Pharmacological Characterization of the Native Store-Operated Calcium Channels of Cortical Neurons from Embryonic Mouse Brain

Abstract

In the murine brain, the first post-mitotic cortical neurons formed during embryogenesis express store-operated channels (SOCs) sensitive to Pyr3, initially proposed as a blocker of the transient receptor potential channel of C type 3 (TRPC3 channel). However, Pyr3 does not discriminate between Orai and TRPC3 channels, questioning the contribution of TRPC3 in SOCs. This study was undertaken to clarify the molecular identity and the pharmacological profile of native SOCs from E13 cortical neurons. The mRNA expression of STIM1-2 and Orai1-3 was assessed by quantitative reverse transcription polymerase chain reaction. E13 cortical neurons expressed STIM1-2 mRNAs, with STIM2 being the predominant isoform. Only transcripts of Orai2 were found but no Orai1 and Orai3 mRNAs. Blockers of Orai and TRPC channels (Pyr6, Pyr10, EVP4593, SAR7334, and GSK-7975A) were used to further characterize the endogenous SOCs. Their activity was recorded using the fluorescent Ca2+ probe Fluo-4. Cortical SOCs were sensitive to the Orai blockers Pyr6 and GSK-7975A, as well as to EVP4593, zinc, copper, and gadolinium ions, the latter one being the most potent SOCs blocker tested (IC50 ∼10 nM). SOCs were insensitive to the TRPC channel blockers Pyr10 and SAR7334. In addition, preventing mitochondrial Ca2+ uptake inhibited SOCs which were unaffected by inhibitors of the Ca2+-independent phospholipase A2. Altogether, Orai2 channels are present at the beginning of the embryonic murine cortico-genesis and form the core component of native SOCs in the immature cortex. This Ca2+ route is likely to play a role in the formation of the brain cortex.

Inhibition of store-operated calcium channels by N-arachidonoyl glycine (NAGly): no evidence for the involvement of lipid-sensing G protein coupled receptors

“…A subsequent re-admission of external Ca 2+ was followed by an intracellular elevation of Ca 2+ (open circles, Fig. 3A) 9,24 . This entry of Ca 2+ was sensitive to the CRAC channel blocker BTP2 45,46 (1 µM, gray up triangles, Fig.…”

“…primary cultures of cortical neurons. Cells were dissociated from cerebral cortices collected from embryonic (E13) mice (with the vaginal plug as E0) according to 9,23,24 . Briefly, tissues were placed in a 1.5 mL Eppendorf tube containing 1 mL of an ice-cold Ca 2+ -and Mg 2+ -free Hank's solution supplemented with 33 mM glucose, 4.2 mM NaHCO 3 , 10 mM HEPES, and 1% penicillin/streptomycin.…”

“…LPAand S1P-induced Ca 2+ responses were analyzed with Fluo-4. Cells were incubated with 5 μM Fluo-4/AM for 20 min following procedures described previously 23,24 . Images were obtained by a CCD CoolSnap HQ2 camera (Princeton Instruments, Roper Scientific, France) mounted on an inverted Zeiss A1 microscope (Carl Zeiss, France).…”

Ca2+signaling driving pacemaker activity in submucosal interstitial cells of Cajal in the colon

“…It could also be suggested that GSK7975A, a well-known antagonist for Orai1 (IC50 = 4 M; [104]), is less effective on Orai2, the dominant isoform expressed in ICC-SM. However, studies on cortical neurons that express only Orai2 showed about 50% block of SOCE by GSK7975A (5 M) [105].…”

Potential Drug Candidates to Treat TRPC6 Channel Deficiencies in the Pathophysiology of Alzheimer’s Disease and Brain Ischemia

“…SAR7334 and the tryptoline derivative 8009-5364 are reported to diminish acute hypoxia-induced pulmonary vasoconstriction and pulmonary arterial pressure in isolated mouse lungs [70,71]. SAR7334 has no effect on SOCE in primary cortical neurons [126].…”