The Ca2+ release-activated Ca2+ current (I(CRAC)) mediates store-operated Ca2+ entry in rat microglia.
Ca2+ signaling plays a central role in microglial activation, and several studies have demonstrated a store-operated Ca2+ entry (SOCE) pathway to supply this ion. Due to the rapid pace of discovery of novel Ca2+ permeable channels, and limited electrophysiological analyses of Ca2+ 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 Ca2+-release-activated Ca2+ (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 Ca2+ entry pathway that was reduced by depolarization and blocked by Gd3+, SKF-96365, diethylstilbestrol (DES), and a high concentration of 2-aminoethoxydiphenyl borate (50 microM 2-APB). The Fura-2 signal was increased by hyperpolarization, and by a low concentration of 2-APB (5 microM), 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 Ca2+ current with the same properties, including high selectivity for Ca2+ 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 microM 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.
Toronto Western Research Institute, University Health Network, Toronto, ON, CA.
Lily
L
Ohana
Evan W
EW
Newell
Elise F
EF
Stanley
Lyanne C
LC
Schlichter
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D004594:Electrophysiology; D020869:Gene Expression Profiling; D017136:Ion Transport; D017628:Microglia / Q000378:metabolism* / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats
D015220:Calcium Channels; D012333:RNA, Messenger; D002118:Calcium