CRAC channel activity in C. elegans is mediated by Orai1 and STIM1 homologues and is essential for ovulation and fertility

Abstract

The Ca(2+) release-activated Ca(2+) (CRAC) channel is a plasma membrane Ca(2+) entry pathway activated by endoplasmic reticulum (ER) Ca(2+) store depletion. STIM1 proteins function as ER Ca(2+) sensors and regulate CRAC channel activation. Recent studies have demonstrated that CRAC channels are encoded by the human Orai1 gene and a homologous Drosophila gene. C. elegans intestinal cells express a store-operated Ca(2+) channel (SOCC) regulated by STIM-1. We cloned a full-length C. elegans cDNA that encodes a 293 amino acid protein, ORAI-1, homologous to human and Drosophila Orai1 proteins. ORAI-1 GFP reporters are co-expressed with STIM-1 in the gonad and intestine. Inositol 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signalling regulates C. elegans gonad function, fertility and rhythmic posterior body wall muscle contraction (pBoc) required for defecation. RNA interference (RNAi) silencing of orai-1 expression phenocopies stim-1 knockdown and causes sterility and prevents intestinal cell SOCC activation, but has no effect on pBoc or intestinal Ca(2+) signalling. Orai-1 RNAi suppresses pBoc defects induced by intestinal expression of a STIM-1 Ca(2+)-binding mutant, indicating that the proteins function in a common pathway. Co-expression of stim-1 and orai-1 cDNAs in HEK293 cells induces large inwardly rectifying cation currents activated by ER Ca(2+) depletion. The properties of this current recapitulate those of the native SOCC current. We conclude that C. elegans expresses bona fide CRAC channels that require the function of Orai1- and STIM1-related proteins. CRAC channels thus arose very early in animal evolution. In C. elegans, CRAC channels do not play obligate roles in all IP(3)-dependent signalling processes and ER Ca(2+) homeostasis. Instead, we suggest that CRAC channels carry out highly specialized and cell-specific signalling roles and that they may function as a failsafe mechanism to prevent Ca(2+) store depletion under pathophysiological and stress conditions.

Figure 1. Amino acid sequence alignment of C. elegans (Ce) ORAI-1 with Drosophila (Dm) Orai and human (Hs) Orai1, Orai2 and Orai3

Yellow and green shading indicates sequence identity and conserved amino acid substitutions, respectively. Transmembrane (TM) domains of ORAI-1 predicted by TMpred (http://www.ch.embnet.org/software/TMPRED_form.html#) are underlined in black. A conserved arginine residue mutated in SCID patients lacking lymphocyte CRAC channel activity (Feske et al. 2006) is denoted by the arrow. Conserved glutamate residues located in TM1 and TM3 that contribute to channel ionic selectivity (Prakriya & Lewis, 2006; Vig et al. 2006a; Yeromin et al. 2006) are denoted by arrowheads. Alignment was performed using Vector NTI software (InforMax, Bethesda, MD, USA). Percentage identity and similarity of C. elegans ORAI-1 to Drosophila Orai, human Orai1, human Orai2 and human Orai3 were 34% and 55%, 34% and 54%, 38% and 59%, and 35% and 59%, respectively. Acidic residues in the extracellular loop between TM1 and TM2 are outlined in black. In Drosophila Orai (Yeromin et al. 2006) and human Orai1 (Vig et al. 2006a), these residues may function to attract polyvalent cations towards the channel pore and control channel selectivity.

Figure 2. ORAI-1 expression pattern

A, low-magnification confocal 3D reconstruction of ORAI-1::GFP expression pattern in an intact worm. Expression was detected throughout the intestine (Int) and appeared to be localized to apical (Ap) and basolateral (Bl) membrane regions. Weaker fluorescence was also detected throughout the hypodermis (Hyp). Scale bar is 50 μm. B, combined DIC and fluorescence confocal micrographs showing ORAI-1::GFP expression in the spermatheca (Sp). Scale bar is 5 μm. Emb, embryo. C, fluorescence micrograph of an isolated gonad dissected from a worm expressing a transcriptional ORAI-1 GFP reporter. P_orai-1::GFP expression is detected in the spermatheca (Sp) and gonadal sheath cells (arrowheads). Scale bar is 10 μm. Oo, oocyte; DG, distal gonad. D, confocal micrograph showing expression of ORAI-1::GFP and STIM-1::DsRed in two cells of the anterior intestine. Bl, basolateral membrane. Scale bar is 5 μm. E and F, confocal 3D reconstructions of ORAI-1::GFP and STIM-1::DsRed expression in the spermatheca. Scale bars are 5 μm.

Figure 3. Effect of orai-1 RNAi on fertility and ovulation

A, brood size in wild-type and rrf-1(pk1417) mutant worms. Brood size is defined as the total number of progeny produced over four days. rrf-1 encodes an RNA-directed RNA polymerase homologue required for RNAi in somatic but not germ cells. pk1417 is a predicted rrf-1 null allele (Sijen et al. 2001). Worms were fed GFP or orai-1 dsRNA-producing bacteria for two generations beginning at the L1 larval stage. Values are means ± s.e.m. (n = 6–12). B, rates of sheath contraction during a single ovulatory cycle. Time 0 is defined as the time at which ovulation was completed in control worms. orai-1(RNAi) worms never ovulated (see Results). Therefore in these animals, time 0 is defined the first time point after peak sheath contraction rate was observed. Values are means ± s.e.m. (n = 6–7). Worms were fed dsRNA-producing bacteria for two generations. C, differential interference contrast micrograph of the gonad of an orai-1(RNAi) worm. The distal spermatheca fails to open during ovulation in orai-1(RNAi) worms, and oocytes are trapped in the proximal gonad arm where they undergo endomitosis. DG, Distal gonad; Oo, normal oocytes; Emo, endomitotic oocytes; Sp, spermatheca; Ut, uterus; Vu, vulva. Note that the uterus in the orai-1(RNAi) worm is empty due to failure of ovulation. worms fed dsRNA-producing bacteria for two generations.

Figure 4. Effect of orai-1 RNAi on ORAI-1 expression, pBoc and intestinal Ca²⁺ signalling

A, effect of orai-1 RNAi on ORAI-1::GFP expression. Top: fluorescence micrographs of wild-type and ORAI-1::GFP-expressing worms fed bacteria containing the empty RNAi feeding empty RNAi feeding vector and ORAI-1::GFP expressing worms fed orai-1 dsRNA producing bacteria [orai-1:: gfp; orai-1 (RNAi)]. Fluorescence in wild-type worms is background autofluorescence. Bottom: relative whole-animal fluorescence in wild-type, orai-1:gfp and orai-1::gfp; orai-1(RNAi) worms. Wild-type and orai-1:gfp worms were fed bacteria containing the empty feeding vector. Values are mean ± s.e.m. (n = 164–532). *P < 0.001 compared to wild-type and orai-1::gfp animals. Whole-worm fluorescence was quantified using a COPAS Biosort and normalized to time-of-flight (i.e. fluorescence/time-of-flight), which is a measure of worm size. RNAi feeding was carried out for one generation. GFP-expressing worms were the integrated strain KbIs18 (Porai-1::ORAI-1::GFP; Pstim-1::STIM-1::DsRed; rol-6(su1006)). B, effect of orai-1 RNAi on pBoc period and coefficient of variance, which is a measure of cycle rhythmicity. Wild-type worms were fed GFP or orai-1 dsRNA-producing bacteria for two generations. Values are means ± s.e.m. (n = 9–15). C, examples of Ca²⁺ oscillations observed in intestines isolated from rde-1(ne219);kbEx200 worms fed bacteria containing the empty feeding vector (control) or orai-1 dsRNA-producing bacteria for two generations. Fluorescent images were acquired at 3 Hz.

Figure 5. Effect of orai-1 RNAi on SOCC activity in cultured intestinal cells

A, examples of whole-cell current–voltage relationships of SOCC currents induced by 5 min of store depletion in a control cell and a cell treated with orai-1 dsRNA. Store depletion was induced using a pipette solution containing 10 μm IP₃, 10 mm BAPTA and 18 nm free-Ca²⁺. Currents were elicited by ramping membrane voltage from –120 mV to +80 mV at 200 mV s⁻¹ every 5 s. B, effect of orai-1 dsRNA on peak I_SOCC measured 5 min after obtaining whole-cell access. Values are means ± s.e.m. (n = 7–10). *P < 0.003 compared to control. Intestinal cells were cultured from rde-1(ne219);kbEx200 worms (Espelt et al. 2005) fed orai-1 dsRNA-producing bacteria for three generations. orai-1 dsRNA was also included in the culture medium beginning at the time of cell plating. Cells were patch clamped 2–3 days after isolation from worm embryos.

Figure 6. Effect of orai-1 RNAi on pBoc period and coefficient of variance in transgenic worms expressing the GFP-tagged STIM-1 EF hand mutant protein STIM-1(D55A; D57A)::GFP

Data on wild-type worms fed GFP dsRNA-producing bacteria (gfp(RNAi)) are reproduced from Fig. 4B. D55A; D57A worms were fed bacteria containing the empty RNAi feeding vector or bacteria producing orai-1 dsRNA. RNAi feeding was carried out over two generations. Values are means ± s.e.m. (n = 13–15). *P < 0.05 compared to gfp(RNAi) worms. †P < 0.01 compared to D55A;D57A worms fed orai-1 dsRNA-producing bacteria. pBoc periods in gfp(RNAi) and D55A; D57A; orai-1(RNAi) worms were not significantly (P > 0.05) different. Coefficients of variance in all three groups of worms were not significantly (P > 0.05) different.

Figure 7. Effect of heterologous expression of ORAI-1 and STIM-1 in HEK293 cells on store-operated currents

A, whole-cell currents in individual HEK293 cells transfected with GFP, STIM-1 and GFP, ORAI-1 and GFP, or STIM-1, ORAI-1 and GFP cDNAs. Solid lines are the mean currents for each of the groups shown. Currents were elicited by ramping membrane voltage from –120 mV to +80 mV at 200 mV s⁻¹ every 5 s. Holding potential was 0 mV. The currents shown were those observed 5 min after obtaining whole-cell access and are leak current subtracted. Mean whole-cell current in STIM-1/ORAI-1-expressing cells with depleted stores was significantly (P < 0.001) different from the other four groups shown. Store depletion was induced by dialysing cells with a pipette solution containing 10 mm BAPTA, 10 μm IP₃ and ∼18 nm free-Ca²⁺. Depletion of stores was prevented using a pipette solution containing 10 mm BAPTA, 2 mm ATP and ∼140 nm free-Ca²⁺. B, examples of time-dependent changes in current activity (left panel) and current–voltage relationships (right panel) of currents observed in an ORAI-1/STIM-1–expressing cell with replete stores and in an ORAI-1/STIM-1-expressing cell in which stores were depleted. Current–voltage relationships were plotted for currents measured 5 min after obtaining whole-cell access. Leak current was subtracted from the current induced by store depletion. C, example of store-operated whole-cell currents elicited by stepping membrane voltage in 20 mV steps from –120 mV to +80 mV in an ORAI-1/STIM-1-expressing cell. Holding potential was 0 mV.

Figure 8. Effect of 2-APB on ORAI-1/STIM-1-induced store-operated currents in HEK293 cells

A, example of the effect of 5 μm (open bar) and 100 μm (filled bar) 2-APB on store-operated current in an ORAI-1/STIM-1-expressing cell. B, effect of 5 μm and 100 μm 2-APB on relative whole-cell current in HEK293 cells co-expressing ORAI-1 and STIM-1. Values are means ± s.e.m. (n = 4–5). *P < 0.0002 compared to control without 2-APB.

Figure 9. Examples of the effects of divalent cation-free (DVF) bath on store-operated whole-cell currents in different ORAI-1/STIM-1-expressing cells

‘Low-current’ (A) and ‘high-current’ (B) cells had current densities of 6–7 pA pF⁻¹ and 30–60 pA pF⁻¹, respectively. The control bath solution for low- and high-current cells contained 10 mm Ca²⁺. ‘Low-Ca²⁺’ (C) cells were bathed in an extracellular solution containing 0.25 mm Ca²⁺ before exposure to the DVF bath. Data shown are for single cells. Current–voltage relationships for currents observed in each of the three groups of cells are shown in the right panels. Numbering of the traces corresponds to the times at which the current–voltage relationships were obtained (i.e. left panel).