Pharmacology of store-operated calcium channels

Abstract

Store-operated calcium entry is a process by which the depletion of calcium from the endoplasmic reticulum activates calcium influx across the plasma membrane. In the past few years, the major players in this pathway have been identified. STIM1 and STIM2 function as calcium sensors in the endoplasmic reticulum and can interact with and activate plasma membrane channels comprised of Orai1, Orai2, or Orai3 subunits. This review discusses recent advances in our understanding of this widespread signaling mechanism as well as the mechanisms by which a number of interesting pharmacological agents modify it.

Figure 1

Measuring store-operated calcium entry and Icrac. A) Receptor agonists and Ca²⁺-depleting agents (e.g., thapsigargin) induce a biphasic increase in cytoplasmic Ca²⁺, most clearly revealed by protocols involving discharge of Ca²⁺ stores in the absence of extracellular Ca²⁺ (dotted line) followed by restoration of extracellular Ca²⁺. B) The current underlying store-operated Ca²⁺ entry, I_crac, is generally measured by means of the whole-cell patch-clamp technique, utilizing IP₃ and a calcium chelator in the patch pipet solution to deplete Ca²⁺ stores. C) Depletion of Ca²⁺ stores while measuring whole-cell currents reveals a relatively slowly developing inward current, I_crac. Depending on the recording configuration and cell types, these currents are typically small, in the range of 0.5 – 3.0 pA/pF. D) I_crac shows strong inward rectification in the current–voltage relationship, as is expected for a Ca²⁺-selective channel as intracellular Ca²⁺ concentrations are extremely low.

Figure 2

STIM1 and Orai1. A) STIM1 is a single-transmembrane protein localized in the plasma membrane and ER. The N terminus is directed to the lumen of the ER and contains an EF-hand domain that acts as a Ca²⁺ sensor, followed by a sterile α-motif [(SAM); i.e., a protein interaction domain] and the transmembrane (TM) domain. In the C terminus, within a region of coiled-coil domains, is a STIM–Orai activating region (SOAR), which is involved in activation of Orai channels. Also shown is a region enriched in acidic residues that appears to be involved in fast inactivation (FI) by Ca²⁺. A regulatory domain contains potential phosphorylation sites that regulate STIM1 function during the cell cycle (61). At the far N terminus is a lysine-rich (K Rich) domain that may contribute to STIM1 localization in near-membrane puncta by interacting with plasma membrane acidic lipids (20). B) Orai channel subunits span the plasma membrane four times, with C and N termini directed to the cytoplasm. Mutations in human Orai1 at positions 106 and 109 alter channel selectivity, providing evidence that Orai proteins are pore-forming units of the CRAC channel. An arginine/lysine rich (R/K Rich) region in the N terminus (99) is involved in coupling Orai to STIM1, as is the coiled-coil C terminus (100). C) Agonism of plasma membrane receptors (R) coupled through a G protein (G) to phospholipase C (PLC) leads to production of IP₃, which causes Ca²⁺ release from the ER through activation of the IP₃ receptor (IP3R). The drop in Ca²⁺ concentration in the ER mobilizes STIM1 to redistribute to near-membrane sites where it activates channels composed of Orai subunits, thereby causing Ca²⁺ entry. A fraction of cellular STIM1 is also located in the plasma membrane (101) where its function is unclear.

Figure 3

Monster Icrac resulting from overexpressed STIM1 and Orai1. In HEK293 cells overexpressing STIM1 and Orai1, IP₃ causes the development of large inward currents with the properties of I_crac. The time course of inward current development is compared to control (A), and the I_crac-like current–voltage relationship is shown (B). Maximum I_crac from the RBL cell line, one of the greatest natural I_crac currents, is shown by the dotted line for comparison. Adapted from (22).

Figure 4

Biphasic effect of 2-APB on Icrac.A) During thapsigargin-(TG)-activated store-operated Ca²⁺ entry in HEK293 cells, addition of 3 or 10 μM 2-APB enhances entry, whereas addition of 30 or 50 μM inhibits. B) I_crac in HEK293 cells overexpressing Orai1 and STIM1 is augmented by 20 μM 2-APB but inhibited by 50 μM. Adapted from (48).