Visualization of localized store-operated calcium entry in mouse astrocytes. Close proximity to the endoplasmic reticulum

Unloading of endoplasmic reticulum (ER) Ca2+ stores activates influx of extracellular Ca2+ through 'store-operated' Ca2+ channels (SOCs) in the plasma membrane (PM) of most cells, including astrocytes. A key unresolved issue concerning SOC function is their spatial relationship to ER Ca2+ stores. Here, using high resolution imaging with the membrane-associated Ca2+ indicator, FFP- 18, it is shown that store-operated Ca2+ entry (SOCE) in primary cultured mouse cortical astrocytes occurs at plasma membrane-ER junctions. In the absence of extracellular Ca2+, depletion of ER Ca2+ stores using cyclopiazonic acid, an ER Ca2+ -ATPase inhibitor, and caffeine transiently increases the sub-plasma-membrane Ca2+ concentration ([Ca2+](SPM)) within a restricted space between the plasma membrane and adjacent ER. Restoration of extracellular Ca2+ causes localized Ca2+ influx that first increases [Ca2+](SPM) in the same restricted regions and then, with a delay, in ER-free regions. Antisense knockdown of the TRPC1 gene, proposed to encode endogenous SOCs, markedly reduces SOCE measured with Fura-2. High resolution immunocytochemistry with anti-TRPC1 antibody reveals that these TRPC-encoded SOCs are confined to the PM microdomains adjacent to the underlying 'junctional' ER. Thus, Ca2+ entry through TRPC-encoded SOCs is closely linked, not only functionally, but also structurally, to the ER Ca2+ stores.