutated and characterized several residues in this domain. Mutation of Trp-176 to Cys (W176C) and Gly-183 to Ala (G183A) had dramatic effects. Unlike wild-type channels, which exhibit little outward current and are activated by STIM1, W176C mutant channels exhibited a large outward current at positive potentials and were constitutively active in the absence of STIM1. G183A mutant channels also exhibited substantial outward currents but were active only in the presence of 2-aminoethoxydiphenyl borate (2-APB), irrespective of STIM1. With W176C mutant channels inward, monovalent currents were blocked by Ca(2+) with a high affinity similar to the wild type, but the Ca(2+)-dependent blocking of outward currents differed in the two cases. Although a 50% block of the WT outward current required 250 μm Ca(2+), more than 6 mm was necessary to have the same effect on W176C mutant channels. In the presence of extracellular Ca(2+), W176C and G183A outward currents developed slowly in a voltage-dependent manner, whereas they developed almost instantaneously in the absence of Ca(2+). These changes in permeation and gating properties mimic the changes induced by mutations of Glu-190 in TMIII and Asp-110/Asp-112 in the TMI/TMII loop. On the basis of these data, we propose that TMIII maintains negatively charged residues at or near the selectivity filter in a conformation that facilitates Ca(2+) inward currents and prevents outward currents of monovalent cations. In addition, to controlling selectivity, TMIII may also stabilize channel gating in a closed state in the absence of STIM1 in a Trp-176-dependent manner.     

Properties of voltage-gated Ca(2+) channels in rabbit ventricular myocytes expressing Ca(2+) channel alpha(1E) cDNA

Using the whole-cell patch-clamp technique, we have studied the properties of alpha(1E) Ca(2+) channel transfected in cardiac myocytes. We have also investigated the effect of foreign gene expression on the intrinsic L-type current (I(Ca,L)). Expression of green fluorescent protein significantly decreased the I(Ca,L). By contrast, expression of alpha(1E) with beta(2b) and alpha(2)/delta significantly increased the total Ca(2+) current, and in these cells a Ca(2+) antagonist, PN-200-110 (PN), only partially blocked the current. The remaining PN-resistant current was abolished by the application of a low concentration of Ni(2+) and was little affected by changing the charge carrier from Ca(2+) to Ba(2+) or by beta-adrenergic stimulation. On the basis of its voltage range for activation, this channel was classified as a high-voltage activated channel. Thus the expression of alpha(1E) did not generate T-like current in cardiac myocytes. On the other hand, expression of alpha(1E) decreased I(Ca,L) and slowed the I(Ca,L) inactivation. This inactivation slowing was attenuated by the beta(2b) coexpression, suggesting that the alpha(1E) may slow the inactivation of I(Ca,L) by scrambling with alpha(1C) for intrinsic auxiliary beta.     

Novel potent and selective Ca2+ release-activated Ca2+ (CRAC) channel inhibitors. Part 3: synthesis and CRAC channel inhibitory activity of 4'-[(trifluoromethyl)pyrazol-1-yl]carboxanilides

From a series of 4'-[(trifluoromethyl)pyrazol-1-yl]carboxanilides derived from 4-methyl-4'-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-1,2,3-thiadiazole-5-carboxanilide, one inhibited thapsigargin-induced Ca2+ influx in Jurkat T cells (IC(50)=77 nM) and exhibited high selectivity for the CRAC channel over the VOC channel (index: >130). Another acted as an inhibitor for both T lymphocyte activation-induced diseases and ovalbumin-induced airway eosinophilia in rats (ED(50)=1.3 mg/kg) p.o.     

Inhibition by calyculin A and okadaic acid of the Ca(2+) release-activated Ca(2+) entry pathway in rat basophilic leukemia cells: evidence for regulation by type 1/2A serine/threonine phosphatase activity

Using a combination of fluorescence measurements of intracellular Ca(2+) ion concentration ([Ca(2+)](i)) and membrane potential we have investigated the sensitivity to serine/threonine phosphatase inhibition of Ca(2+) entry stimulated by activation of the Ca(2+) release-activated Ca(2+) (CRAC) entry pathway in rat basophilic leukemia cells. In both suspension and adherent cells, addition of the type 1/2A phosphatase inhibitor calyculin A, during activation of CRAC uptake, resulted in a fall in [Ca(2+)](i) to near preactivation levels. Pre-treatment with calyculin A abolished the component of the Ca(2+) rise associated with activation of CRAC uptake and inhibited Mn(2+) entry, consistent with a requirement of phosphatase activity for activation of the pathway. Depletion of intracellular Ca(2+) stores is accompanied by a large depolarisation which is absolutely dependent upon Ca(2+) entry via the CRAC uptake pathway. Application of calyculin A or okadaic acid, a structurally unrelated phosphatase antagonist inhibits this depolarisation. Taken in concert, these data demonstrate a marked sensitivity of the CRAC entry pathway to inhibition by calyculin A and okadaic acid.     

S-nitrosation controls gating and conductance of the alpha 1 subunit of class C L-type Ca(2+) channels

Modulation of smooth muscle, L-type Ca(2+) channels (class C, Ca(V)1.2b) by thionitrite S-nitrosoglutathione (GSNO) was investigated in the human embryonic kidney 293 expression system at the level of whole-cell and single-channel currents. Extracellular administration of GSNO (2 mM) rapidly reduced whole-cell Ba(2+) currents through channels derived either by expression of alpha1C-b or by coexpression of alpha1C-b plus beta2a and alpha2-delta. The non-thiol nitric oxide (NO) donors 2,2-diethyl-1-nitroso-oxhydrazin (2 mM) and 3-morpholinosydnonimine-hydrochloride (2 mM), which elevated cellular cGMP levels to a similar extent as GSNO, failed to affect Ba(2+) currents significantly. Intracellular administration of copper ions, which promote decomposition of the thionitrite, antagonized its inhibitory effect, and loading of cells with high concentrations of dithiothreitol (2 mM) prevented the effect of GSNO on alpha1C-b channels. Intracellular loading of cells with oxidized glutathione (2 mM) affected neither alpha1C-b channel function nor their modulation by GSNO. Analysis of single-channel behavior revealed that GSNO inhibited Ca(2+) channels mainly by reducing open probability. The development of GSNO-induced inhibition was associated with the transient occurrence of a reduced conductance state of the channel. Our results demonstrate that GSNO modulates the alpha1 subunit of smooth muscle L-type Ca(2+) channels by an intracellular mechanism that is independent of NO release and stimulation of guanylyl cyclase. We suggest S-nitrosation of intracellularly located sulfhydryl groups as an important determinant of Ca(2+) channel gating and conductance.     

Small-conductance, Ca(2+) -activated K+ channel 2 is the key functional component of SK channels in mouse urinary bladder

Small-conductance Ca(2+)-activated K(+) (SK) channels play an important role in regulating the frequency and in shaping urinary bladder smooth muscle (UBSM) action potentials, thereby modulating contractility. Here we investigated a role for the SK2 member of the SK family (SK1-3) utilizing: 1) mice expressing beta-galactosidase (beta-gal) under the direction of the SK2 promoter (SK2 beta-gal mice) to localize SK2 expression and 2) mice lacking SK2 gene expression (SK2(-/-) mice) to assess SK2 function. In SK2 beta-gal mice, UBSM staining was observed, but staining was undetected in the urothelium. Consistent with this, urothelial SK2 mRNA was determined to be 4% of that in UBSM. Spontaneous phasic contractions in wild-type (SK2(+/+)) UBSM strips were potentiated (259% of control) by the selective SK channel blocker apamin (EC(50) = 0.16 nM), whereas phasic contractions of SK2(-/-) strips were unaffected. Nerve-mediated contractions of SK2(+/+) UBSM strips were also increased by apamin, an effect absent in SK2(-/-) strips. Apamin increased the sensitivity of SK2(+/+) UBSM strips to electrical field stimulation, since pretreatment with apamin decreased the frequency required to reach a 50% maximal contraction (vehicle, 21 +/- 4 Hz, n = 6; apamin, 12 +/- 2 Hz, n = 7; P < 0.05). In contrast, the sensitivity of SK2(-/-) UBSM strips was unaffected by apamin. Here we provide novel insight into the molecular basis of SK channels in the urinary bladder, demonstrating that the SK2 gene is expressed in the bladder and that it is essential for the ability of SK channels to regulate UBSM contractility.     

Allosteric linkage between voltage and Ca(2+)-dependent activation of BK-type mslo1 K(+) channels

The activation of BK type Ca(2+)-activated K(+) channels depends on both voltage and Ca(2+). We studied three point mutations in the putative voltage sensor S4 or S4-S5 linker regions in the mslo1 BK channels to explore the relationship between voltage and Ca(2+) in activating the channel. These mutations reduced the steepness of the open probability - voltage (P(o) - V) relation and increased the shift of the P(o) - V relations on the voltage axis in response to increases in the calcium concentration. It is striking that these two effects were reciprocally related for all three mutations, despite different effects of the mutations on other aspects of the voltage dependence of channel gating. This reciprocal relationship suggests strongly that the free energy contributions to channel activation provided by voltage and by calcium binding are simply additive. We conclude that the Ca(2+) binding sites and the voltage sensors do not directly interact. Rather they both affect the mslo1 channel opening through an allosteric mechanism, by influencing the conformational change between the closed and open conformations. The mutations changed the channel's voltage dependence with little effect on its Ca(2+) affinitiy.     

ATP regulation of type 1 inositol 1,4,5-trisphosphate receptor channel gating by allosteric tuning of Ca(2+) activation.

Inositol 1,4,5-trisphosphate (InsP(3)) mobilizes intracellular Ca(2+) by binding to its receptor (InsP(3)R), an endoplasmic reticulum-localized Ca(2+) release channel. Patch clamp electrophysiology of Xenopus oocyte nuclei was used to study the effects of cytoplasmic ATP concentration on the cytoplasmic Ca(2+) ([Ca(2+)](i)) dependence of single type 1 InsP(3)R channels in native endoplasmic reticulum membrane. Cytoplasmic ATP free-acid ([ATP](i)), but not the MgATP complex, activated gating of the InsP(3)-liganded InsP(3)R, by stabilizing open channel state(s) and destabilizing the closed state(s). Activation was associated with a reduction of the half-maximal activating [Ca(2+)](i) from 500 +/- 50 nM in 0 [ATP](i) to 29 +/- 4 nM in 9.5 mM [ATP](i), with apparent ATP affinity = 0.27 +/- 0.04 mM, similar to in vivo concentrations. In contrast, ATP was without effect on maximum open probability or the Hill coefficient for Ca(2+) activation. Thus, ATP enhances gating of the InsP(3)R by allosteric regulation of the Ca(2+) sensitivity of the Ca(2+) activation sites of the channel. By regulating the Ca(2+)-induced Ca(2+) release properties of the InsP(3)R, ATP may play an important role in shaping cytoplasmic Ca(2+) signals, possibly linking cell metabolic state to important Ca(2+)-dependent processes.     

Competitive and synergistic interactions of G protein beta(2) and Ca(2+) channel beta(1b) subunits with Ca(v)2.1 channels, revealed by mammalian two-hybrid and fluorescence resonance energy transfer measurements

Presynaptic Ca2+ channels are inhibited by metabotropic receptors. A possible mechanism for this inhibition is that G protein betagamma subunits modulate the binding of the Ca2+ channel beta subunit on the Ca2+ channel complex and induce a conformational state from which channel opening is more reluctant. To test this hypothesis, we analyzed the binding of Ca2+ channel beta and G protein beta subunits on the two separate binding sites, i.e. the loopI-II and the C terminus, and on the full-length P/Q-type alpha12.1 subunit by using a modified mammalian two-hybrid system and fluorescence resonance energy transfer (FRET) measurements. Analysis of the interactions on the isolated bindings sites revealed that the Ca2+ channel beta1b subunit induces a strong fluorescent signal when interacting with the loopI-II but not with the C terminus. In contrast, the G protein beta subunit induces FRET signals on both the C terminus and loopI-II. Analysis of the interactions on the full-length channel indicates that Ca2+ channel beta1b and G protein beta subunits bind to the alpha1 subunit at the same time. Coexpression of the G protein increases the FRET signal between alpha1/beta1b FRET pairs but not for alpha1/beta1b FRET pairs where the C terminus was deleted from the alpha1 subunit. The results suggest that the G protein alters the orientation and/or association between the Ca2+ channel beta and alpha12.1 subunits, which involves the C terminus of the alpha1 subunit and may corresponds to a new conformational state of the channel.