The Role of Lipids in CRAC Channel Function

Abstract

The composition and dynamics of the lipid membrane define the physical properties of the bilayer and consequently affect the function of the incorporated membrane transporters, which also applies for the prominent Ca²⁺ release-activated Ca²⁺ ion channel (CRAC). This channel is activated by receptor-induced Ca²⁺ store depletion of the endoplasmic reticulum (ER) and consists of two transmembrane proteins, STIM1 and Orai1. STIM1 is anchored in the ER membrane and senses changes in the ER luminal Ca²⁺ concentration. Orai1 is the Ca²⁺-selective, pore-forming CRAC channel component located in the plasma membrane (PM). Ca²⁺ store-depletion of the ER triggers activation of STIM1 proteins, which subsequently leads to a conformational change and oligomerization of STIM1 and its coupling to as well as activation of Orai1 channels at the ER-PM contact sites. Although STIM1 and Orai1 are sufficient for CRAC channel activation, their efficient activation and deactivation is fine-tuned by a variety of lipids and lipid- and/or ER-PM junction-dependent accessory proteins. The underlying mechanisms for lipid-mediated CRAC channel modulation as well as the still open questions, are presented in this review.

Keywords: CRAC channel; ER-PM junctions; Orai1; STIM1; lipids; modulatory proteins; protein-lipid interactions.

Figure 1

Lipid transport and phosphoinositide (PI) cycle at the ER-PM contact sites. PI is consecutively transformed at the PM into PI4P by PIP4K and PIP₂ by PIP5K (PIP₂ can be converted to PI4P or PI by phosphatases). PM-receptor stimulation can trigger PLC mediated hydrolysis of PIP₂ to form DAG and IP₃. While DAG can activate PKC or some ion channels, IP₃ activates the ER located IP₃R. Non-vesicular transport mechanisms (see Section 5) can transport DAG to the ER membrane, where it is converted to PI. PITPs transport PI back to the plasma membrane. Furthermore, sterols, PS, and PI4P are transported between the two membranes via non-vesicular transport (see Section 5).

Figure 2

STIM1, Orai1, and their interplay with lipids. In the resting state, STIM1, located in the ER membrane, captures a quiescent and folded state, while Orai1, located in the PM, possesses a closed pore. After store-depletion STIM1 undergoes a conformational change, oligomerizes, and couples to Orai1. Although the interaction and function of the two proteins is sufficient for CRAC channel activation, their machinery is modulated by a variety of lipids including PIP₂, PI4P, cholesterol, sphingomyelin and possibly also ER-phospholipids as outlined in detail in Section 4.

Figure 3

ER-PM spanning proteins involved in the modulation of the CRAC channel complex. In addition to the PI cycle, phospholipids can also be transported by ER-PM spanning protein, allowing non-vesicular transport. The localization of ER-PM spanning proteins depends mainly on the lipid composition in the PM, in particular the PIP₂ levels. E-Syt proteins are anchored in the ER membrane and are either Ca²⁺-dependently (E-Syt1) or -independently (E-Syt2, E-Syt3) bound to PIP₂ in the PM. E-Syt proteins can transport DAG from the PM to the ER membrane. Furthermore, E-Syt1 co-localizes with Nir2 to modulate PIP₂ levels in the PM. VAP proteins exchange PI and PA between the ER and PM. Moreover, VAP proteins associate with Nir2 or ORP3/6 to span from the ER to the PM. Distinct ORP variants, ORP5 and ORP8, reside in the ER and bridge the distance to the PM without additional proteins. VAP/ORP proteins and complexes exchange phosphatidylserine (PS) and sterols in the ER for PI4P in the PM. ORP proteins supply PI4P to Sac1 phosphatase in the ER membrane. GRAMD proteins contain a single TM domain located in the ER, bind to PIP₂ in the PM, and are involved in the transport of sterols. ANO8 is multi-transmembrane domain protein in the ER that binds PIP₂ in the PM.

Figure 4

Modulatory proteins at the ER-PM contact sites involved in modulating the interplay of STIM1/Orai1. Schematic of all modulatory proteins located at the ER-PM contact sites reported to be critical for modulation of the STIM1/Orai1 complex. Junctate, junctophilin, STIMATE, and SARAF are located in the ER. RASSF4 and septins are located close to and Cav1 is anchored within the PM.

Figure 5

Complex interplay of ER-PM proteins and lipids with the STIM1/Orai1 channel. GRAMD, Ano8 and E-Syt proteins modulate the ER-PM contact site to facilitate the interplay of STIM1 and Orai1 in an indirect manner. STIMATE, junctate, and junctophilin 4, together with PIP₂, directly interact with STIM1 to support its oligomerization, association near the PM, and coupling with Orai1. SARAF contributes to efficient restorage of the closed state of STIM1 through direct interaction. RASSF4 and septins modulate PIP₂ levels in the PM and are thus indirectly involved in STIM1 activation. Cav-1 is thought to move Orai1 into cholesterol-rich regions.