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Density of functional Ca2+ release-activated Ca2+ (CRAC) channels declines after T-cell activation.

ABSTRACT: CRAC channel-mediated Ca(2+) entry plays a crucial role in T lymphocyte activation. Activated T cells display enhanced Ca(2+) signaling compared with resting T cells; this is partially attributed to activation-induced upregulation of CRAC channel expression. Orai and Stim family genes encode CRAC channel structural elements and regulatory proteins, respectively, but studies of their expression in T cells have led to controversial results. We re-examined Orai and Stim gene expression in resting, activated, and Jurkat T cells. Levels of Orai1 transcripts, encoding the human T cell CRAC channel subunit, were not significantly different between resting T and activated T cells. The total amount of all Orai transcripts was 2-fold higher in activated T cells than in resting T cells. Orai1 and total Orai transcript levels were significantly higher in Jurkat T cells than those in resting T cells. Stim expression did not vary significantly among cell types. Maximal whole-cell CRAC current amplitudes were 1.4-fold and 2.3-fold higher in activated and Jurkat T cells, respectively, than in resting T cells. Due to the small size of resting T cells, the surface CRAC channel density was 2.5-fold and 1.6-fold higher in resting T cells than in activated and Jurkat T cells, respectively. Predicted the rates of cytosolic Ca(2+) elevation calculated using the average values of CRAC channel currents and cell volumes showed that < 2-fold increase in the functional CRAC channel expression level cannot account for the enhanced rate of store-operated Ca(2+) entry in activated T cells compared with resting T cells.

SUBMITTER: Thakur P

PROVIDER: S-EPMC3265799 | biostudies-other | 2011 Nov-Dec

REPOSITORIES: biostudies-other

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Density of functional Ca2+ release-activated Ca2+ (CRAC) channels declines after T-cell activation.

Thakur Pratima P Fomina Alla F AF

Channels (Austin, Tex.) 20111101 6

CRAC channel-mediated Ca(2+) entry plays a crucial role in T lymphocyte activation. Activated T cells display enhanced Ca(2+) signaling compared with resting T cells; this is partially attributed to activation-induced upregulation of CRAC channel expression. Orai and Stim family genes encode CRAC channel structural elements and regulatory proteins, respectively, but studies of their expression in T cells have led to controversial results. We re-examined Orai and Stim gene expression in resting, ...[more]

PMID: 22172731

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Project description: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.

Project description:STUDY QUESTION:Are the transient receptor potential cation channels vanilloid 3 (TRPV3) present and able to mediate strontium (Sr2+) induced artificial activation in human oocytes? SUMMARY ANSWER:Sr2+ did not induce Ca2+ rises or provoke activation in human oocytes, however, mRNA for the TRPV3 channel was present in metaphase II (MII) human oocytes after IVM and TRPV3 agonists induced Ca2+ rises and oocyte activation, demonstrating the channels were functional. WHAT IS KNOWN ALREADY:Selective activation of TRPV3 by agonists induces Ca2+ entry and promotes mouse oocyte activation, and the absence of TRPV3 channels in mouse oocytes prevents Sr2+ mediated artificial activation. Sr2+ is sometimes used to overcome fertilization failure after ICSI in the clinic, but its efficiency is still controversial and the mechanism(s) of how it mediates the Ca2+ flux has not been studied yet in human. STUDY DESIGN SIZE DURATION:The protein distribution (n = 10) and mRNA expression level (n = 19) of the TRPV3 channels was investigated in human MII oocytes after IVM. The Sr2+ (10 mM) and TRPV3 agonists (200 ?M 2-aminoethoxydiphenyl borate [2-APB] and 200 ?M carvacrol)-induced Ca2+ response was analyzed in human (n = 15, n = 16 and n = 16, respectively) and mouse oocytes (n = 15, n = 19 and n = 26, respectively). The subsequent embryonic developmental potential following the parthenogenetic activation using these three agents was recorded in human (n = 10, n = 9 and n = 9, respectively) and mouse (n = 20 per agent) oocytes, by determining pronucleus, or 2-cell and blastocyst formation rates. PARTICIPANTS/MATERIALS SETTING METHODS:MII oocytes from B6D2F1 mice (6-10 weeks old) as well as human IVM oocytes and IVO oocytes (from patients aged 25-38 years old) with aggregates of smooth endoplasmic reticulum clusters were used. The expression of TRPV3 channels was determined by immunofluorescence staining with confocal microscopy and RT-PCR, and the temporal evolution of intracellular Ca2+ concentration was measured by time-lapse imaging after exposure to Sr2+ and TRPV3 agonists (2-APB and carvacrol). Artificial activation efficiency was assessed using these agents. MAIN RESULTS AND THE ROLE OF CHANCE:Sr2+ did not promote Ca2+ oscillations or provoke activation in human oocytes. Transcripts of TRPV3 channels were present in IVM MII human oocytes. TRPV3 protein was expressed and distributed throughout the ooplasm of human oocytes, rather than particularly concentrated in plasma membrane as observed in mouse MII oocytes. Both agonists of TRPV3 (2-APB and carvacrol), promoted a single Ca2+ transient and activated a comparable percentage of more than half of the exposed human oocytes (P > 0.05). The agonist 2-APB was also efficient in activating mouse oocytes, however, significantly fewer mouse oocytes responded to carvacrol than 2-APB in both the Ca2+ analysis and activation test (P < 0.001). LIMITATIONS REASONS FOR CAUTION:The availability of fresh IVO matured oocytes in human was limited. Data from TRPV3 knockout model are not included. WIDER IMPLICATIONS OF THE FINDINGS:The benefit of clinical application using Sr2+ to overcome fertilization failure after ICSI requires further validation. STUDY FUNDING/COMPETING INTERESTS:This study was supported by FWO-Vlaanderen, China Scholarship Council and Special Research Fund from Ghent University (Bijzonder Onderzoeksfonds, BOF). No competing interests are declared.

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Density of functional Ca2+ release-activated Ca2+ (CRAC) channels declines after T-cell activation.

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Density of functional Ca2+ release-activated Ca2+ (CRAC) channels declines after T-cell activation.

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