Pool Of STIM1 Research Articles

Complex Function of Stim1 in the Activation of Store-Independent Orai Channels

Orai proteins contribute to Ca2+ entry pathways through store-dependent, Ca2+ release-activated Ca2+ (CRAC) channels (Orai1), and store-independent, arachidonic acid (AA)-regulated Ca2+ (ARC) or LeukotrieneC4-regulated Ca2+ (LRC) channels (Orai1 and Orai3). Remarkably, although activated by fundamentally different mechanisms, both CRAC and ARC/LRC channels share a requirement for STIM1 expression. To date the role of endoplasmic reticulum-resident STIM1 (ER-STIM1) in the activation of CRAC channels is well appreciated. There is a minor pool of STIM1 at the plasma membrane (PM-STIM1) that was shown to be necessary for ARC current activation in HEK293 cells. Using pharmacological tools targeting AA synthesis and metabolism, Ca2+ imaging, whole-cell and perforated patch clamp electrophysiological recordings we demonstrate that both Orai1 and Orai3 are required for ARC and LRC current activation in both primary vascular smooth muscle cells (VSMCs) and HEK293 cells. Surprisingly, while PM-STIM1 is required for ARC and LRC current activation under whole cell patch clamp recordings in both cell types, ER-STIM1 is sufficient for both ARC and LRC channel activation when intact cells are considered. These results are first to demonstrate ARC channel function in primary VSMCs, highlight the complexity of STIM1 regulation of store-independent Orai channels and demonstrate that ARC and LRC currents are mediated by the same channels.

A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels, an arachidonate-regulated store-independent Orai channel

The Orai family of calcium channels includes the store-operated CRAC channels and store-independent, arachidonic acid (AA)-regulated ARC channels. Both depend on STIM1 for their activation but, whereas CRAC channel activation involves sensing the depletion of intracellular calcium stores via a luminal N terminal EF-hand of STIM1 in the endoplasmic reticulum (ER) membrane, ARC channels are exclusively activated by the pool of STIM1 that constitutively resides in the plasma membrane (PM). Here, the EF-hand is extracellular and unlikely to ever lose its bound calcium, suggesting that STIM1-dependent activation of ARC channels is very different from that of CRAC channels. We now show that attachment of the cytosolic portion of STIM1 to the inner face of the PM via an N terminal Lck-domain sequence is sufficient to enable normal AA-dependent activation of ARC channels, while failing to allow activation of store-operated CRAC channels. Introduction of a point mutation within the Lck-domain resulted in the loss of both PM localization and ARC channel activation. Reversing the orientation of the PM-anchored STIM1 C terminus via a C-terminal CAAX-box fails to support either CRAC or ARC channel activation. Finally, the Lck-anchored STIM1 C-terminal domain also enabled the exclusive activation of the ARC channels following physiological agonist addition. These data demonstrate that simple tethering of the cytosolic C-terminal domain of STIM1 to the inner face of the PM is sufficient to allow the full, normal and exclusive activation of ARC channels, and that the N-terminal regions of STIM1 (including the EF-hand domain) play no significant role in this activation.

STIM/Orai signalling complexes in vascular smooth muscle

Stromal interaction molecules (STIM1 and STIM2) are single pass transmembrane proteins located mainly in the endoplasmic reticulum (ER). STIM proteins contain an EF-hand in their N-termini that faces the lumen side of the ER allowing them to act as ER calcium (Ca(2+)) sensors. STIM1 has been recognized as central to the activation of the highly Ca(2+) selective store-operated Ca(2+) (SOC) entry current mediated by the Ca(2+) release-activated Ca(2+) (CRAC) channel; CRAC channels are formed by tetramers of the plasma membrane (PM) protein Orai1. Physiologically, the production of inositol 1,4,5-trisphosphate (IP(3)) upon stimulation of phospholipase C-coupled receptors and the subsequent emptying of IP(3)-sensitive ER Ca(2+) stores are sensed by STIM1 molecules which aggregate and move closer to the PM to interact physically with Orai1 channels and activate Ca(2+) entry. Orai1 has two homologous proteins encoded by separate genes, Orai2 and Orai3. Other modes of receptor-regulated Ca(2+) entry into cells are store-independent; for example, arachidonic acid activates a highly Ca(2+) selective store-independent channel formed by heteropentamers of Orai1 and Orai3 and regulated by the PM pool of STIM1. Here, I will discuss results pertaining to the roles of STIM and Orai proteins in smooth muscle Ca(2+) entry pathways and their role in vascular remodelling.

Activation of ARC Channels, a Noncapacitative Orai Channel, is Independent of the N-Terminal Domains of STIM1

The Orai family of Ca2+ channels includes the store-operated CRAC channels and store-independent, arachidonic acid (AA)-activated ARC channels. Although both channels are dependent on STIM1 for their activation, they differ in the pool of STIM1 responsible. Physiologically, CRAC channel activation relies on a loss of Ca2+ from a luminal N-terminal EF-hand domain of STIM1 resident in the ER membrane. However, ARC channels are exclusively regulated by the pool of STIM1 that is constitutively located in the plasma membrane (PM). Here, the EF-hand is extracellular and is unlikely to ever lose its bound Ca2+ - suggesting that STIM1-dependent activation of the ARC channels is very different from that of the CRAC channels.Expression of just the cytosolic C-terminal region of STIM1 in HEK293 cells resulted in the complete loss of significant CRAC or ARC channel currents following store-depletion or AA addition, respectively. However, attachment of the same STIM1 C-terminus to the inner face of the PM via an N-terminal sequence based on the SH4 domain of Lck, resulted in the full restoration of normal AA-activated ARC channel currents. Store-operated CRAC channel currents, however, were not restored - an effect confirmed in RBL cells, whose endogenous CRAC currents are ∼4-5 times larger than those in HEK293 cells. Finally, introduction of a point mutation within the Lck sequence that blocks myristoylation/palmitoylation of this domain, with the consequent loss of PM localization, resulted in the failure of effective ARC channel activation. These data indicate that N-terminal regions of STIM1 (including the EF-hand and SAM domains) play no significant role in AA-dependent activation of the ARC channels, and that the simple location of the C-terminal domain to the cytosolic face of the PM is all that is required.

STIM and Orai proteins and the non-capacitative ARC channels

The ARC channel is a small conductance, highly Ca²⁺-selective ion channel whose activation is specifically dependent on low concentrations of arachidonic acid acting at an intracellular site. They are widely distributed in diverse cell types where they provide an alternative, store-independent pathway for agonist-activated Ca²⁺ entry. Although biophysically similar to the store-operated CRAC channels, these two conductances function under distinct conditions of agonist stimulation, with the ARC channels providing the predominant route of Ca²⁺ entry during the oscillatory signals generated at low agonist concentrations. Despite these differences in function, like the CRAC channel, activation of the ARC channels is dependent on STIM1, but it is the pool of STIM1 that constitutively resides in the plasma membrane that is responsible. Similarly, both channels are formed by Orai proteins but, whilst the CRAC channel pore is a tetrameric assembly of Orai1 subunits, the ARC channel pore is formed by a heteropentameric assembly of three Orai1 subunits and two Orai3 subunits. There is increasing evidence that the activity of these channels plays a critical role in a variety of different cellular activities.

Orai3 and the Selective Activation of the Arc Channel by Arachidonic Acid

The store-operated CRAC channels and the store-independent, arachidonic acid-activated ARC channels represent the founding members of a new family of biophysically similar, highly Ca2+-selective, Ca2+ entry channels - the “Orai channels”. Both of these channels are dependent on STIM1 for their activation, but they differ in the pool of STIM1 responsible. Thus, whereas STIM1 in the ER regulates the CRAC channels on store-depletion, ARC channels are exclusively regulated by the pool of STIM1 that constitutively resides in the PM.Recent studies have shown that the functional CRAC channel pore is formed by a tetrameric arrangement of Orai1 units. In contrast, a heteropentameric assembly of three Orai1 subunits and two Orai3 subunits forms the functional ARC channel pore (Mignen et al. J. Physiol. 587: 4181). Importantly, this inclusion of Orai3 subunits in the channel structure has been shown to play a specific, and unique, role in determining the selectivity of the ARC channels for activation by arachidonic acid. Using an approach based on the generation and expression of various concatenated constructs, we examined the basis for this Orai3-dependent effect on selectivity for arachidonic acid. These studies revealed that, whilst heteropentamers containing only one Orai3 subunit are sensitive to arachidonic acid, specific selectivity for activation by this fatty acid is only achieved on inclusion of the second Orai3 subunit in the pentamer. Further studies identified the cytosolic N-terminal domain as the region of the Orai3 molecule that is specifically responsible for this switch in selectivity. Substitution of just this domain into an otherwise complete Orai1 subunit within a concatenated 31111 pentamer is sufficient to change the resulting channel from one that is predominantly store-operated, to one that is essentially exclusively activated by arachidonic acid.

STIM1 and the noncapacitative ARC channels

Our understanding of the nature and regulation of receptor-activated Ca 2+ entry in nonexcitable cells has recently undergone a radical change that began with the identification of the stromal interacting molecule proteins (e.g., STIM1) as playing a critical role in the regulation of the capacitative, or store-operated, Ca 2+ entry. As such, current models emphasize the role of STIM1 located in the endoplasmic reticulum membrane, where it senses the status of the intracellular Ca 2+ stores via a luminal N-terminal Ca 2+-binding EF-hand domain. Dissociation of Ca 2+ from this domain induces the clustering of STIM1 to regions of the ER that lie close to the plasma membrane, where it regulates the activity of the store-operated Ca 2+ channels (e.g., CRAC channels). Thus, the specific dependence on store-depletion, and the role of the Ca 2+-binding EF-hand domain in this process, are critical to all current models of the action of STIM1 on Ca 2+ entry. However, until recently, the effects of STIM1 on other modes of receptor-activated Ca 2+ entry have not been examined. Surprisingly, we found that STIM1 exerts similar, although not identical, actions on the arachidonic acid-regulated Ca 2+-selective (ARC) channels—a widely expressed mode of agonist-activated Ca 2+ entry whose activation is completely independent of Ca 2+ store depletion. Regulation of the ARC channels by STIM1 is not only independent of store depletion, but also of the Ca 2+-binding function of the EF-hand, and translocation of STIM1 to the plasma membrane. Instead, it is the pool of STIM1 that constitutively resides in the plasma membrane that is critical for the regulation of the ARC channels. Thus, ARC channel activity is selectively inhibited by exposure of intact cells to an antibody targeting the extracellular N-terminal domain of STIM1. Similarly, introducing mutations in STIM1 that prevent the N-linked glycosylation-dependent constitutive expression of the protein in the plasma membrane specifically inhibits the activity of the ARC channels without affecting the CRAC channels. These studies demonstrate that STIM1 is a far more universal regulator of Ca 2+ entry pathways than previously assumed, and has multiple, and entirely distinct, modes of action. Precisely how this same protein can act in such separate and specific ways on these different pathways of agonist-activated Ca 2+entry remains an intriguing, yet currently unresolved, question.