Pharmacology of Store-Operated Calcium Entry Channels

In general, calcium signaling in non-excitable cells is primarily initiated by the activation of surface membrane receptors coupled to phospholipase C (PLC) and stimulates a calcium signaling process that is complex both spatially and temporally, involving the interplay of calcium channels and calcium pumps [1]. Receptor activation of PLC leads to a breakdown of phosphatidylinositol 4,5-bisphosphate in the plasma membrane and production of diacylglycerol and inositol 1,4,5-trisphosphate (IP) [2]. Fundamentally, receptor activation results in a biphasic process of calcium mobilization composed of the release of intracellular calcium ions from an intracellular organelle, which is coupled to and activates the entry of calcium ions across the plasma membrane of the cell. This second phase of calcium entry is known as store-operated calcium entry (SOCE). Our ability to identify and define underlying calcium signaling processes and mechanisms is greatly facilitated and influenced by two chief experimental approaches to monitor and characterize the mobilization and movement of Ca ions. Fluorescence-based techniques using calcium-sensitive ion probes provide the ability to measure calcium signals with high temporal and spatial resolution, and simultaneously in multiple cells. However, the measured “fluorescent calcium signal” is the result of multiple processes involving calcium pumps and calcium channels that contribute to a steady-state flux of Ca ions. To identify and discern specific calcium signaling pathways using this technique, it has been very important to employ pharmacological manipulations that help define, or rule out, specific mechanisms. As will be described later, these approaches can help define the underlying calcium entry process as SOCE or non-SOCE, and the potential involvement of Orai and canonical transient receptor potential (TRPC) family proteins. The other major and complementary technique for defining calcium signaling processes involves the electrophysiological measurement of ion movement. Importantly, this technique can define and distinguish the biophysical properties of underlying calcium channel activities. In concert with pharmacological manipulations, this technique can be used to identify PLC-activated SOCE ion currents either as I or I and distinguish this from the PLC-activated and non-SOCE ion current I (see Chapters 1 and 11). Much is known about the regulation of intracellular Ca stores by IP [3] and the nature of the SOCE process [4–6]. Discoveries within the past decade have helped identify the molecular players underlying PLC-coupled Ca entry, the Ca sensors STIM1 and STIM2, and the SOCE channel subunit proteins Orai1, Orai2, and Orai3 [7]. Indeed, one can describe three types of channels, I, I, and I. I represents the most extensively characterized store-operated channel and is composed of the pore-forming subunit Orai1, Orai2, or Orai3. I is characterized as a less Ca-selective SOCE channel compared to I that, in addition to the Orai subunit, combines in an incompletely understood manner with TRPC family members (see Chapter 10). As mentioned earlier, there is also a non-store-operated current, I, which is gated by arachidonic acid and involves Orai1, Orai3, and STIM1. Since I and I are composed of Orai subunits, they share some similar properties, yet it is possible to clearly distinguish these calcium entry pathways by both biophysical and pharmacological techniques. I is a small, strongly inwardly rectifying current activated by Ca store depletion and inhibited by the drug 2-APB (discussed later). I is a similarly small and strongly inwardly rectifying current, activated by a ligand (not by store depletion), has a different pH sensitivity, exhibits reduced or lacks fast Ca-dependent inactivation (CDI), does not rapidly depotentiate, and is not inhibited by 2-APB. In addition, Orai1 was recently discovered to be expressed as two isoforms due to alternative translation initiation, Orai1α (long) and Orai1β (short) [8]. Channels composed of either Orai1α and Orai1β can associate with STIM1 and form CRAC or SOC channels. However, only Orai1α, and not Orai1β, undergoes CDI, and only Orai1α appears to form channels underlying I [9] (see Chapter 11). Today, our ability to pharmacologically dissect and manipulate the SOCE calcium signaling pathway remains a readily accessible way to understand receptor-regulated calcium signaling in a wide variety of biological systems. This is particularly useful in systems where molecular biological strategies are difficult to employ. However, it always remains a challenge to ensure these pharmacological approaches provide some degree of specificity and control.