Visualization of localized store-operated calcium entry in mouse astrocytes. Close proximity to the endoplasmic reticulum.
Unloading of endoplasmic reticulum (ER) Ca(2+) stores activates influx of extracellular Ca(2+) through 'store-operated' Ca(2+) 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 Ca(2+) stores. Here, using high resolution imaging with the membrane-associated Ca(2+) indicator, FFP-18, it is shown that store-operated Ca(2+) entry (SOCE) in primary cultured mouse cortical astrocytes occurs at plasma membrane-ER junctions. In the absence of extracellular Ca(2+), depletion of ER Ca(2+) stores using cyclopiazonic acid, an ER Ca(2+)-ATPase inhibitor, and caffeine transiently increases the sub-plasma-membrane Ca(2+) concentration ([Ca(2+)](SPM)) within a restricted space between the plasma membrane and adjacent ER. Restoration of extracellular Ca(2+) causes localized Ca(2+) influx that first increases [Ca(2+)](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, Ca(2+) entry through TRPC-encoded SOCs is closely linked, not only functionally, but also structurally, to the ER Ca(2+) stores.
Golovina
Vera A
VA
Department of Physiology, University of Maryland School of Medicine, 685 W. Baltimore Street, Baltimore, MD 21201, USA. vgolovin@umaryland.edu
D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence / Q000379:methods*; D050052:TRPC Cation Channels
D015220:Calcium Channels; D005456:Fluorescent Dyes; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D002118:Calcium; D016257:Fura-2
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