Diseases caused by mutations in ORAI1 and STIM1

Abstract

Ca(2+) release-activated Ca(2+) (CRAC) channels mediate a specific form of Ca(2+) influx called store-operated Ca(2+) entry (SOCE) that contributes to the function of many cell types. CRAC channels are composed of ORAI1 proteins located in the plasma membrane, which form its ion-conducting pore. ORAI1 channels are activated by stromal interaction molecule (STIM) 1 and STIM2 located in the endoplasmic reticulum. Loss- and gain-of-function gene mutations in ORAI1 and STIM1 in human patients cause distinct disease syndromes. CRAC channelopathy is caused by loss-of-function mutations in ORAI1 and STIM1 that abolish CRAC channel function and SOCE; it is characterized by severe combined immunodeficiency (SCID)-like disease, autoimmunity, muscular hypotonia, and ectodermal dysplasia, with defects in sweat gland function and dental enamel formation. The latter defect emphasizes an important role of CRAC channels in tooth development. By contrast, autosomal dominant gain-of-function mutations in ORAI1 and STIM1 result in constitutive CRAC channel activation, SOCE, and increased intracellular Ca(2+) levels that are associated with an overlapping spectrum of diseases, including nonsyndromic tubular aggregate myopathy (TAM) and York platelet and Stormorken syndromes. The latter two syndromes are defined, besides myopathy, by thrombocytopenia, thrombopathy, and bleeding diathesis. The fact that myopathy results from both loss- and gain-of-function mutations in ORAI1 and STIM1 highlights the importance of CRAC channels for Ca(2+) homeostasis in skeletal muscle function. The cellular dysfunction and clinical disease spectrum observed in mutant patients provide important information about the molecular regulation of ORAI1 and STIM1 proteins and the role of CRAC channels in human physiology.

Keywords: CRAC channel; Ca2+; ORAI1; SOCE; STIM1; Stormorken syndrome; York platelet syndrome; ameloblast; autoimmunity; calcium; channelopathy; disease; enamel; muscular hypotonia; mutation; platelets; skeletal muscle; thrombocytopenia; tubular aggregate myopathy.

Figure 1

Model of STIM1 activation and effects of p.R429C mutation. (A) STIM1 and ORAI1 domain organization. ORAI1 is the pore-forming subunit of the CRAC channel in the plasma membrane. It contains 4 alphahelical transmembrane domains (M1–4) and cytoplasmic N- and C-termini that interact with STIM1. M1 lines the ion conducting pore of the channel. STIM1 is a single pass transmembrane protein located in the ER membrane. Its N terminus is located in the ER lumen and contains canonical and non-canonical EF hand (cEFh, nEFh) domains and a sterile alpha motif (SAM). The cytoplasmic C-terminus of STIM1 contains 3 coiled-coil (CC) domains and a lysine-rich (K) domain, which mediate STIM1 binding to ORAI1 and plasma membrane phospholipids, respectively. STIM1 binding to ORAI1 is mediated by the CRAC activation domain (CAD, also called SOAR or CCb9) in STIM1 that encompasses CC2 and CC3. (B) Stepwise activation of ORAI1-CRAC channels by STIM1. In cells with filled ER Ca²⁺ stores, the cytosolic STIM1 domain is in a closed, inactive conformation and forms a dimer with another STIM1 molecule. By contrast, the Ca²⁺-bound EF-SAM domain of STIM1 located in the ER lumen is monomeric (1). Upon stimulation of cell surface receptors (R) that induce activation of PLCγ1 or PLCγ2 and production of IP₃, Ca²⁺ is released from the ER through IP₃ receptors that are non-selective Ca²⁺ channels. The decreased Ca²⁺ concentration in the ER results in dissociation of Ca²⁺ from the canonical EF hand (cEFh) domain in the N-terminus of STIM1 and dimerization of EF-SAM domains. This causes a change in the conformation of the STIM1 C terminus into an extended, active structure in which the CAD and polybasic domains (K) are exposed (2). In the extended conformation, STIM1 dimers oligomerize mediated by CC domains including CC3 (3). STIM1 is recruited to ER-PM junctions through interactions of the K-rich polybasic domain with membrane phospholipids (4). Oligomerized STIM1 proteins form puncta in ER-PM junctions and bind to ORAI1, thereby recruiting it into ER-PM junctions and puncta. STIM1 binding results in opening of ORAI1 (CRAC) channels and SOCE (5). Abbreviations: CC1-CC3, coiled-coil; K, lysine-rich polybasic domain; PIP₂, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PM, plasma membrane.

Figure 2

Disease phenotypes associated with mutations in ORAI1 and STIM1. Null and loss-of-function (LoF) mutations in ORAI1 and STIM1 cause CRAC channelopathy, which is defined by (i) SCID-like immunodeficiency with recurrent and chronic infections, (ii) autoimmunity due to autoantibody-mediated hemolytic anemia and thrombocytopenia, (iii) muscular hypotonia and (iv) ectodermal dysplasia characterized by anhidrosis and defects in dental enamel development (left). Gain-of-function (GoF) mutations in STIM1 and ORAI1 cause a spectrum of disease entities with partially overlapping symptoms: tubular aggregate myopathy (TAM), York platelet syndrome and Stormorken syndrome. The arrows and white rectangles indicate organs and cell types affected by both LoF and GoF mutations in ORAI1 and STIM1 that result in similar disease manifestations although their pathophysiology differs. Abbreviations: AIHA, autoimmune hemolytic anemia; SCID, severe combined immunodeficiency.

Figure 3

Loss- and gain-of-function mutations in ORAI1 and STIM1. (A) Loss-of-function (LoF) mutations in ORAI1 and STIM1 abolish SOCE and cause CRAC channelopathy. Patients are homozygous either for null mutations that abolish protein expression of ORAI1 (p.A88SfsX25, p.A103E, p.L194P, p.H165PfsX1) and STIM1 (p.E128RfsX9, c.1538-1G>A) or LoF mutations that disrupt the function of ORAI1 (p.R91W) and STIM1 (p.R429C, p.P165Q, p.R426C?). The ORAI1 p.R91W mutation does not interfere with channel expression but blocks permeation of Ca²⁺ through the CRAC channel pore. The STIM1 p.R429C mutation has complex effects on STIM1 function and impairs CRAC channel activation at multiple steps (as shown in Fig. 4). How STIM1 p.P165Q and p.R426C mutations inhibit SOCE has not been established. (B) Gain-of-function (GoF) mutations in ORAI1 and STIM1 result in constitutive Ca²⁺ influx and cause three clinically overlapping disease syndromes. Patients with Stormorken syndrome are heterozygous for a single autosomal dominant STIM1 p.R304W mutation located in CC1. The same mutation was described to cause the clinically very similar York platelet syndrome. Tubular aggregate myopathy (TAM) is caused by a variety of autosomal dominant mutations in the EFh of STIM1 or in transmembrane domains of ORAI1 that result in constitutive CRAC channel activation. Thin, dashed arrows through ORAI1 indicate abrogated SOCE; thick, solid arrows indicate constitutive Ca²⁺ influx. Abbreviations: CCb9, Coiled-coiled fragment b9; fs, frameshift; IP₃, inositol 1,4,5-triphosphate; M, transmembrane domain; ncEFh, non-canonical EF hand; PLC, phospholipase C; SAM, sterile alpha motif; SOAR, STIM-ORAI activation region.

Figure 4

The STIM1 p.R429C LoF mutation interferes with CRAC channel activation. The STIM1 p.R429C mutation has complex effects on STIM1 function and interferes with CRAC channel activation at multiple steps. The mutation results in constitutive opening of the STIM1-CT independent of the filling state of ER Ca²⁺ stores (1). In the extended conformation, the K-rich polybasic domain is exposed and mutant STIM1 is constitutively recruited to ER-PM junctions (2). Because R429 is required for the CC3 domain-mediated oligomerization of STIM1 dimers, mutant STIM1 cannot form puncta (3). Mutation of R429 abolishes the binding of STIM1 to ORAI1 (4). As a consequence, mutant STIM1 p.R429C fails to open ORAI1 channels and induce SOCE. For details see text. Blue circles represent Ca²⁺ ions. Dashed lines in the PM represent negatively charged phospholipids including PIP₂. Abbreviations: EFh, EF hand; CC1-CC3, coiled-coil; K, lysine-rich polybasic domain; PIP₂, phosphatidylinositol 4,5-bisphosphate; PM, plasma membrane; SAM, sterile alpha motif; STIM1-CT, STIM1 C-terminus.

Figure 5

Hypocalcified amelogenesis imperfecta in a patient with ORAI1 loss-of-function mutation. Photograph of the teeth of a patient with ORAI1 p.R91W mutation that abolishes SOCE (originally reported in Refs.^, ). Shown here is the lower jaw at approximately 6 years of age. All erupted teeth have lost their enamel capping on the occlusal surfaces and only remnants can be seen on the lateral margins of the teeth (arrows). The yellowish appearance of the teeth is related to dentine exposure.

Figure 6

Schematic representation of a tooth, enamel cells and the Ca²⁺ transport model. (A) Schematic section of a human molar showing the location of enamel forming cells, ameloblasts, in relation to other structures and cells. The main structures found in a tooth are the outer enamel layer, the dentine (formed by odontoblasts) and the innermost dental pulp. Secretory ameloblasts are epithelium-derived cells arising from pre-ameloblasts (PA) at the cervical loop (CL). The cells undergo morphological changes including increases in height, and develop a specialized secretory process at the distal (apical) end of the cell. Secretory ameloblasts reorganize into maturation stage ameloblasts once the thickness (volume) of enamel tissue has been completed. Maturation stage ameloblasts are shorter, loose the secretory process (Tomes’ process) and switch their functional properties to become cells specialized in transport. (B) Representative model of maturation stage ameloblasts showing the localization of ORAI1 and STIM1 in the plasma membrane and ER, respectively. This working model of Ca²⁺ transport in ameloblasts builds on the model proposed by Hubbard . It postulates that Ca²⁺ enters through PM Ca²⁺ channels and is taken up into the ER at the basolateral pole of the cell, and released into the cytoplasm via IP₃R or RyR at the apical pole, where it is secreted into the extracellular enamel matrix. The emerging details and molecular components of this Ca²⁺ transport need to be investigated further, but our recent data suggest that STIM1 and ORAI1 mediate Ca²⁺ uptake whereas NCKX/NCX exchangers mediate Ca²⁺ extrusion. Abbreviations: BV, blood vessels; DP (dental pulp); NCKX, Na⁺/Ca²⁺-K⁺ exchanger; NCX, Na⁺/Ca²⁺ exchanger; OEE (outer enamel epithelium); PL (papillary layer); SERCA, Sarco/endoplasmic reticulum Ca²⁺ ATPase; SI (stratum intermedium); SR (stellate reticulum).