Tatiana Kilch

Universität des Saarlandes, Saarbrücken, Saarland, Germany

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Publications (9)43.94 Total impact

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    ABSTRACT: Although ICRAC and other store-operated currents are often analyzed by Ca(2+) imaging, whole-cell patch clamp, described here, is the preferred technique to analyze ICRAC whenever possible. The whole-cell patch-clamp protocol can even be used to record endogenous ICRAC in primary cells. The small endogenous current size of ICRAC requires some precautions: First, it is important to inhibit potential interferences from other channels in the cell by carefully choosing the combination of pipette and bath solutions. Second, the noise should be <150 fA root mean square (RMS) when the pipette holder (with its wire) with or without a patch pipette is adjusted over (but not in!) the solution using a high amplification gain (50 mV/pA or higher) of the patch-clamp amplifier. In addition, this protocol draws attention to measures that should be considered when recording ICRAC currents from an overexpression system. This protocol also suggests sets of solutions that can be used for distinguishing ICRAC from potentially interfering currents. In addition to the solutions, the identity of ICRAC can be confirmed by the characteristic inward rectification, its high Ca(2+) selectivity, and the reversal potential of more than +50 mV. A few (mostly nonspecific) CRAC channel blockers are known, which can also be applied for characterization purposes.
    Cold Spring Harbor Protocols 06/2014; 2014(6):pdb.prot073254. DOI:10.1101/pdb.prot073254 · 4.63 Impact Factor
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    ABSTRACT: 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.
    Cold Spring Harbor Protocols 06/2014; 2014(6):pdb.prot073262. DOI:10.1101/pdb.prot073262 · 4.63 Impact Factor
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    ABSTRACT: 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.
    Cold Spring Harbor Protocols 06/2014; 2014(6):pdb.top066795. DOI:10.1101/pdb.top066795 · 4.63 Impact Factor
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    Biophysical Journal 01/2014; 106(2):317a. DOI:10.1016/j.bpj.2013.11.1831 · 3.97 Impact Factor
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    ABSTRACT: Labelled 5α-dihydrotestosterone (DHT) binding experiments have shown that expression levels of (yet unidentified) membrane androgen receptors (mAR) are elevated in prostate cancer and correlate with a negative prognosis. However, activation of these receptors which mediate a rapid androgen response can counteract several cancer hallmark functions such as unlimited proliferation, enhanced migration, adhesion and invasion and the inability to induce apoptosis. Here, we investigate the downstream signaling pathways of mAR and identify rapid DHT induced activation of store-operated Ca2+ entry (SOCE) in primary cultures of human prostate epithelial cells (hPEC) from non-tumorous tissue. Consequently, down-regulation of Orai1, the main molecular component of Ca2+ release-activated Ca2+ (CRAC) channels results in an almost complete loss of DHT induced SOCE. We demonstrate that this DHT induced Ca2+ influx via Orai1 is important for rapid androgen triggered prostate specific antigen (PSA) release. We furthermore identified alterations of the molecular components of CRAC channels in prostate cancer. Three lines of evidence indicate that prostate cancer cells down-regulate expression of the Orai1 homolog Orai3: First, Orai3 mRNA expression levels are significantly reduced in tumorous tissue when compared to non-tumorous tissue from prostate cancer patients. Second, mRNA expression levels of Orai3 are decreased in prostate cancer cell lines LNCaP and DU145 when compared to hPEC from healthy tissue. Third, the pharmacological profile of CRAC channels in prostate cancer cell lines and hPEC differ and siRNA based knock-down experiments indicate changed Orai3 levels are underlying the altered pharmacological profile. The cancer-specific composition and pharmacology of CRAC channels identifies CRAC channels as putative targets in prostate cancer therapy.
    Oncotarget 10/2013; 4(11). · 6.63 Impact Factor
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    ABSTRACT: A drop of endoplasmic reticulum (ER) Ca(2+) concentration triggers its Ca(2+) sensor protein STIM1 to oligomerize and accumulate within ER-plasma membrane (PM) junctions where it activates Orai1 channels, providing store operated Ca(2+) entry (SOCE). To elucidate the functional significance of N-glycosylation sites of STIM1, we created different mutations of N131 and N171. STIM1 NN/DQ resulted in a strong gain-of-function. Patch-clamp, TIRF and FRAP analyses revealed that expression of STIM1 DQ mutants increases the number of active Orai1 channels and the rate of STIM1 translocation to ER-PM junctions with a decrease in current latency. Surprisingly, coexpression 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 Ca(2+) homeostasis.
    Journal of Biological Chemistry 12/2012; 288(3). DOI:10.1074/jbc.M112.417246 · 4.57 Impact Factor
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    Ivan Bogeski · Tatiana Kilch · Barbara A Niemeyer
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    ABSTRACT: Abstract  Store-operated Ca(2+) entry (SOCE) is a widespread mechanism in cells to raise cytosolic Ca(2+) and to refill Ca(2+) 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 Ca(2+) homeostasis mechanisms. Exposure to environmental or cell-intrinisic reactive oxygen species (ROS) can affect several components involved in Ca(2+) 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.
    The Journal of Physiology 05/2012; 590(Pt 17):4193-200. DOI:10.1113/jphysiol.2012.230565 · 4.54 Impact Factor
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    ABSTRACT: 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.
    Journal of Investigative Dermatology 02/2012; 132(5):1443-51. DOI:10.1038/jid.2011.478 · 6.37 Impact Factor
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    Biophysical Journal 01/2012; 102(3):680-. DOI:10.1016/j.bpj.2011.11.3700 · 3.97 Impact Factor