The action of selective CRAC channel blockers is affected by the Orai pore geometry

Abstract

As the molecular composition of calcium-release activated calcium (CRAC) channels has been unknown for two decades, elucidation of selective inhibitors has been considerably hampered. By the identification of the two key components of CRAC channels, STIM1 and Orai1 have emerged as promising targets for CRAC blockers. The aim of this study was to thoroughly characterize the effects of two selective CRAC channel blockers on currents derived from STIM1/Orai heterologoulsy expressed in HEK293 cells. The novel compounds GSK-7975A and GSK-5503A were tested for effects on STIM1 mediated Orai1 or Orai3 currents by whole-cell patch-clamp recordings and for the effects on STIM1 oligomerisation or STIM1/Orai coupling by FRET microscopy. To investigate their site of action, inhibitory effects of these molecules were explored using Orai pore mutants. The GSK blockers inhibited Orai1 and Orai3 currents with an IC(50) of approximately 4μM and exhibited a substantially slower rate of onset than the typical pore blocker La(3+), together with almost no current recovery upon wash-out over 4min. For the less Ca(2+)-selective Orai1 E106D pore mutant, I(CRAC) inhibition was significantly reduced. FRET experiments indicated that neither STIM1-STIM1 oligomerization nor STIM1-Orai1 coupling was affected by these compounds. These CRAC channel blockers are acting downstream of STIM1 oligomerization and STIM1/Orai1 interaction, potentially via an allosteric effect on the selectivity filter of Orai. The elucidation of these CRAC current blockers represents a significant step toward the identification of CRAC channel-selective drug compounds.

Fig. 1

Structures of GSK-CRAC channel inhibitors in comparison to Synta-66.

Fig. 2

Inhibitory profiles of known and GSK-CRAC channel blockers on STIM1/Orai1 currents. (A) Time-course of whole cell inward currents at −74 mV maximally activated upon passive store-depletion of HEK293 cells co-expressing CFP-STIM1 with YFP-Orai1 upon perfusion of 10 μM La³⁺, 10 μM GSK-5503A and 10 μM GSK-7975A. Time-axes in (A and B) were shifted to superimpose time-points of drug administration. (B) Time-course of normalized whole cell inward currents at −74 mV, maximally activated upon passive store-depletion of HEK293 cells co-expressing CFP-STIM1 with YFP-Orai1 upon perfusion of 10 μM La³⁺, 10 μM GSK-5503A and 10 μM GSK-7975A in comparison to 75 μM 2-APB, 10 μM DES and 10 μM Synta-66 (t = 0 s was shifted to a time-point where currents had already reached their maximum). (C and D) Corresponding I/V relationships to (A, B: 1, 2, 3) of STIM1/Orai1 currents after maximal activation (1), after ∼half maximal block (2) as well as complete block (3) by 10 μM GSK-7975 (C) or 10 μM GSK-5503A (D). (E) Block diagram representing half-maximal inhibition time t_1/2 of 10 μM La³⁺, 75 μM 2-APB, 10 μM DES, 10 μM Synta-66, 10 μM GSK-5503A and 10 μM GSK-7975A.

Fig. 3

Inhibitory profiles of known and GSK-CRAC channel blockers on STIM1/Orai3 currents. (A) Time-course of whole cell inward currents at −74 mV maximally activated upon passive store-depletion of HEK293 cells co-expressing CFP-STIM1 with YFP-Orai3 upon perfusion of 10 μM La³⁺, 10 μM GSK-5503A and 10 μM GSK-7975A. Time-axes in (A and B) were shifted to superimpose time-points of drug administration. (B) Time-course of normalized whole cell inward currents at −74 mV maximally activated upon passive store-depletion of HEK293 cells co-expressing CFP-STIM1 with YFP-Orai3 upon perfusion of 10 μM La³⁺, 10 μM GSK-5503A and 10 μM GSK-7975A in comparison to 10 μM Synta-66 (t = 0 s was shifted to a time-point where currents had already reached their maximum). (C and D) Corresponding I/V relationships to (A, B: 1, 2, 3) of STIM1/Orai3 currents after maximal activation (1), after ∼half maximal block (2) as well as complete block (3) by 10 μM GSK-7975 (C) or 10 μM GSK-5503A (D). (E) Block diagram representing half-maximal inhibition time t_1/2 of 10 μM La³⁺, 10 μM Synta-66, 10 μM GSK-5503A and 10 μM GSK-7975A.

Fig. 4

Wash-out of GSK-CRAC channel blockers following inhibition of STIM1/Orai1 and STIM1/Orai3 currents. Time-course of whole cell inward currents at −74 mV maximally activated upon passive store-depletion of HEK293 cells co-expressing CFP-STIM1 with YFP-Orai1 (A and B) or YFP-Orai3 (E and F). Upon full blockade of STIM1/Orai currents by 10 μM GSK-7975A (A and E) or 10 μM GSK-5503A (B and F) 10 mM Ca²⁺ solution was perfused for wash-out (t = 0 s was shifted to a time-point where currents had already reached their maximum). Corresponding I/V relationships to (A, B, E, F: 1, 2, 3) of STIM1/Orai1 (C and D) and STIM1/Orai3 (G and H) currents upon maximal store-operated activation (1), upon complete inhibition (2) of 10 μM GSK-7975A (C and G) or 10 μM GSK-5503A (D and H) and after washout (3) of the respective blocker.

Fig. 5

Dose-response relationships of the CRAC channel blocker GSK-7975A on STIM1/Orai1 and STIM1/Orai3 currents. (A and B) Time-course of whole cell inward currents at −74 mV maximally activated upon passive store-depletion of HEK293 cells co-expressing CFP-STIM1 with YFP-Orai1 (A) or YFP-Orai3 (B) upon perfusion of 0.3 μM, 1 μM, 3 μM, 10 μM GSK-7975A (t = 0 s was shifted to a time-point where currents had already reached their maximum). (C and D) Corresponding I/V relationships to (A and B) of STIM1/Orai1 (C) and STIM1/Orai3 (D) currents upon maximal inhibition by 0, 0.3, 1, 3, 10 μM GSK-7975A. (E and F) Concentration–response relationship depicting the inhibitory effect of GSK-7975A on CFP-STIM1-mediated YFP-Orai1 (E) and YFP-Orai3 (F) currents.

Fig. 6

GSK-7975A impaired neither STIM1 oligomerization nor STIM1/Orai1 coupling. (A) Left: time course of relative FRET between CFP-STIM1 and YFP-STIM1 with or without perfusion of GSK-7975A. Right: localization, overlay and calculated FRET life cell image series of YFP-STIM1 and CFP-STIM1 (1) in the absence and (2) presence of GSK-7975A and (3) following store-depletion in the presence of GSK-7975A. (B) Left: time course of relative FRET between STIM1-CFP and Orai1-YFP upon store-depletion and after application of GSK-7975A. Right: localization, overlay and calculated FRET life cell image series of STIM1-CFP and Orai1-YFP (1) after store-depletion and (2) after perfusion of GSK-7975A.

Fig. 7

The less Ca²⁺-selective Orai3 current elicited by 2-APB is substantially less susceptible to GSK-7975A inhibition. (A–C) Time-courses of 2-APB-stimulated Orai3 currents, which were treated after maximal activation with 10 μM (A), 50 μM (B) and 75 μM (C) GSK-7975A. (D) I/V-relationship of 2-APB stimulated Orai3 currents upon addition of 10 μM, 50 μM or 100 μM GSK-7975A.

Fig. 8

Inhibitory profile of GSK-7975A on less Ca²⁺-selective or monovalent Orai1 currents. (A and B) Time course of whole cell inward currents at −74 mV maximally activated upon passive store-depletion of HEK293 cells co-expressing CFP-STIM1 with YFP-Orai1 E106D upon perfusion of 10 μM (A) and 100 μM (B) GSK-7975A. (C and D) Corresponding I/V relationships to (A, B: 1, 2) of STIM1/Orai1 E106D currents after maximal store-operated activation (1) and upon addition (2) of 10 μM (C) or 100 μM (D) GSK-7975A. (E) Time course of whole cell inward currents at −74 mV maximally activated upon passive store-depletion of HEK293 cells co-expressing CFP-STIM1 with YFP-Orai1 D110/112/114A upon perfusion of 10 μM GSK-7975A (t = 0 s was shifted to a time-point where currents had already reached their maximum). (F) Time course of whole cell inward currents at −74 mV maximally activated upon passive store-depletion of HEK293 cells co-expressing CFP-STIM1 with YFP-Orai1 in 10 mM Ca²⁺ solution. Afterwards 10 mM Ca²⁺ solution was exchanged by a DVF solution and blocked by 10 μM GSK-7975A. (G) Corresponding I/V relationships to (E: 1, 2) of STIM1/Orai1 D110/112/114A currents after maximal store-operated activation (1) and upon addition (2) of 10 μM GSK-7975A. (H) Corresponding I/V relationships to (F: 1, 2, 3) of maximal activated STIM1/Orai1 current in 10mM Ca²⁺ containing solution (1), DVF solution (2) and upon addition (3) of 10 μM GSK-7975A in DVF solution.

Fig. 9

Inhibitory profile of GSK-7975A on endogenous CRAC currents and Ca²⁺ entry of RBL cells. (A) Time course of whole cell inward currents at −74 mV maximally activated upon passive store-depletion of RBL-2H3 cells upon perfusion of 10 μM GSK-7975A in comparison to 10 μM La³⁺. (B) Concentration-dependent inhibition of thapsigargin-evoked Ca²⁺ influx from Fura-4 loaded RBL cells expressed as fold over unstimulated cells.

Fig. 10

Inhibitory profile of GSK-7975A on other Ca²⁺-selective channels. (A) Time course of whole cell inward currents at −74 mV of TRPV6 expressing HEK293 cells upon perfusion of 10 μM GSK-7975A in comparison to 10 μM La³⁺. (B) Time course of whole cell inward currents at −74 mV of l-type Ca²⁺-channel expressing HEK293 cells upon perfusion of 10 μM GSK-7975A in comparison to 10 μM La³⁺.