The effects of the IP3-receptor antagonist 2-aminoethyldiphenyl borate (2-APB) on the Ca2+ releas... more The effects of the IP3-receptor antagonist 2-aminoethyldiphenyl borate (2-APB) on the Ca2+ release-activated Ca2+ current (ICRAC) in Jurkat human T cells, DT40 chicken B cells and rat basophilic leukaemia (RBL) cells were examined.2-APB elicited both stimulatory and inhibitory effects on Ca2+ influx through CRAC channels. At concentrations of 1–5 μm, 2-APB enhanced Ca2+ entry in intact cells and increased ICRAC amplitude by up to fivefold. At levels ≥ 10 μm, 2-APB caused a transient enhancement of ICRAC followed by inhibition.2-APB altered the kinetics of fast Ca2+-dependent inactivation of ICRAC. At concentrations of 1–5 μm, 2-APB increased the rate of fast inactivation. In contrast, 2-APB at higher concentrations (≥ 10 μm) reduced or completely blocked inactivation.2-APB inhibited Ca2+ efflux from mitochondria.2-APB inhibited ICRAC more potently when applied extracellularly than intracellularly. Furthermore, increased protonation of 2-APB at low pH did not affect potentiation or inhibition. Thus, 2-APB may have an extracellular site of action.Neither ICRAC activation by passive store depletion nor the effects of 2-APB were altered by intracellular dialysis with 500 μg ml−1 heparin.ICRAC is present in wild-type as well as mutant DT40 B cells lacking all three IP3 receptor isoforms. 2-APB also potentiates and inhibits ICRAC in both cell types, indicating that 2-APB exerts its effects independently of IP3 receptors.Our results show that CRAC channel activation does not require physical interaction with IP3 receptors as proposed in the conformational coupling model. Potentiation of ICRAC by 2-APB may be a useful diagnostic feature for positive identification of putative CRAC channel genes, and provides a novel tool for exploring the physiological functions of store-operated channels.
Store-operated Ca2+ release-activated Ca2+ (CRAC) channels are a widespread mechanism for generat... more Store-operated Ca2+ release-activated Ca2+ (CRAC) channels are a widespread mechanism for generating cellular Ca2+ signals and regulate many Ca2+-dependent functions, including transcription, motility and proliferation. The opening of CRAC channels in response to depletion of intracellular Ca2+ stores involves a cascade of cellular events that culminate in direct interactions between STIM1, the endoplasmic reticulum Ca2+ sensor, and the channels composed of Orai proteins. Evidence gathered over the last two decades indicates that CRAC channels display a unique functional pore fingerprint characterized by exquisite Ca2+ selectivity, low unitary conductance, and low permeability to large cations. Here, we review the key pore properties of CRAC channels and discuss recent progress in addressing the molecular foundations of these properties. Structure–function and cysteine-scanning studies have revealed the identity and organization of pore-lining residues, including those that form the selectivity filter, providing a structural framework for understanding CRAC channel pore properties. Recent studies in pore mutants that produce STIM1-independent constitutive channel activation indicate that exquisite Ca2+ selectivity in CRAC channels is not hardwired into Orai proteins, but is instead manifested only following the binding of STIM1 to the intrinsically poorly Ca2+-selective Orai channels. These findings reveal new functional aspects of CRAC channels and suggest that the selectivity filter of the CRAC channel is a dynamic structure whose conformation and functional properties are powerfully regulated by the channel activation stimulus.
Astrocytes contribute to brain inflammation in neurological disorders but the molecular mechanism... more Astrocytes contribute to brain inflammation in neurological disorders but the molecular mechanisms controlling astrocyte reactivity and their relationship to neuroinflammatory endpoints are complex and poorly understood. In this study, we assessed the role of the calcium channel, Orai1, for astrocyte reactivity and inflammation-evoked depression behaviors in mice. Transcriptomics and metabolomics analysis indicated that deletion of Orai1 in astrocytes downregulates genes in inflammation and immunity, metabolism, and cell cycle pathways, and reduces cellular metabolites and ATP production. Systemic inflammation by peripheral lipopolysaccharide (LPS) increases hippocampal inflammatory markers in WT but not in astrocyte Orai1 knockout mice. Loss of Orai1 also blunts inflammation-induced astrocyte Ca2+ signaling and inhibitory neurotransmission in the hippocampus. In line with these cellular changes, Orai1 knockout mice showed amelioration of LPS-evoked depression-like behaviors includi...
The effects of the IP3-receptor antagonist 2-aminoethyldiphenyl borate (2-APB) on the Ca2+ releas... more The effects of the IP3-receptor antagonist 2-aminoethyldiphenyl borate (2-APB) on the Ca2+ release-activated Ca2+ current (ICRAC) in Jurkat human T cells, DT40 chicken B cells and rat basophilic leukaemia (RBL) cells were examined.2-APB elicited both stimulatory and inhibitory effects on Ca2+ influx through CRAC channels. At concentrations of 1–5 μm, 2-APB enhanced Ca2+ entry in intact cells and increased ICRAC amplitude by up to fivefold. At levels ≥ 10 μm, 2-APB caused a transient enhancement of ICRAC followed by inhibition.2-APB altered the kinetics of fast Ca2+-dependent inactivation of ICRAC. At concentrations of 1–5 μm, 2-APB increased the rate of fast inactivation. In contrast, 2-APB at higher concentrations (≥ 10 μm) reduced or completely blocked inactivation.2-APB inhibited Ca2+ efflux from mitochondria.2-APB inhibited ICRAC more potently when applied extracellularly than intracellularly. Furthermore, increased protonation of 2-APB at low pH did not affect potentiation or inhibition. Thus, 2-APB may have an extracellular site of action.Neither ICRAC activation by passive store depletion nor the effects of 2-APB were altered by intracellular dialysis with 500 μg ml−1 heparin.ICRAC is present in wild-type as well as mutant DT40 B cells lacking all three IP3 receptor isoforms. 2-APB also potentiates and inhibits ICRAC in both cell types, indicating that 2-APB exerts its effects independently of IP3 receptors.Our results show that CRAC channel activation does not require physical interaction with IP3 receptors as proposed in the conformational coupling model. Potentiation of ICRAC by 2-APB may be a useful diagnostic feature for positive identification of putative CRAC channel genes, and provides a novel tool for exploring the physiological functions of store-operated channels.
Store-operated Ca2+ release-activated Ca2+ (CRAC) channels are a widespread mechanism for generat... more Store-operated Ca2+ release-activated Ca2+ (CRAC) channels are a widespread mechanism for generating cellular Ca2+ signals and regulate many Ca2+-dependent functions, including transcription, motility and proliferation. The opening of CRAC channels in response to depletion of intracellular Ca2+ stores involves a cascade of cellular events that culminate in direct interactions between STIM1, the endoplasmic reticulum Ca2+ sensor, and the channels composed of Orai proteins. Evidence gathered over the last two decades indicates that CRAC channels display a unique functional pore fingerprint characterized by exquisite Ca2+ selectivity, low unitary conductance, and low permeability to large cations. Here, we review the key pore properties of CRAC channels and discuss recent progress in addressing the molecular foundations of these properties. Structure–function and cysteine-scanning studies have revealed the identity and organization of pore-lining residues, including those that form the selectivity filter, providing a structural framework for understanding CRAC channel pore properties. Recent studies in pore mutants that produce STIM1-independent constitutive channel activation indicate that exquisite Ca2+ selectivity in CRAC channels is not hardwired into Orai proteins, but is instead manifested only following the binding of STIM1 to the intrinsically poorly Ca2+-selective Orai channels. These findings reveal new functional aspects of CRAC channels and suggest that the selectivity filter of the CRAC channel is a dynamic structure whose conformation and functional properties are powerfully regulated by the channel activation stimulus.
Astrocytes contribute to brain inflammation in neurological disorders but the molecular mechanism... more Astrocytes contribute to brain inflammation in neurological disorders but the molecular mechanisms controlling astrocyte reactivity and their relationship to neuroinflammatory endpoints are complex and poorly understood. In this study, we assessed the role of the calcium channel, Orai1, for astrocyte reactivity and inflammation-evoked depression behaviors in mice. Transcriptomics and metabolomics analysis indicated that deletion of Orai1 in astrocytes downregulates genes in inflammation and immunity, metabolism, and cell cycle pathways, and reduces cellular metabolites and ATP production. Systemic inflammation by peripheral lipopolysaccharide (LPS) increases hippocampal inflammatory markers in WT but not in astrocyte Orai1 knockout mice. Loss of Orai1 also blunts inflammation-induced astrocyte Ca2+ signaling and inhibitory neurotransmission in the hippocampus. In line with these cellular changes, Orai1 knockout mice showed amelioration of LPS-evoked depression-like behaviors includi...
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Papers by Murali Prakriya