Tatiana Kilch

Mutations of the Ca2+-sensing Stromal Interaction Molecule STIM1 regulate Ca2+ influx by altered oligomerization of STIM1 and by destabilization of the Ca2+ channel Orai1

Background: Calcium influx (ICRAC) is important for proper cell function. Results: A novel STIM1 mutant increases ICRAC, Ca2+-dependently destabilizes Orai1, and alters clustering. A new mathematical model explains the phenotype. Conclusion: The molecular kinetics of STIM1 and Orai1 are major determinants of ICRAC. Significance: The diffusion trap model and alteration of Orai1 stability provide a tool for understanding ICRAC regulation. A drop of endoplasmic reticulum Ca2+ concentration triggers its Ca2+ ssensor protein stromal interaction molecule 1 (STIM1) to oligomerize and accumulate within endoplasmic reticulum-plasma membrane junctions where it activates Orai1 channels, providing store-operated Ca2+ entry. To elucidate the functional significance of N-glycosylation sites of STIM1, we created different mutations of asparagine-131 and asparagine-171. STIM1 NN/DQ resulted in a strong gain of function. Patch clamp, Total Internal Reflection Fluorescent (TIRF) microscopy, and fluorescence recovery after photobleaching (FRAP) analyses revealed that expression of STIM1 DQ mutants increases the number of active Orai1 channels and the rate of STIM1 translocation to endoplasmic reticulum-plasma membrane junctions with a decrease in current latency. Surprisingly, co-expression of STIM1 DQ decreased Orai1 protein, altering the STIM1:Orai1 stoichiometry. We describe a novel mathematical tool to delineate the effects of altered STIM1 or Orai1 diffusion parameters from stoichiometrical changes. The mutant uncovers a novel mechanism whereby “superactive” STIM1 DQ leads to altered oligomerization rate constants and to degradation of Orai1 with a change in stoichiometry of activator (STIM1) to effector (Orai1) ratio leading to altered Ca2+ homeostasis.

ORAI1 Ca2+ Channels Control Endothelin-1-Induced Mitogenesis and Melanogenesis in Primary Human Melanocytes

UV radiation of the skin triggers keratinocytes to secrete endothelin-1 (ET-1) that binds to endothelin receptors on neighboring melanocytes. Melanocytes respond with a prolonged increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), which is necessary for proliferation and melanogenesis. A major fraction of the Ca(2+) signal is caused by entry through Ca(2+)-permeable channels of unknown identity in the plasma membrane. ORAI Ca(2+) channels are molecular determinants of Ca(2+) release-activated Ca(2+) (CRAC) channels and are expressed in many tissues. Here, we show that ORAI1-3 and their activating partners stromal interaction molecules 1 and 2 (STIM1 and STIM2) are expressed in human melanocytes. Although ORAI1 is the predominant ORAI isoform, STIM2 mRNA expression exceeds STIM1. Inhibition of ORAI1 by 2-aminoethoxydiphenyl borate (2-APB) or downregulation of ORAI1 by small interfering RNA (siRNA) reduced Ca(2+) entry and CRAC current amplitudes in activated melanocytes. In addition, suppression of ORAI1 caused reduction in the ET-1-induced cellular viability, melanin synthesis, and tyrosinase activity. Our results imply a role for ORAI1 channels in skin pigmentation and their potential involvement in UV-induced stress responses of the human skin.

ROS and SOCE: recent advances and controversies in the regulation of STIM and Orai

Abstract  Store‐operated Ca2+ entry (SOCE) is a widespread mechanism in cells to raise cytosolic Ca2+ and to refill Ca2+ stores. T cells critically rely on SOCE mediated by stromal interaction molecules (STIM) and Orai molecules for their activation and regulation of gene transcription; cells such as muscle cells, neurons or melanocytes probably utilize SOCE for the transmission of inducible receptor‐mediated function as well as for generalized Ca2+ homeostasis mechanisms. Exposure to environmental or cell‐intrinisic reactive oxygen species (ROS) can affect several components involved in Ca2+ homeostasis and thus alter multiple pathways. While all cells have a capacity to produce intracellular ROS, exposure of immune and skin cells to extracellular oxidative stress is particularly high during inflammation and/or with UV exposure. This review briefly summarizes cell‐intrinsic sources of ROS and focuses on current findings and controversies regarding the regulation of STIM and Orai by oxidative modifications. We also introduce melanocytes as a new model system to study the function of STIM and Orai isoforms under physiological conditions that include exposure to UV light as an activating stimulus.

Differential Redox Regulation of Ca2+ Signaling and Viability in Normal and Malignant Prostate Cells

In prostate cancer, reactive oxygen species (ROS) are elevated and Ca(2+) signaling is impaired. Thus, several novel therapeutic strategies have been developed to target altered ROS and Ca(2+) signaling pathways in prostate cancer. Here, we investigate alterations of intracellular Ca(2+) and inhibition of cell viability caused by ROS in primary human prostate epithelial cells (hPECs) from healthy tissue and prostate cancer cell lines (LNCaP, DU145, and PC3). In hPECs, LNCaP and DU145 H2O2 induces an initial Ca(2+) increase, which in prostate cancer cells is blocked at high concentrations of H2O2. Upon depletion of intracellular Ca(2+) stores, store-operated Ca(2+) entry (SOCE) is activated. SOCE channels can be formed by hexameric Orai1 channels; however, Orai1 can form heteromultimers with its homolog, Orai3. Since the redox sensor of Orai1 (Cys-195) is absent in Orai3, the Orai1/Orai3 ratio in T cells determines the redox sensitivity of SOCE and cell viability. In prostate cancer cells, SOCE is blocked at lower concentrations of H2O2 compared with hPECs. An analysis of data from hPECs, LNCaP, DU145, and PC3, as well as previously published data from naive and effector TH cells, demonstrates a strong correlation between the Orai1/Orai3 ratio and the SOCE redox sensitivity and cell viability. Therefore, our data support the concept that store-operated Ca(2+) channels in hPECs and prostate cancer cells are heteromeric Orai1/Orai3 channels with an increased Orai1/Orai3 ratio in cells derived from prostate cancer tumors. In addition, ROS-induced alterations in Ca(2+) signaling in prostate cancer cells may contribute to the higher sensitivity of these cells to ROS.

Transient receptor potential melastatin 4 channel contributes to migration of androgen-insensitive prostate cancer cells

Impaired Ca2+ signaling in prostate cancer contributes to several cancer hallmarks, such as enhanced proliferation and migration and a decreased ability to induce apoptosis. Na+ influx via transient receptor potential melastatin 4 channel (TRPM4) can reduce store-operated Ca2+ entry (SOCE) by decreasing the driving force for Ca2+. In patients with prostate cancer, gene expression of TRPM4 is elevated. Recently, TRPM4 was identified as a cancer driver gene in androgen-insensitive prostate cancer. We investigated TRPM4 protein expression in cancer tissue samples from 20 patients with prostate cancer. We found elevated TRPM4 protein levels in prostatic intraepithelial neoplasia (PIN) and prostate cancer tissue compared to healthy tissue. In primary human prostate epithelial cells (hPEC) from healthy tissue and in the androgen-insensitive prostate cancer cell lines DU145 and PC3, TRPM4 mediated large Na+ currents. We demonstrated significantly increased SOCE after siRNA targeting of TRPM4 in hPEC and DU145 cells. In addition, knockdown of TRPM4 reduced migration but not proliferation of DU145 and PC3 cells. Taken together, our data identify TRPM4 as a regulator of SOCE in hPEC and DU145 cells, demonstrate a role for TRPM4 in cancer cell migration and suggest that TRPM4 is a promising potential therapeutic target.

Cell type–specific glycosylation of Orai1 modulates store-operated Ca2+ entry

T cells and mast cells may use differential glycosylation of Orai1 to regulate store-operated calcium signaling. Sugary coating on Orai1 Glycosylation is the addition of sugars to proteins and the chemical modification of these sugars to form highly complex structures. Glycosylation can affect protein stability, protein-protein interactions, and protein function. Human Orai1 is the channel-forming subunit of a complex that mediates calcium entry into cells when calcium in the endoplasmic reticulum becomes depleted (a process called SOCE). Orai1 has a single N-glycosylation site. Dörr et al. found that cells, including several types of immune cells, exposed to conditions that altered Orai1 glycosylation specifically or cellular glycosylation globally exhibited cell-specific effects on SOCE. In many cases, disruption of glycosylation enhanced SOCE without affecting Orai1 abundance or presence at the cell surface, suggesting that glycosylation limited Orai1 activity. Excess SOCE and altered glycosylation are associated with various immune diseases and differences in Orai1 function may be an important molecular connection between these two cellular events. N-glycosylation of cell surface proteins affects protein function, stability, and interaction with other proteins. Orai channels, which mediate store-operated Ca2+ entry (SOCE), are composed of N-glycosylated subunits. Upon activation by Ca2+ sensor proteins (stromal interaction molecules STIM1 or STIM2) in the endoplasmic reticulum, Orai Ca2+ channels in the plasma membrane mediate Ca2+ influx. Lectins are carbohydrate-binding proteins, and Siglecs are a family of sialic acid–binding lectins with immunoglobulin-like repeats. Using Western blot analysis and lectin-binding assays from various primary human cells and cancer cell lines, we found that glycosylation of Orai1 is cell type–specific. Ca2+ imaging experiments and patch-clamp experiments revealed that mutation of the only glycosylation site of Orai1 (Orai1N223A) enhanced SOCE in Jurkat T cells. Knockdown of the sialyltransferase ST6GAL1 reduced α-2,6–linked sialic acids in the glycan structure of Orai1 and was associated with increased Ca2+ entry in Jurkat T cells. In human mast cells, inhibition of sialyl sulfation altered the N-glycan of Orai1 (and other proteins) and increased SOCE. These data suggest that cell type–specific glycosylation influences the interaction of Orai1 with specific lectins, such as Siglecs, which then attenuates SOCE. In summary, the glycosylation state of Orai1 influences SOCE-mediated Ca2+ signaling and, thus, may contribute to pathophysiological Ca2+ signaling observed in immune disease and cancer.

Regulation of Ca(2+) signaling in prostate cancer cells.

AbstractPrecise intracellular Ca2+ signaling is crucial to ensure proper cellular functions. Subsequently, imbalances in Ca2+ signaling, particularly in store-operated Ca2+ entry (SOCE), contribute to several pathophysiological malfunctions in immune diseases and cancer. We lately reported on heteromeric Orai1/Orai3 SOCE channels and transient receptor potential melastatin-4 (TRPM4) channels in prostate cancer cells. We here sum up our findings and provide a model for the regulation of Ca2+ signaling in prostate cancer cells.

Regulation of Ca2+ signaling in prostate cancer cells

AbstractPrecise intracellular Ca2+ signaling is crucial to ensure proper cellular functions. Subsequently, imbalances in Ca2+ signaling, particularly in store-operated Ca2+ entry (SOCE), contribute to several pathophysiological malfunctions in immune diseases and cancer. We lately reported on heteromeric Orai1/Orai3 SOCE channels and transient receptor potential melastatin-4 (TRPM4) channels in prostate cancer cells. We here sum up our findings and provide a model for the regulation of Ca2+ signaling in prostate cancer cells.

The Minimal Requirements to Use Calcium Imaging to Analyze ICRAC

Endogenous calcium release-activated channel (CRAC) currents are usually quite small and not always easy to measure using the patch-clamp technique. While we have, for instance, successfully recorded very small CRAC currents in primary human effector T cells, we have not yet managed to record CRAC in naïve primary human T cells. Many groups, including ours, therefore use Ca(2+) imaging technologies to analyze CRAC-dependent Ca(2+) influx. However, Ca(2+) signals are quite complex and depend on many different transporter activities; thus, it is not trivial to make quantitative statements about one single transporter, in this case CRAC channels. Therefore, a detailed patch-clamp analysis of ICRAC is always preferred. Since many laboratories use Ca(2+) imaging for ICRAC analysis, we detail here the minimal requirements for reliable measurements. Ca(2+) signals not only depend on the net Ca(2+) influx through CRAC channels but also depend on other Ca(2+) influx mechanisms, K(+) channels or Cl(-) channels (which determine the membrane potential), Ca(2+) export mechanisms like plasma membrane Ca(2+) ATPase (PMCA), sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) or Na(+)-Ca(2+) exchangers, and (local) Ca(2+) buffering often by mitochondria. In this protocol, we summarize a set of experiments that allow (quantitative) statements about CRAC channel activity using Ca(2+) imaging experiments, including the ability to rule out Ca(2+) signals from other sources.

Patch-Clamp Measurement of ICRAC and ORAI Channel Activity

Depletion of internal Ca(2+) stores activates store-operated Ca(2+) channels. The most prominent members of this class of channels are Ca(2+) release-activated Ca(2+) (CRAC) channels, which are present in a variety of cell types including immune cells. CRAC channels are composed of ORAI proteins, which are activated by endoplasmic reticulum-bound STIM proteins on Ca(2+) store depletion. The underlying Ca(2+) current is called ICRAC, which is required for many cellular functions including T-cell activation, mast cell activation, Ca(2+)-dependent gene expression, and refilling of internal Ca(2+) stores. To analyze ICRAC or the Ca(2+) current through heterologously expressed ORAI channels, whole-cell patch clamp is the technique of choice. It allows the direct analysis of ion currents through CRAC/ORAI channels. The patch-clamp technique has been used to determine selectivity, permeability, rectification, inactivation, and several other biophysical and pharmacological properties of the channels, and is the most direct and reliable technique to analyze ICRAC.