Hannes Schleifer

PKC-dependent coupling of calcium permeation through transient receptor potential canonical 3 (TRPC3) to calcineurin signaling in HL-1 myocytes

Cardiac transient receptor potential canonical (TRPC) channels are crucial upstream components of Ca2+/calcineurin/nuclear factor of activated T cells (NFAT) signaling, thereby controlling cardiac transcriptional programs. The linkage between TRPC-mediated Ca2+ signals and NFAT activity is still incompletely understood. TRPC conductances may govern calcineurin activity and NFAT translocation by supplying Ca2+ either directly through the TRPC pore into a regulatory microdomain or indirectly via promotion of voltage-dependent Ca2+ entry. Here, we show that a point mutation in the TRPC3 selectivity filter (E630Q), which disrupts Ca2+ permeability but preserves monovalent permeation, abrogates agonist-induced NFAT signaling in HEK293 cells as well as in murine HL-1 atrial myocytes. The E630Q mutation fully retains the ability to convert phospholipase C-linked stimuli into L-type (CaV1.2) channel-mediated Ca2+ entry in HL-1 cells, thereby generating a dihydropyridine-sensitive Ca2+ signal that is isolated from the NFAT pathway. Prevention of PKC-dependent modulation of TRPC3 by either inhibition of cellular kinase activity or mutation of a critical phosphorylation site in TRPC3 (T573A), which disrupts targeting of calcineurin into the channel complex, converts cardiac TRPC3-mediated Ca2+ signaling into a transcriptionally silent mode. Thus, we demonstrate a dichotomy of TRPC-mediated Ca2+ signaling in the heart constituting two distinct pathways that are differentially linked to gene transcription. Coupling of TRPC3 activity to NFAT translocation requires microdomain Ca2+ signaling by PKC-modified TRPC3 complexes. Our results identify TRPC3 as a pivotal signaling gateway in Ca2+-dependent control of cardiac gene expression.

Regulation of Gene Expression through a Transcriptional Repressor that Senses Acyl-Chain Length in Membrane Phospholipids

Summary Membrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis. Acc1 activity is attenuated by AMPK/Snf1-dependent phosphorylation, which is required to maintain an appropriate acyl-chain length distribution. Moreover, we find that the transcriptional repressor Opi1 preferentially binds to C16 over C18 phosphatidic acid (PA) species: thus, C16-chain containing PA sequesters Opi1 more effectively to the ER, enabling AMPK/Snf1 control of PA acyl-chain length to determine the degree of derepression of Opi1 target genes. These findings reveal an unexpected regulatory link between the major energy-sensing kinase, membrane lipid composition, and transcription.

Novel pyrazole inhibitors for discrimination between receptor-operated and store-operated Ca2+ entry

Background Calcium governs a wide range of cellular processes. Specifically, control of gene transcription involves Ca entry channels that are activated by either voltage, second messengers or depletion of intracellular stores. The family of classical transient receptor potential channels (TRPC) has been implicated in both the receptor/second messenger as well as in store-operated Ca entry pathway, and represents an attractive target for therapeutic intervention.

Molecular engineering of the TRPC3 pore structure identifies Ca2+ permeation through TRPC3 channels as a key determinant of cardiac calcineurin/NFAT signaling.

Results Elimination of Ca permeation through TRPC3 abrogated its ability to trigger NFAT translocation in both HEK293 cells and in HL-1 atrial myocytes. Wild-type TRPC3 was found capable of initiating NFAT translocation in atrial myocytes by a small, homogenous elevation of cytoplasmic Ca that was independent of voltagegated CaV1.2 channels. By contrast, a Ca 2+ impermeant TRPC3 mutant strongly promoted endothelin-induced Ca signals in HL1 cells via enhanced activity of CaV1.2 channels without concomitant NFAT translocation. Conclusions Our results demonstrate two strictly separated Ca signaling functions of cardiac TRPC3 channels as well as a tight and efficient link between TRPC3-mediated Ca permeation and calcineurin/NFAT signaling.

Store-operated calcium entry into rat basophil leukaemia cells: contribution of TRPC3 and Orai1

Background Before the discovery of STIM and Orai proteins, mammalian TRPC channels, including TRPC3, were considered as candidates for mediating store-operated Ca2+ entry (SOCE). This calcium entry pathway governs diverse cellular processes from exocytosis, cellular remodelling to gene transcription. Although a prominent role of Orai1 is now well established for immune cells, the role of TRPC proteins and the possible crosstalk between these two ion channels families to the overall store operated calcium entry into cells of the immune system is still elusive.

Identification of amino acid residues relevant for gating and permeation of the cation channel TRPC3

Background and methods Mammalian TRPC3 cation channels are activated through phospholipase type C (PLC)-dependent pathways and play a fundamental role in a variety of physiological functions. So far, only little information is available on structural determinants of channel function, especially domains involved in channel gating and permeation. Therefore, we set out to modify putative permeation-relevant residues of this ion channel by site-directed mutagenesis and analyzed the impact of these mutations on channel functions using a HEK293 expression system and the patch clamp technique.