Store-operated Ca2+ channels and Stromal Interaction Molecule 1 (STIM1) are targets for the actions of bile acids on liver cells

School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 06/2008; 1783(5):874-85. DOI: 10.1016/j.bbamcr.2008.02.011
Source: PubMed


Cholestasis is a significant contributor to liver pathology and can lead to primary sclerosis and liver failure. Cholestatic bile acids induce apoptosis and necrosis in hepatocytes but these effects can be partially alleviated by the pharmacological application of choleretic bile acids. These actions of bile acids on hepatocytes require changes in the release of Ca2+ from intracellular stores and in Ca2+ entry. However, the nature of the Ca2+ entry pathway affected is not known. We show here using whole cell patch clamp experiments with H4-IIE liver cells that taurodeoxycholic acid (TDCA) and other choleretic bile acids reversibly activate an inwardly-rectifying current with characteristics similar to those of store-operated Ca2+ channels (SOCs), while lithocholic acid (LCA) and other cholestatic bile acids inhibit SOCs. The activation of Ca2+ entry was observed upon direct addition of the bile acid to the incubation medium, whereas the inhibition of SOCs required a 12 h pre-incubation. In cells loaded with fura-2, choleretic bile acids activated a Gd3+-inhibitable Ca2+ entry, while cholestatic bile acids inhibited the release of Ca2+ from intracellular stores and Ca2+ entry induced by 2,5-di-(tert-butyl)-1,4-benzohydro-quinone (DBHQ). TDCA and LCA each caused a reversible redistribution of stromal interaction molecule 1 (STIM1, the endoplasmic reticulum Ca2+ sensor required for the activation of Ca2+ release-activated Ca2+ channels and some other SOCs) to puncta, similar to that induced by thapsigargin. Knockdown of STIM1 using siRNA caused substantial inhibition of Ca2+-entry activated by choleretic bile acids. It is concluded that choleretic and cholestatic bile acids activate and inhibit, respectively, the previously well-characterised Ca2+-selective hepatocyte SOCs through mechanisms which involve the bile acid-induced redistribution of STIM1.

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Available from: Edoardo C Aromataris, Dec 12, 2013
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    • "The long-standing mystery of the molecular composition of CRAC channels has hampered the identification of specific inhibitors. Nevertheless a variety of compounds that strongly inhibit CRAC currents has been identified including divalent and trivalent cations such as La3+ and Gd3+, diverse imidazoles, 2-APB (2-aminoethoxydiphenylborate), capsaicin [2], NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid) [3–5], BTP2 (a bistrifluoromethyl-pyrazole derivative) [6–8], DES (diethylstilbestrol) [9], BEL (bromenol lactone) [10], bile acids [11] and ML-9 (1-(5-chloronaphthalene-1-sulfonyl)homopiperazine) [12]. La3+ and Gd3+ represent general inhibitors of Ca2+-selective influx pathways comprising voltage-dependent Ca2+, TRPV5/6 and also CRAC channels [13,14]. "
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    ABSTRACT: As the molecular composition of calcium-release activated calcium (CRAC) channels has been unknown for two decades, elucidation of selective inhibitors has been considerably hampered. By the identification of the two key components of CRAC channels, STIM1 and Orai1 have emerged as promising targets for CRAC blockers. The aim of this study was to thoroughly characterize the effects of two selective CRAC channel blockers on currents derived from STIM1/Orai heterologoulsy expressed in HEK293 cells. The novel compounds GSK-7975A and GSK-5503A were tested for effects on STIM1 mediated Orai1 or Orai3 currents by whole-cell patch-clamp recordings and for the effects on STIM1 oligomerisation or STIM1/Orai coupling by FRET microscopy. To investigate their site of action, inhibitory effects of these molecules were explored using Orai pore mutants. The GSK blockers inhibited Orai1 and Orai3 currents with an IC50 of approximately 4 μM and exhibited a substantially slower rate of onset than the typical pore blocker La3+, together with almost no current recovery upon wash-out over 4 min. For the less Ca2+-selective Orai1 E106D pore mutant, ICRAC inhibition was significantly reduced. FRET experiments indicated that neither STIM1–STIM1 oligomerization nor STIM1–Orai1 coupling was affected by these compounds. These CRAC channel blockers are acting downstream of STIM1 oligomerization and STIM1/Orai1 interaction, potentially via an allosteric effect on the selectivity filter of Orai. The elucidation of these CRAC current blockers represents a significant step toward the identification of CRAC channel-selective drug compounds.
    Full-text · Article · Dec 2012 · Cell calcium
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    ABSTRACT: Background: Ca(2+) release-activated Ca(2+) (CRAC) channels, a subfamily of store-operated channels, play an essential role in various diseases such as immune disorders and allergic responses. Objective: The successful treatment of these diseases requires the identification of specific inhibitors. So far, a variety of chemical compounds blocking CRAC have been identified; however, they have all turned out to be less specific. Recently two proteins, STIM1 and ORAI1, have been identified as the essential components that fully reconstitute CRAC currents with a similar biophysical fingerprint. Method: These two proteins and their activation process represent direct targets for the application of specific CRAC inhibitors. Results/conclusion: For drug development, fluorescence microscopy adaptable for high-throughput screening will provide a powerful assay to mechanistically identify potential CRAC inhibitors that act on various stages within the STIM1/ORAI1 activation pathway visualized by fluorescent-tagged proteins.
    No preview · Article · Jul 2008 · Expert Opinion on Drug Discovery
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