CRACM1 is a plasma membrane protein essential for store-operated Ca2+ entry

Abstract

Store-operated Ca2+ entry is mediated by Ca2+ release-activated Ca2+ (CRAC) channels following Ca2+ release from intracellular stores. We performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that inhibit store-operated Ca2+ influx. A secondary patch-clamp screen identified CRACM1 and CRACM2 (CRAC modulators 1 and 2) as modulators of Drosophila CRAC currents. We characterized the human ortholog of CRACM1, a plasma membrane-resident protein encoded by gene FLJ14466. Although overexpression of CRACM1 did not affect CRAC currents, RNAi-mediated knockdown disrupted its activation. CRACM1 could be the CRAC channel itself, a subunit of it, or a component of the CRAC signaling machinery.

Fig. 1

Identification of CRACM1 and CRACM2 as crucial regulators of store-operated Ca²⁺ entry in Drosophila. (A) Ca²⁺ signals measured in Drosophila S2R+ cells in the primary high-throughput screen using FLIPR. Representative FLIPR raw data file showing 384 minigraphs, each of which represents fluo-4 fluorescence change in an individual well with respect to time. Each plate contained the negative control dsRNA Rho1 in well A1 and the positive control dsRNA stim1 in well B1. (B) Fluo-4 fluorescence changes in relative fluorescence units (r.f.u.) obtained from cells treated with the indicated dsRNAs. Cells were kept in Ca²⁺-free solution and exposed to thapsigargin (2 µM), followed by addition of 2 mM Ca²⁺. The traces are representative of two independent repeats of the primary screen. (C) Same protocol as in (B) but for cells treated with CRACM2 dsRNA. (D) Normalized average time course of IP₃-induced (20 µM) I_CRAC measured in Drosophila Kc cells. Currents of individual cells were measured at −80 mV, normalized by their respective cell size, averaged and plotted versus time (±SEM). Cytosolic calcium was clamped to 150 nM with 10 mM BAPTA and 4 mM CaCl². Traces correspond to untreated control [wild type (wt), black filled circles, n = 10); Rho1 dsRNA (mock, open circles, n = 8); CRACM1 dsRNA (red circles, n = 6); and CRACM2 dsRNA (green circles, n = 9)]. (E) Averaged current-voltage (I/V) data traces of I_CRAC extracted from representative cells at 60 s for currents evoked by 50-ms voltage ramps from −100 to +100 mV with leak currents subtracted and normalized to cell size (pF). Traces correspond to untreated control (wt, n = 9); CRACM1 dsRNA (n = 5); and CRACM2 dsRNA (n = 6).

Fig. 2

Suppression of store-operated Ca²⁺ entry and I_CRAC by CRACM1 siRNA. (A) (Left) RT-PCR of CRACM1 mRNA from HEK293 cells infected with the indicated CRACM1-specific siRNAs and a scrambled sequence control. (Right) Control with primers specific for small ribosomal protein. (B) Fura 2–AM (pentaacetoxymethyl ester) fluorescence measurements of [Ca²⁺]_i in cells treated with scramble (control) or the two CRACM1-specific siRNAs in HEK293 cells. Cells were kept in Ca²⁺-free solution and exposed to thapsigargin (2 µM), followed by addition of 2 mM Ca²⁺. The traces are representative of three independent experiments. (C) Same protocol as in (B), but for Jurkat cells. The traces are averages of three independent experiments. (D) Normalized average time course of IP₃-induced (20 µM) I_CRAC measured in HEK293 cells treated with the indicated siRNAs (n = 9 to 13 for each group). [Ca²⁺]_i was clamped to near zero by 10 mM BAPTA. (E) Current-voltage (I/V) data traces of I_CRAC from representative cells at 60 s for currents evoked by 50-ms voltage ramps from −100 to +100 mV in cells treated with the indicated siRNAs (n = 7 to 10). (F and G) Same as panel (D) and (E), but for Jurkat cells (n = 8 to 9).

Fig. 3

Overexpression of CRACM1. (A) Analysis of HEK293 cells for overexpression of CRACM1 by immunoprecipitation with antibodies against Myc or C-terminal His and immunoblotting with antibody against Myc. Control immunoprecipitation from empty vector-transfected cells did not show any bands. (B) Normalized average time course of IP₃-induced (20 µM) I_CRAC measured in HEK293 cells. Currents of individual cells were measured at −80 mV, normalized by their respective cell size, averaged, and plotted against time (± SEM). Cytosolic calcium was clamped to near zero by using 10 mM BAPTA. Traces correspond to cells transfected with GFP alone (control, black circles, n = 13) and cells transfected with GFP plus CRACM1 (red circles, n = 14). (C) Immunofluorescence localization of CRACM1 in HEK293 cells visualized by confocal microscopy. Immunostaining for CRACM1–flag–N terminus (top) or CRACM1–Myc–C terminus (bottom) in intact (left) and permeabilized cells (right). (D) Same as bottom right panel of (C), but at higher magnification of selected cells to illustrate plasma membrane staining.