CRACM1, CRACM2, and CRACM3 are store-operated Ca2+ channels with distinct functional properties

Abstract

STIM1 in the endoplasmic reticulum and CRACM1 in the plasma membrane are essential molecular components for controlling the store-operated CRAC current. CRACM1 proteins multimerize and bind STIM1, and the combined overexpression of STIM1 and CRACM1 reconstitutes amplified CRAC currents. Mutations in CRACM1 determine the selectivity of CRAC currents, demonstrating that CRACM1 forms the CRAC channel's ion-selective pore, but the CRACM1 homologs CRACM2 and CRACM3 are less well characterized. Here, we show that both CRACM2 and CRACM3, when overexpressed in HEK293 cells stably expressing STIM1, potentiate I(CRAC) to current amplitudes 15-20 times larger than native I(CRAC). A nonconducting mutation of CRACM1 (E106Q) acts as a dominant negative for all three CRACM homologs, suggesting that they can form heteromultimeric channel complexes. All three CRACM homologs exhibit distinct properties in terms of selectivity for Ca(2+) and Na(+), differential pharmacological effects in response to 2-APB, and strikingly different feedback regulation by intracellular Ca(2+). Each of the CRAC channel proteins' specific functional features and the potential heteromerization provide for flexibility in shaping Ca(2+) signals, and their characteristic biophysical and pharmacological properties will aid in identifying CRAC-channel species in native cells that express them.

Figure 1

All CRACM Homologs Produce Store-Operated Currents (A) Average CRAC current densities at −80 mV induced by IP₃ (20 μM) in stable STIM1-expressing HEK293 cells transiently overexpressing CRACM1 (black, n = 12), CRACM2 (blue, n = 7), and CRACM3 (red, n = 9). Open symbols represent cells that were cotransfected with the WT constructs of the three homologs plus the dominant negative E106Q mutant of CRACM1 (CRACM1-E106Q + CRACM1, n = 6; + CRACM2, n = 6; + CRACM3, n = 7). [Ca²⁺]_i was clamped to near zero with 20 mM BAPTA. Error bars indicate SEM. (B) Average current-voltage (I/V) relationships of CRAC currents extracted from representative HEK293 cells shown in (A) obtained at 120 s. Data represent leak-subtracted current densities (pA/pF) evoked by 50 ms voltage ramps from −150 to +150 mV corresponding to CRACM1 (black, n = 11), CRACM2 (blue, n = 6), and CRACM3 (red, n = 9). (C) Average CRAC current densities at −80 mV in cells expressing STIM1 alone (n = 13) or additionally with CRACM1-E106Q + CRACM1/CRACM2/CRACM 3; data points correspond to currents analyzed from (A) at 120 s. Error bars indicate SEM. (D) Coimmunoprecipitation of CRACM1 with CRACM2 and CRACM3. Wild-type HEK293 cells were cotransfected with CRACM1-Myc in combination with HA-CRACM1, HA-CRACM2, or HA-CRACM3. Lanes 1 and 2 show nontransfected HEK293 cells. Lanes 3 and 4 show that CRACM1-Myc can co-IP HA-CRACM3, HA-CRACM2 (lanes 5 and 6), and HA-CRACM1 (lanes 7 and 8). The resulting immune complexes were immunoblotted with HA antibody, thus revealing bands with molecular weights of ~33, ~28, and ~31 kDa for CRACM1, CRACM2, and CRACM3, respectively.

Figure 2

CRACM Homologs Have Distinct Fast and Slow Ca²⁺-Dependent-Inactivation Properties (A) Average CRAC current densities at −80 mV induced by IP₃ (20 μM) with 10 mM EGTA in stable STIM1-expressing HEK293 cells transiently overexpressing CRACM1 (n = 3; total n = 8 and three cells with the smallest current densities were averaged to approximate the lower current densities of CRACM2 and CRACM3). CRAC currents were monitored continuously by voltage ramps spanning −100 mV to +100 mV over 50 ms delivered at a rate of 0.5 Hz. After CRAC currents were fully activated (120 s), rectangular voltage pulses of 2 s duration were delivered to various negative voltages (see [D]–[F]) interspaced by 10 ramps. Error bars indicate SEM. (B) Experimental protocol as described in (A), but for CRACM2-expressing cells. Note the minor fast inactivation and virtual absence of slow inactivation. Error bars indicate SEM. (C) Experimental protocol as described in (A), but for CRACM3-expressing cells. Note the significant fast inactivation and virtual absence of slow inactivation. Error bars indicate SEM. (D) Average CRAC currents evoked by step pulses (2 s duration) to −20 mV (green), −40 mV (blue), −60 mV (red), and −80 mV (black) in cells expressing CRACM1 (n = 3, same cells as in [A]). At the beginning of each pulse, 2.5 ms were blanked out so that residual capacitative artifacts could be eliminated. (E) Average CRAC currents evoked by step pulses from −20 mV to −80 mV in cells expressing CRACM2 (n = 4, same cells as in [B]). (F) Average CRAC currents evoked by step pulses from −20 mV to −80 mV in cells expressing CRACM3 (n = 5, same cells as in [C]).

Figure 3

CRACM1, but Not CRACM2 or CRACM3, Is Inhibited by Increased [Ca²⁺]_i (A) Average CRAC-current densities at −80 mV induced by IP₃ (20 μM) in stable STIM1-expressing HEK293 cells transiently overexpressing CRACM1 and perfused with increasing [Ca²⁺]_i (n = 5–12). Error bars indicate SEM. (B) Experimental protocol as described in (A), but for CRACM2-expressing cells (n = 5–8). Error bars indicate SEM. (C) Experimental protocol as described in (A), but for CRACM3-expressing cells (n = 9–15). Error bars indicate SEM. (D) Average current densities of CRACM1 (black), CRACM2 (blue), and CRACM3 (red) at −80 mV extracted at 120 s (150 s for CRACM3) from the cells shown in (A)–(C) and plotted versus [Ca²⁺]_i. Error bars indicate SEM. (E) Half-maximal activation time of CRACM1 (black, n = 5–12), CRACM2 (blue, n = 5–8), and CRACM3 (red, n = 9–15) plotted versus [Ca²⁺]_i. Data were derived from the cells shown in (A)–(C). All cells had similar series resistances in the range of 4–6 MU. Error bars indicate SEM. (F) Average changes in [Ca²⁺]_i induced by store depletion in stable STIM1-expressing HEK293 cells transfected with empty vector (green, n = 14), or transiently overexpressing CRACM1 (black, n = 23), CRACM2 (blue, n = 39), or CRACM3 (red, n = 27). The arrows indicate application of thapsigargin (2 μM) in Ca²⁺-free solution to induce store depletion and readmission of 2 mM Ca²⁺ to probe Ca²⁺ entry. The inset represents rates of [Ca²⁺]_i obtained by differentiating the trace segment enclosed by the rectangle.

Figure 4

CRACM Homologs Have Distinct Ion Selectivity and Pharmacology (A) Average normalized CRAC currents at −80 mV induced by IP₃ (20 μM) in stable STIM1-expressing HEK293 cells transiently overexpressing CRACM1 (black, n = 12, data taken from [5]) CRACM2 (blue, n = 8), or CRACM3 (red, n = 10). Currents of individual cells were normalized to the current before solution change at 120 s (I/I_120s). [Ca²⁺]_i was clamped to near zero with 20 mM BAPTA. The bar indicates application of nominally Ca²⁺-free external solution. Error bars indicate SEM. (B) Average I/V relationships of CRACM2 currents extracted from representative cells shown in (A) obtained at 120 s and 180 s (n = 7). Data represent leak-subtracted current densities (pA/pF) evoked by 50 ms voltage ramps from −150 to +150 mV. (C) Average I/V relationships of CRACM3 currents extracted from representative cells shown in (A) at 120 s and 180 s into the experiment (n = 9). (D) Average normalized CRAC currents (I/I_120s) at −80 mV induced by IP₃ (20 μM) in stable STIM1-expressing HEK293 cells transiently overexpressing CRACM1 (black, n = 5), CRACM2 (blue, n = 7), or CRACM3 (red, n = 10). The bar indicates application of an external solution containing 10 mM Ba²⁺ in the presence of Na⁺. Error bars indicate SEM. (E) Average normalized CRAC currents (I/I_120s) at −80 mV induced by IP₃ (20 μM) in stable STIM1-expressing HEK293 cells transiently overexpressing CRACM1 (black, n = 9; data taken from [5]), CRACM2 (blue, n = 6), or CRACM3 (red, n = 6). The bar indicates application of an external solution containing 10 mM Ba²⁺ with external Na⁺ being replaced by TEA⁺. Error bars indicate SEM. (F) Average normalized CRAC currents (I/I_120s) at −80 mV induced by IP₃ (20 μM) in stable STIM1-expressing HEK293 cells transiently overexpressing CRACM1 (black, n = 3, data taken from [5]), CRACM2 (blue, n = 5), or CRACM3 (red, n = 10). The bar indicates application of divalent-free external solution. Error bars indicate SEM. (G) Average normalized CRAC currents (I/I_120s) at −80 mV induced by IP₃ (20 μM) in stable STIM1-expressing HEK293 cells transiently over-expressing CRACM1 (black, n = 8), CRACM2 (blue, n = 4), or CRACM3 (n = 9). The bar indicates application of external solution containing 50 μM 2-APB. Error bars indicate SEM.