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

Abstract

The means by which Ca(2+) store depletion evokes the opening of store-operated Ca(2+) channels (SOCs) in the plasma membrane of excitable and non-excitable cells has been a longstanding mystery. Indirect evidence has supported local interactions between the ER and SOCs as well as long-range interactions mediated through a diffusible activator. The recent molecular identification of the ER Ca(2+) sensor (STIM1) and a subunit of the CRAC channel (Orai1), a prototypic SOC, has now made it possible to visualize directly the sequence of events that links store depletion to CRAC channel opening. Following store depletion, STIM1 moves from locations throughout the ER to accumulate in ER subregions positioned within 10-25nm of the plasma membrane. Simultaneously, Orai1 gathers at discrete sites in the plasma membrane directly opposite STIM1, resulting in local CRAC channel activation. These new studies define the elementary units of store-operated Ca(2+) entry, and reveal an unprecedented mechanism for channel activation in which the stimulus brings a channel and its activator/sensor together for interaction across apposed membrane compartments. We discuss the implications of this choreographic mechanism with regard to Ca(2+) dynamics, specificity of Ca(2+) signaling, and the existence of a specialized ER subset dedicated to the control of the CRAC channel.

Figure 1

Redistribution of STIM1 following store depletion. Two Jurkat cells expressing GFP-STIM1 were imaged by wide-field epifluorescence microscopy (A) or TIRF microscopy (B) at rest and after treatment with 8 μM cyclopiazonic acid (a SERCA inhibitor) for > 2 min (store depleted). After store depletion, STIM1 moves from being diffusely distributed throughout the ER to accumulate in junctional ER located close to the PM. Scale bars, 5 μm.

Figure 2

The elementary unit of store-operated Ca²⁺ entry. Electron micrograph and TIRF images of individual Jurkat cells treated with TG to deplete stores and exposed to cytochalasin D to disperse puncta. (A) HRP-STIM1 accumulates in ER tubules (arrows) located 10-25 nm from the plasma membrane (pm). (B) TIRF images of Cherry-STIM1 fluorescence at the cell footprint (left) and at a single punctum within the boxed area. Ca²⁺ influx (middle) at the same punctum is expressed as pseudocolored ΔF/F₀ ratios from minimum (blue; −0.01) to maximum (red; 1.05) levels. Pseudocolored contour lines of Ca²⁺ influx density overlaid on the Cherry-STIM1 image (right) show a close association between the Ca²⁺ influx site and the STIM1 punctum. (C) TIRF images of Cherry-STIM1 fluorescence at the cell footprint (left) and at a single punctum within the boxed area. Cherry-STIM1 (red) and GFP-myc-Orai1 (green) colocalize (merged image). Reproduced from [59]. Copyright 2006 Rockefeller University Press.

Figure 3

A current model for store-operated Ca²⁺ entry. (A) In cells with full ER Ca²⁺ stores, Ca²⁺-bound STIM1 and Orai1 are distributed throughout the endoplasmic reticulum and the PM, respectively. (B) Store depletion promotes functional coupling of Ca²⁺-free STIM1 and Orai1 by driving the coordinated redistribution of both proteins to closely apposed sites in the ER and PM, and by increasing the number of these sites. (C) The parallel accumulation of both proteins allows STIM1 and Orai1 to interact, causing the local activation of CRAC channels at individual junctions. Reproduced from [53].