Calcium release and influx colocalize to the endoplasmic reticulum

Abstract

Intracellular Ca2+ is released from intracellular stores in the endoplasmic reticulum (ER) in response to the second messenger inositol (1,4,5) trisphosphate (InsP3) [1,2]. Then, a poorly understood cellular mechanism, termed capacitative Ca2+ entry, is activated [3,4]; this permits Ca2+ to enter cells through Ca(2+)-selective Ca(2+)-release-activated ion channels [5,6] as well as through less selective store-operated channels [7]. The level of stored Ca2+ is sensed by Ca(2+)-permeant channels in the plasma membrane, but the identity of these channels, and the link between them and Ca2+ stores, remain unknown. It has been argued that either a diffusible second messenger (Ca2+ influx factor; CIF) [8] or a physical link [9,10] connects the ER Ca(2+)-release channel and store-operated channels; strong evidence for either mechanism is lacking, however [7,10]. Petersen and Berridge [11] showed that activation of the lysophosphatidic acid receptor in a restricted region of the oocyte membrane results in stimulation of Ca2+ influx only in that region, and concluded that a diffusible messenger was unlikely. To investigate the relationship between ER stores and Ca2+ influx, we used centrifugation to redistribute into specific layers the organelles inside intact Xenopus laevis oocytes, and used laser scanning confocal microscopy with the two-photon technique to 'uncage' InsP3 while recording intracellular Ca2+ concentration. Ca2+ release was localized to the stratified ER layer and Ca2+ entry to regions of the membrane directly adjacent to this layer. We conclude that Ca2+ depletion and entry colocalize to the ER and that the mechanism linking Ca2+ stores to Ca2+ entry is similarly locally constrained.

Calcium influx factor is synthesized by yeast and mammalian cells depleted of organellar calcium stores

“…Fig. 3A shows a photomicrograph of the centrifuged oocytes, in which [Ca 2ϩ ] i increases in response to IP 3 and thapsigargin were previously localized to the portion of the oocyte enriched for the ER (17,30). Our observations also showed maximal [Ca 2ϩ ] i increases near this band and were faithfully mimicked by a model based on a diffusible CIF produced only by the ER-enriched band (A.B., P.C., R.B.M., and J.E.K., manuscript in preparation).…”

Calcium Influx Factor Directly Activates Store-operated Cation Channels in Vascular Smooth Muscle Cells

“…Strong arguments against a CIF-based model of SOC activation have been previously raised by some experimental data that were apparently inconsistent with a freely diffusible and long lived messenger traveling from Ca 2ϩ stores to the plasma membranes (19,25,26). Other recent data have provided compelling evidence for the importance of secretory processes and vesicle docking and/or membrane fusion in the activation of store-operated Ca 2ϩ influx (19,27).…”

Some assembly required: Constructing the elementary units of store-operated Ca2+ entry

“…The ability of intracellular BAPTA to slow reloading, and of Ba 2+ and Mn 2+ , which are not transported by Ca 2+ pumps, to enter the cytosolic pool following store depletion, was taken as evidence that Ca 2+ entering the cell traverses a narrow gap of cytosol before being actively pumped into the ER [17]. Later studies of Xenopus oocytes, endothelial cells and astrocytes showed that local receptor stimulation evoked Ca 2+ entry (as detected by Cl − channel activation or Ca 2+ -dependent dye signals) that was confined to within 10's to 100's of μm from where Ca 2+ release was thought to occur [18][19][20][21].…”

“…The only available marker for active CRAC channels is the elevated [Ca 2+ ] i microdomains that form near open channels. Several prior studies using Ca 2+ -sensitive dyes have shown that SOCE occurs within 10-100 μM of the ER [19][20][21], but the applied imaging techniques lacked sufficient resolution to pinpoint the relationship between individual influx sites and junctional ER structures. Much higher resolution is possible with TIRF microscopy, which has been used to image Ca 2+ microdomains near voltage-gated Ca 2+ (Ca V ) channels on a submicron scale [67,68].…”