Activating mutations in STIM1 and ORAI1 cause overlapping syndromes of tubular myopathy and congenital miosis

Abstract

Signaling through the store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel regulates critical cellular functions, including gene expression, cell growth and differentiation, and Ca(2+) homeostasis. Loss-of-function mutations in the CRAC channel pore-forming protein ORAI1 or the Ca(2+) sensing protein stromal interaction molecule 1 (STIM1) result in severe immune dysfunction and nonprogressive myopathy. Here, we identify gain-of-function mutations in the cytoplasmic domain of STIM1 (p.R304W) associated with thrombocytopenia, bleeding diathesis, miosis, and tubular myopathy in patients with Stormorken syndrome, and in ORAI1 (p.P245L), associated with a Stormorken-like syndrome of congenital miosis and tubular aggregate myopathy but without hematological abnormalities. Heterologous expression of STIM1 p.R304W results in constitutive activation of the CRAC channel in vitro, and spontaneous bleeding accompanied by reduced numbers of thrombocytes in zebrafish embryos, recapitulating key aspects of Stormorken syndrome. p.P245L in ORAI1 does not make a constitutively active CRAC channel, but suppresses the slow Ca(2+)-dependent inactivation of the CRAC channel, thus also functioning as a gain-of-function mutation. These data expand our understanding of the phenotypic spectrum of dysregulated CRAC channel signaling, advance our knowledge of the molecular function of the CRAC channel, and suggest new therapies aiming at attenuating store-operated Ca(2+) entry in the treatment of patients with Stormorken syndrome and related pathologic conditions.

Keywords: calcium signaling; human genetics.

Fig. 1.

p.R304W in STIM1 causes Stormorken syndrome. (A and B) Pedigrees of the two families affected by Stormorken syndrome. Arrows indicate the probands in each family. Asterisk indicates individuals whose whole exome was sequenced. (C) Diagram of human STIM1_R304W and sequence alignment of the Cα2 of coiled coil domain 1 (CC1) of WT human, mouse, and zebrafish STIM1 and STIM2. Cα1–3, α-helix 1–3 in CC1; EF, EF hand; ID, inhibitory domain; TM, transmembrane domain.

Fig. 2.

Store-operated Ca²⁺ entry and I_CRAC is enhanced in patients with Stormorken syndrome. (A) Store-operated Ca²⁺ entry in lymphocytes obtained from an unaffected individual (control; black, n = 127 cells) or a patient with Stormorken syndrome (patient; red, n = 288 cells) using single-cell Ca²⁺ imaging. Cells were loaded with 2 µM Fura-2/AM and placed in an extracellular solution (ECS) containing 0 mM Ca²⁺. Stores were depleted with 2 µM TG and Ca²⁺ influx was stimulated by the addition of 2 mM Ca²⁺ in the ECS. (B) Time course of I_CRAC in lymphocytes from a control subject (black, n = 11 cells) and patient with Stormorken syndrome (red, n = 16 cells) induced by 10 mM EGTA in the recording pipette and blocked by 20 µM La³⁺ in the ECS. (C) Current–voltage curves taken at 50 s or 400 s after “break-in” in control cells and cells from a patient with Stormorken syndrome. (D) Quantification of inactivation as determined by the ratio (R195ms) of the peak current at the beginning of a hyperpolarizing pulse (I₀) to tail current at the end of the pulse (I₁₉₅) in cells from a healthy individual (black, n = 11 cells) and a patient with Stormorken syndrome (red, n = 16 cells). Representative step currents generated from hyperpolarizing pulses at the indicated test potentials at 400 s following break-in in a control subject and a patient (Stormorken) cell. Duration of the pulse was 200 ms.

Fig. 3.

STIM1_R304W enhances CRAC channel activity in transfected cells. (A) Time course of I_CRAC in HEK 293 cells transiently cotransfected with ORAI1 plus STIM1_WT (OS_WT; black, n = 5), ORAI1 plus STIM1_R304W (OS_R304W; red, n = 7), ORAI1 plus STIM1_K(384-6)Q [OS_K(384-6)Q; dark yellow, n = 5], ORAI1 plus STIM1_R304W_K(384-6)Q [OS_R304W_K(384-6)Q; blue, n = 6], ORAI1 plus STIM1_D76A (OS_D76A; green, n = 7), or ORAI1 plus a 1:1 mix of STIM1_WT and STIM1_R304W (OS_WT/R304W; burgundy, n = 8) induced by 10 mM EGTA and suppressed by 20 µM La³⁺. (B and C) Current–voltage curves taken at 50 (B) or 200 s (C) after break-in in cells transfected with plasmid combinations shown in A. (D) Representative step currents generated from hyperpolarizing pulses at the indicated test potentials at 200 s following break-in in cells transfected with OS_WT, OS_R304W, OS_D76A, or OS_WT/R304W in 10 mM extracellular Ca²⁺. Duration of the pulse was 200 ms. (E) Quantification of inactivation as determined by the ratio (R195ms) of the peak current at the beginning of the pulse (I₀) to tail current at the end of the pulse (I₁₉₅) in cells transfected with OS_WT (n = 6), OS_R304W (n = 7), D76A (n = 8), or OS_WT/R304W (n = 8).

Fig. 4.

p.245L in ORAI1 causes a Stormorken-like syndrome with aggregate tubular myopathy. (A) Pedigree of the two branches of an extended family with common ancestry in mid-19th century with Stormorken-like syndrome. Branch 1 [Left branch (below generation II)] was described by Shahrizaila et al. (38). The disorder follows an autosomal-dominant inheritance pattern. The arrows indicate the proband in each branch of this family (patient 1, left arrow; patient 2, right arrow). Asterisk indicates individuals whose whole exome was sequenced. (B) Cartoon showing the location of P245 in transmembrane segment M4, and a sequence alignment of the M4 α-helix of Drosophila ORAI1 and human ORAI1-3.

Fig. 5.

ORAI1_P245L suppresses slow CDI of I_CRAC. (A) Time course of I_CRAC in HEK293 cells transiently cotransfected with STIM1_WT plus WT ORAI1 (S_WT/O_WT; black, n = 5) or STIM1_WT plus ORAI1_P245L (S_WT/O_P245L; dark cyan, n = 8) induced by 10 mM EGTA and suppressed by 20 µM La³⁺. (B) Normalized whole-cell currents measured at −80 mV of cells transfected with STIM1_WT plus ORAI1_WT (black, n = 10) or STIM1_WT plus ORAI1_P245L (dark cyan, n = 14) in the presence of 2 mM EGTA in the pipette solution. (C) Normalized whole-cell currents measured at −80 mV of cells transfected with STIM1_WT plus ORAI1_WT (black, n = 8) or STIM1_WT plus ORAI1_P245L (dark cyan, n = 9) in the presence of 10 mM BAPTA in the pipette solution. (D) Store-operated Ca²⁺ entry is enhanced in a patient with congenital miosis and TAM. Store-operated Ca²⁺ entry in lymphocytes obtained from an unaffected individual (control; black, n = 146 cells) or a patient (patient; red, n = 302 cells) using single-cell Ca²⁺ imaging. Cells were loaded with 2 µM Fura-2/AM and placed in an ECS containing 0 mM Ca²⁺. Stores were depleted with 2 µM TG, and Ca²⁺ influx was stimulated by the addition of 2 mM Ca²⁺ in the ECS.

Fig. 6.

Expression of STIM1_R304W, but not ORAI1_P245L, results in bleeding and reduced expression and defective flow of thrombocyte progenitors in zebrafish embryos. (A–F) Whole-body lateral views of a control (A) and STIM1_R304W (D) injected embryos at 48 h post fertilization stained with ο-dianisidine. B and E are magnifications of the cephalic boxed area in A in all respective embryos, showing brain hemorrhages (white arrows) in the STIM1_R304W mRNA injected embryos (E). Panels C and F show magnified views of the boxed caudal area in A in the respective conditions. Areas of intrasomitic and caudal bleeding are highlighted with black arrows. (G) Percent distribution of normal embryos vs. embryos with spontaneous bleeding episodes (**P < 0.001, ***P < 0.0001). (Scale bar: 500 µm.) Whole-body lateral views of control (H), STIM1_WT- (J), STIM1_D76A- (L), and STIM1_R304W- (N) injected Tg(CD41:GFP) embryos at 72 h post fertilization. I, K, M, and O are magnifications of the ventral boxed area in H in the respective embryos, showing expression of GFP in thrombocytes. The boxed area corresponds to the site where early hematopoiesis takes place in zebrafish. (P) Percent distribution of normal embryos vs. embryos with reduced or no expression of thrombocyte progenitors (*P < 0.05, **P < 0.001, and ***P < 0.0001).