A number of important second messengers, used in several signal transduction pathways, are derived from the membrane lipid phosphatidylinositol (PI; Figure 15-33). The inositol group in this phospholipid, which always faces the cytosol, can be reversibly phosphorylated at one or more positions by the combined actions of various kinases and phosphatases discussed in Chapter 16. One derivative of PI, the lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂], is made by stepwise addition of two phosphates to PI. PI(4,5)P₂ is then cleaved by activated phospholipase C into two important second messengers: 1,2-diacylglycerol (DAG), a lipophilic molecule that remains associated with the membrane, and inositol 1,4,5-trisphosphate (IP₃), which can freely diffuse in the cytosol (see Figure 15-33). We refer to downstream events involving these two second messengers collectively as the IP₃/DAG pathway.

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Phospholipase C is activated by G proteins containing either G_αo or G_αq subunits. In response to hormone activation of their associated GPCR, the G_αo or G_αq subunits bound to GTP separate from G_βγ and bind to and activate phospholipase C in the membrane (Figure 15-34a, step 1). In turn, activated phospholipase C cleaves PI(4,5)P₂ into DAG and IP₃ (see Figure 15-34a, step 2). The two second messengers trigger distinct, but sometimes interrelated, downstream effects.

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Ca²⁺ Release from the ER Triggered by IP₃ Ligand binding to G protein–coupled receptors that activate phospholipase C induces an elevation in cytosolic Ca²⁺ even when Ca²⁺ ions are absent from the surrounding extracellular fluid. In this case, Ca²⁺ is released into the cytosol from the ER lumen (where, as noted above, Ca²⁺ can accumulate to almost millimolar concentrations) through the operation of IP₃-gated Ca²⁺ channels in the ER membrane, as depicted in Figure 15-34a (steps 3 and 4). (This family of channel proteins is similar in structure to the family of voltage-sensitive Ca²⁺ channels that are called ryanodine receptors in muscle cell sarcoplasmic reticulum membranes; see page 805.) Each of these large IP₃-gated channels is composed of four identical subunits, each of which contains an IP₃-binding site in its N-terminal cytosolic domain. IP₃ binding induces opening of the channel, allowing Ca²⁺ to flow down its concentration gradient from the ER lumen into the cytosol. When different phosphorylated inositols normally found in cells were added to preparations of ER vesicles, only IP₃ caused release of Ca²⁺ ions from the vesicles. This simple experiment demonstrates the specificity of the IP₃ effect.

The IP₃-mediated rise in the cytosolic Ca²⁺ level is transient because Ca²⁺ pumps located in the plasma membrane and in the ER membrane actively transport Ca²⁺ from the cytosol to the cell exterior and back into the ER lumen, respectively. Furthermore, within a second of its generation, the phosphate linked to carbon 5 of IP₃ is hydrolyzed, yielding inositol 1,4-bisphosphate (see Figure 15-33). This compound cannot bind to the IP₃-gated Ca²⁺ channel and thus does not stimulate Ca²⁺ release from the ER.

Ca²⁺ Transport from the ER to the Mitochondrial Matrix Triggered by IP₃ In Chapter 12, we learned that direct contacts between specialized regions of the ER membrane, called mitochondria-associated membranes (MAMs), and the outer mitochondrial membrane affect mitochondrial structure, dynamics, and function. Regulated movement of Ca²⁺ from the ER lumen through the MAMs into the mitochondrial matrix is a major part of this regulation (Figure 15-35a). IP₃-gated Ca²⁺ channels in the MAMs open in response to a rise in cytosolic IP₃ (step 1a, Figure 15-35a). Voltage-dependent anion channels (VDACs) in the outer mitochondrial membrane adjacent to the MAMs, which are physically linked to these IP₃-gated Ca²⁺ channels by the GRP75 protein, efficiently pass the Ca²⁺ released from the ER lumen into the intermembrane space (step 2). A mitochondrial calcium uniporter (MCU) in the inner mitochondrial membrane then transports Ca²⁺ into the mitochondrial matrix, where it increases ATP synthesis as well as enhancing mitochondrial activity in other ways.

MCUs open only in the presence of a high Ca²⁺ concentration in the intermembrane space. Regulatory subunits of the MCU that face the intermembrane space (Figure 15-35b) have Ca²⁺-binding EF hands (see Chapter 3) with relatively low binding affinity for Ca²⁺; these subunits must bind calcium for the MCU to open. Individuals with mutations in the gene encoding one of these subunits have skeletal muscle defects and learning disabilities, symptoms that can accompany other mitochondrial disorders, attesting to the importance of these uniporters in mitochondrial metabolism.

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To avoid the buildup of excess, potentially toxic, intramitochondrial calcium, the mitochondrial matrix gradually releases Ca²⁺ into the cytosol. The calcium first moves across the inner mitochondrial membrane into the intermembrane space via Na⁺/Ca²⁺ and H⁺/Ca²⁺ antiporters, then crosses the outer mitochondrial membrane, most likely via VDACs (step 4). The calcium transport cycle is completed when cytosolic calcium enters the ER via an ATP-powered Ca²⁺ pump (step 5; see Figure 11-10) or is pumped out of the cell by plasma-membrane ATP-powered Ca²⁺ pumps.

The Store-Operated Plasma-Membrane Ca²⁺ Channel Continued opening of the IP₃-gated Ca²⁺ channel in the ER membrane, coupled with operation of the plasma-membrane Ca²⁺ pump, would eventually deplete the intracellular stores of Ca²⁺, and a cell would soon be unable to increase the cytosolic Ca²⁺ level in response to hormone-induced IP₃. Patch-clamping studies (see Figure 11-22) have revealed that a plasma-membrane Ca²⁺ channel called the store-operated channel opens in response to depletion of ER Ca²⁺ stores and admits extracellular Ca²⁺ into the cytosol. Studies in which each potential Ca²⁺ channel protein was knocked down one at a time with shRNAs (see Figure 6-42) established the identity of this channel protein as Orai1; the sequence and three-dimensional structure of Orai1 is unlike that of any other known ion-channel protein (see Figure 15-34c), which partly explains why it took so long to identify it as the store-operated channel.

The ER Ca²⁺-sensing protein is STIM, a transmembrane protein in the ER membrane (see Figure 15-34b). An EF hand, similar to that in calmodulin (see Figure 3-33), on the luminal side of the ER membrane binds Ca²⁺ when its level in the lumen is high. As stores of Ca²⁺ in the ER are depleted, the STIM proteins lose their bound Ca²⁺, oligomerize, and in an unknown manner, relocalize to areas of the ER membrane near the plasma membrane (see Figure 15-34b). There the CAD domains of the STIM proteins bind to and trigger the opening of Orai1, allowing the influx of extracellular Ca²⁺ (see Figure 15-34c). Combined overexpression of Orai and STIM in cultured cells leads to a marked increase in Ca²⁺ influx, establishing that these two proteins are the key components of the store-operated Ca²⁺ pathway.

Feedback Loops That Trigger Spikes in the Cytosolic Ca²⁺ Concentration Continuous activation of certain G protein–coupled receptors induces rapid, repeated spikes in the level of cytosolic Ca²⁺ (Figure 15-36). These oscillations in cytosolic Ca²⁺ levels are caused by a complex feedback interaction between the cytosolic Ca²⁺ concentration and the IP₃-gated Ca²⁺-channels. The submicromolar level of cytosolic Ca²⁺ in the resting cell potentiates the opening of these channels via a rise in IP₃, thus facilitating the rapid rise in cytosolic Ca²⁺ that follows hormone stimulation of cell-surface G protein–coupled receptors. However, the high cytosolic Ca²⁺ levels reached at the peak of the spike inhibit further IP₃-induced release of Ca²⁺ from ER stores by decreasing the affinity of the Ca²⁺ channels for IP₃. As a result, the IP₃-gated Ca²⁺ channels close, and the cytosolic Ca²⁺ level drops rapidly as Ca²⁺ is pumped into the ER lumen or out of the cell. Thus cytosolic Ca²⁺ is a feedback inhibitor of the IP₃-gated Ca²⁺ channels that, when open, trigger a rise in cytosolic Ca²⁺. As an example, this mechanism produces calcium ion oscillations occur in the pituitary gland cells that secrete luteinizing hormone (LH), which plays an important role in controlling ovulation and thus female fertility. LH secretion is induced by the binding of luteinizing hormone–releasing hormone (LHRH) to its G protein–coupled receptors on the surfaces of these cells; LHRH binding induces repeated Ca²⁺ spikes. Each Ca²⁺ spike induces exocytosis of a few LH-containing secretory vesicles, presumably those close to the plasma membrane.