The Ca²⁺release-activated Ca²⁺current (I_CRAC) mediates store-operated Ca²⁺entry in rat microglia

Abstract

Channels 129 I CRAC mediates SOCE in microglia [Channels 3:2, 129-139; March/April 2009]; ©2009 Landes BioscienceCa 2+ signaling plays a central role in microglial activation, and several studies have demonstrated a store-operated Ca 2+ entry (SOCE) pathway to supply this ion. Due to the rapid pace of discovery of novel Ca 2+ permeable channels, and limited electrophysiological analyses of Ca 2+ currents in microglia, characterization of the SOCE channels remains incomplete. At present, the prime candidates are 'transient receptor potential' (TRP) channels and the recently cloned Orai1, which produces a Ca 2+ -release-activated Ca 2+ (CRAC) current. We used cultured rat microglia and real-time RT-PCR to compare expression levels of Orai1, Orai2, Orai3, TRPM2, TRPM7, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 channel genes. Next, we used Fura-2 imaging to identify a store-operated Ca 2+ entry pathway that was reduced by depolarization and blocked by Gd 3+ , SKF-96365, diethylstilbestrol (DES), and a high concentration of 2-aminoethoxydiphenyl borate (50 μM 2-APB). The Fura-2 signal was increased by hyperpolarization, and by a low concentration of 2-APB (5 μM), and exhibited Ca 2+ -dependent potentiation. These properties are entirely consistent with Orai1/CRAC, rather than any known TRP channel and this conclusion was supported by patch-clamp electrophysiological analysis. We identified a store-operated Ca 2+ current with the same properties, including high selectivity for Ca 2+ over monovalent cations, pronounced inward rectification and a very positive reversal potential, Ca 2+ -dependent current potentiation, and block by SKF-96365, DES and 50 μM 2-APB. Determining the contribution of Orai1/CRAC in different cell types is crucial to future mechanistic and therapeutic studies; this comprehensive multi-strategy analysis demonstrates that Orai1/CRAC channels are responsible for SOCE in primary microglia.

Physiological and Pathophysiological Roles of Transient Receptor Potential Channels in Microglia-Related CNS Inflammatory Diseases

“…Quantitative comparisons of mRNA expression have shown that TRPC1, TRPC3, and TRPC6 are highly expressed in cultured microglia. 17) In line with this finding, double-label immunofluorescence studies show that TRPC3 and TRPC6 are weakly localized in microglia in vivo. 18,19) Recently, Liu et al reported that amyloid β-protein (Aβ) upregulates TRPC6 via nuclear factorkappaB (NF-κB) and promotes the production of cyclooxygenase-2 (COX-2) in BV-2 microglia, which could be involved in microglial activation-induced hippocampus neuronal damage.…”

Physiology of Microglia

“…The qRT-PCR performed on neonatal cultured neonatal rat microglia revealed the following rank of mRNAs expression: (671). The TRPM7 expression was very high and comparable with expression of housekeeping gene HPRT-1 (671).…”

“…The Na ϩ currents through store-operated channels demonstrated very slow activation kinetics; the unitary channel conductance was 42.5 pS, and the channels were positively modulated by PKA and negatively by PKC (344). Likely, these currents reflected the activation of TRPM7 channels (671).…”

“…The qRT-PCR revealed expression of all three Orai mRNAs with maximal expression of Orai3 (671). Further electrophysiological and pharmacological investigations allowed the authors to suggest that highly Ca 2ϩ -selective (P Ca /P Na Ͼ1,000) Orai1/CRAC channels are responsible for I CRAC generation in microglia (671).…”

Inhibition of the Ca2+-Dependent K+Channel,KCNN4/KCa3.1, Improves Tissue Protection and Locomotor Recovery after Spinal Cord Injury

“…Fura-2 signaling was used to monitor intracellular Ca 2ϩ (Ca 2ϩ i ), according to our standard procedures (Ohana et al, 2009). Astrocytes were loaded for ϳ45 min at room temperature with 3.5 g/ml Fura-2-AM (Invitrogen) diluted in standard bath solution containing the following (in mM): 135 NaCl, 5 KCl, 1 MgCl 2 , 1 CaCl 2 , 5 glucose, 10 HEPES, adjusted to pH 7.4 with NaOH, and to ϳ300 mOsm with sucrose.…”