Barbara E. Ehrlich

Polycystin-2 is an intracellular calcium release channel

Polycystin-2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease (ADPKD), is the prototypical member of a subfamily of the transient receptor potential (TRP) channel superfamily, which is expressed abundantly in the endoplasmic reticulum (ER) membrane. Here, we show by single channel studies that polycystin-2 behaves as a calcium-activated, high conductance ER channel that is permeable to divalent cations. Epithelial cells overexpressing polycystin-2 show markedly augmented intracellular calcium release signals that are lost after carboxy-terminal truncation or by the introduction of a disease-causing missense mutation. These data suggest that polycystin-2 functions as a calcium-activated intracellular calcium release channel in vivo and that polycystic kidney disease results from the loss of a regulated intracellular calcium release signalling mechanism.

The pharmacology of intracellular Ca2+-release channels

Abstract Two classes of intracellular Ca 2+ -release channels, the ryanodine reccptor and the mositol (1,4,5)-trisphosphate (IP 3 ) receptor, are essential for spatiotemporal Ca 2+ signalling in cells. Heparin and caffeine have been widely used to study these channels. It was originally thought that caffeine acts solely as an agonist for the ryanodine receptoi and heparin acts solely as an inhibitor for the IP 3 receptor. However, recent experiments indicate that these compounds have multiple effects, and are discussed in this review by Barbara Ehrlich and colleagues . In the same concentration range, caffeine activates the ryanodine receptor and inhibits the IP 3 receptor, and hepaun inhibits the IP 3 receptor and activates the ryanodine leceptor. More specific pharmacological tools that are suitable for studies of ryanodine and IP 3 receptors are now beginning to emerge.

Type III InsP3 receptor channel stays open in the presence of increased calcium

The inositol 1,4,5-trisphosphate receptor (InsP3R) is the main calcium(Ca2+) release channel in most tissues. Three isoforms have been identified, but only types I and II InsP3R have been characterized,. Here we examine the functional properties of the type III InsP3R because this receptor is restricted to the trigger zone from which Ca2+ waves originate and it has distinctive InsP3-binding properties,. We find that type III InsP3R forms Ca2+ channels with single-channel currents that are similar to those of type I InsP3R; however, the open probability of type III InsP3R isoform increases monotonically with increased cytoplasmic Ca2+ concentration, whereas the type I isoform has a bell-shaped dependence on cytoplasmic Ca2+. The properties of type III InsP3R provide positive feedback as Ca2+ is released; the lack of negative feedback allows complete Ca2+ release from intracellular stores. Thus, activation of type III InsP3R in cells that express only this isoform results in a single transient, but global, increase in the concentration of cytosolic Ca2+. The bell-shaped Ca2+-dependence curve of type I InsP3R is ideal for supporting Ca2+ oscillations, whereas the properties of type III InsP3R are better suited to signal initiation.

Nuclear and cytosolic calcium are regulated independently

Nuclear calcium (Ca2+) regulates a number of important cellular processes, including gene transcription, growth, and apoptosis. However, it is unclear whether Ca2+ signaling is regulated differently in the nucleus and cytosol. To investigate this possibility, we examined subcellular mechanisms of Ca2+ release in the HepG2 liver cell line. The type II isoform of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) was expressed to a similar extent in the endoplasmic reticulum and nucleus, whereas the type III InsP3R was concentrated in the endoplasmic reticulum, and the type I isoform was not expressed. Ca2+ signals induced by low InsP3 concentrations started earlier or were larger in the nucleus than in the cytosol, indicating higher sensitivity of nuclear Ca2+ stores for InsP3. Nuclear InsP3R channels were active at lower InsP3 concentrations than InsP3R from cytosol. Enriched expression of type II InsP3R in the nucleus results in greater sensitivity of the nucleus to InsP3, thus providing a mechanism for independent regulation of Ca2+-dependent processes in this cellular compartment.

Cilia at the node of mouse embryos sense fluid flow for left-right determination via Pkd2.

Distinguishing Right from Left In most vertebrates during embryonic development, rotational movement of the cilia within a structure in the embryo, known as the node, generates unidirectional flow required for future left-right asymmetry of the internal organs. The flow may transport a determinant molecule or provide mechanical force. However, it is not clear how the flow is sensed. Yoshiba et al. (p. 226, published online 13 September; see the Perspective by Norris and Grimes) show that nodal flow in mouse embryos is sensed by the cilia of perinodal cells located at the edge of the node, in a manner dependent on Pkd2, a Ca2+-permeable cation channel that has been implicated in polycystic kidney disease in humans. A Ca2+ channel implicated in polycystic kidney disease helps to establish the left-right body axis of the mammalian embryo. Unidirectional fluid flow plays an essential role in the breaking of left-right (L-R) symmetry in mouse embryos, but it has remained unclear how the flow is sensed by the embryo. We report that the Ca2+ channel Polycystin-2 (Pkd2) is required specifically in the perinodal crown cells for sensing the nodal flow. Examination of mutant forms of Pkd2 shows that the ciliary localization of Pkd2 is essential for correct L-R patterning. Whereas Kif3a mutant embryos, which lack all cilia, failed to respond to an artificial flow, restoration of primary cilia in crown cells rescued the response to the flow. Our results thus suggest that nodal flow is sensed in a manner dependent on Pkd2 by the cilia of crown cells located at the edge of the node.

The Inositol 1,4,5-Trisphosphate Receptor (IP3R) and Its Regulators: Sometimes Good and Sometimes Bad Teamwork

In both nonexcitable and excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is the primary cytosolic target responsible for the initiation of intracellular calcium (Ca2+) signaling. To fulfill this function, the IP3R depends on interaction with accessory subunits and regulatory proteins. These include proteins that reside in the lumen of the endoplasmic reticulum (ER), such as chromogranin A and B and ERp44, and cytosolic proteins, such as neuronal Ca2+ sensor 1, huntingtin, cytochrome c, IP3R-binding protein released with inositol 1,4,5-trisphosphate, Homer, and 4.1N. Specific interactions between these modulatory proteins and the IP3R have been described, making it clear that the controlled modulation of the IP3R by its binding partners is necessary for physiological cell regulation. The functional coupling of these modulators with the IP3R can control apoptosis, intracellular pH, the initiation and regulation of neuronal Ca2+ signaling, exocytosis, and gene expression. The pathophysiological relevance of IP3R modulation is apparent when the functional interaction of these proteins is enhanced or abolished by mutation or overexpression. The subsequent deregulation of the IP3R leads to pathological changes in Ca2+ signaling, signal initiation, the amplitude and frequency of Ca2+ signals, and the duration of the Ca2+ elevation. Consequences of this deregulation include abnormal growth and apoptosis. Complex regulation of Ca2+ signaling is required for the cell to live and function, and this difficult task can only be managed when the IP3R teams up and acts properly with its numerous binding partners. Changes in the amount of calcium inside cells initiate many cellular events, including muscle contraction, hormone secretion, and cell growth. These changes can be modulated in time and space by the cell to tailor its response to the prevailing conditions. A major pathway used for the release of calcium from intracellular stores involves opening the inositol 1,4,5-trisphosphate receptor (IP3R), a calcium channel that is part of a signaling complex with multiple binding partners. This Review, which contains 2 tables, 6 figures, and 119 references, discusses the role of specific binding partners in regulating the function of the IP3R. These studies provide information on how these channels work and form the background for the investigation of disease-induced changes in calcium release channel function. The initiation of intracellular calcium signals can depend on factors as diverse as the nonuniform distribution of a binding partner in the lumen of the endoplasmic reticulum or the activation of a calcium-binding protein in the cytoplasm. Once calcium signals start, they can be transient, sustained, or oscillating; these temporal aspects of the signal are regulated by similarly diverse factors. Changes in intracellular calcium regulation occur in many diseases. As more and more new regulators of the IP3R are discovered, it will be important to understand the teamwork needed to regulate and modulate this complex system.

Small molecule Wnt inhibitors enhance the efficiency of BMP-4-directed cardiac differentiation of human pluripotent stem cells

Human induced pluripotent stem (iPS) cells potentially provide a unique resource for generating patient-specific cardiomyocytes to study cardiac disease mechanisms and treatments. However, existing approaches to cardiomyocyte production from human iPS cells are inefficient, limiting the application of iPS cells in basic and translational cardiac research. Furthermore, strategies to accurately record changes in iPS cell-derived cardiomyocyte action potential duration (APD) are needed to monitor APD-related cardiac disease and for rapid drug screening. We examined whether modulation of the bone morphogenetic protein 4 (BMP-4) and Wnt/β-catenin signaling pathways could induce efficient cardiac differentiation of human iPS cells. We found that early treatment of human iPS cells with BMP-4 followed by late treatment with small molecule Wnt inhibitors led to a marked increase in production of cardiomyocytes compared to existing differentiation strategies. Using immunocytochemical staining and real-time intracellular calcium imaging, we showed that these induced cardiomyocytes expressed typical sarcomeric markers, exhibited normal rhythmic Ca(2+) transients, and responded to both β-adrenergic and electric stimulation. Furthermore, human iPS cell-derived cardiomyocytes demonstrated characteristic changes in action potential duration in response to cardioactive drugs procainamide and verapamil using voltage-sensitive dye-based optical recording. Thus, modulation of the BMP-4 and Wnt signaling pathways in human iPS cells leads to highly efficient production of cardiomyocytes with typical electrophysiological function and pharmacologic responsiveness. The use of human iPS cell-derived cardiomyocytes and the application of calcium- and voltage-sensitive dyes for the direct, rapid measurement of iPS cell-derived cardiomyocyte activity promise to offer attractive platforms for studying cardiac disease mechanisms and therapeutics.

Regulation of ryanodine receptor-dependent calcium signaling by polycystin-2

Mutations in polycystin-2 (PC2) cause autosomal dominant polycystic kidney disease. A function for PC2 in the heart has not been described. Here, we show that PC2 coimmunoprecipitates with the cardiac ryanodine receptor (RyR2) from mouse heart. Biochemical assays showed that the N terminus of PC2 binds the RyR2, whereas the C terminus only binds to RyR2 in its open state. Lipid bilayer electrophysiological experiments indicated that the C terminus of PC2 functionally inhibited RyR2 channel activity in the presence of calcium (Ca2+). Pkd2−/− cardiomyocytes had a higher frequency of spontaneous Ca2+ oscillations, reduced Ca2+ release from the sarcoplasmic reticulum stores, and reduced Ca2+ content compared with Pkd2+/+ cardiomyocytes. In the presence of caffeine, Pkd2−/− cardiomyocytes exhibited decreased peak fluorescence, a slower rate of rise, and a longer duration of Ca2+ transients compared with Pkd2+/+. These data suggest that PC2 is important for regulation of RyR2 function and that loss of this regulation of RyR2, as occurs when PC2 is mutated, results in altered Ca2+ signaling in the heart.

Three‐dimensional structure of the type 1 inositol 1,4,5‐trisphosphate receptor at 24 Å resolution

We report here the first three‐dimensional structure of the type 1 inositol 1,4,5‐trisphosphate receptor (IP3R). From cryo‐electron microscopic images of purified receptors embedded in vitreous ice, a three‐dimensional structure was determined by use of standard single particle reconstruction techniques. The structure is strikingly different from that of the ryanodine receptor at similar resolution despite molecular similarities between these two calcium release channels. The 24 Å resolution structure of the IP3R takes the shape of an uneven dumbbell, and is ∼170 Å tall. Its larger end is bulky, with four arms protruding laterally by ∼50 Å and, in comparison with the receptor topology, probably corresponds to the cytoplasmic domain of the receptor. The lateral dimension at the height of the protruding arms is ∼155 Å. The smaller end, whose lateral dimension is ∼100 Å, has structural features indicative of the membrane‐spanning domain. A central opening in this domain, which is occluded on the cytoplasmic half, outlines a pathway for calcium flow in the open state of the channel.

Elevated testosterone induces apoptosis in neuronal cells

Testosterone plays a crucial role in neuronal function, but elevated concentrations can have deleterious effects. Here we show that supraphysiological levels of testosterone (micromolar range) initiate the apoptotic cascade. We used three criteria, annexin V labeling, caspase activity, and DNA fragmentation, to determine that apoptotic pathways were activated by testosterone. Micromolar, but not nanomolar, testosterone concentrations increased the response in all three assays of apoptosis. In addition, testosterone induced different concentration-dependent Ca2+ signaling patterns: at low concentrations of testosterone (100 nm), Ca2+ oscillations were produced, whereas high concentrations (1-10 μm) induced a sustained Ca2+ increase. Elevated testosterone concentrations increase cell death, and this effect was abolished in the presence of either inhibitors of caspases or the inositol 1,4,5-trisphosphate receptor (InsP3R)-mediated Ca2+ release. Knockdown of InsP3R type 1 with specific small interfering RNA also abolished the testosterone-induced cell death and the prolonged Ca2+ signals. In contrast, knockdown of InsP3R type 3 modified neither the apoptotic response nor the Ca2+ signals. These results support our hypothesis that elevated testosterone alters InsP3R type 1-mediated intracellular Ca2+ signaling and that the prolonged Ca2+ signals lead to apoptotic cell death. These effects of testosterone on neurons will have long term effects on brain function.