Through studies with ligand binding to the acetylcholine binding protein (AChBP), we previously identified a series of 4,6-substituted 2-aminopyrimidines that associate with this soluble surrogate of the nicotinic acetylcholine receptor (nAChR) in a cooperative fashion, not seen for classical nicotinic agonists and antagonists. To examine receptor interactions of this structural family on ligand-gated ion channels, we employed HEK cells transfected with cDNAs encoding three requisite receptor subtypes: α7-nAChR, α4β2-nAChR, and a serotonin receptor (5-HTR), along with a fluorescent reporter. Initial screening of a series of over 50 newly characterized 2-aminopyrimidines with affinity for AChBP showed only two to be agonists on the α7-nAChR below 10 μM concentration. Their unique structural features were incorporated into design of a second subset of 2-aminopyrimidines yielding several congeners that elicited α7 activation with EC values of 70 nM and K values for AChBP in a similar range. Several compounds within this series exhibit specificity for the α7-nAChR, showing no activation or antagonism of α4β2-nAChR or 5-HT3AR at concentrations up to 10 μM, while others were weaker antagonists (or partial agonists) on these receptors. Analysis following cocrystallization of four ligand complexes with AChBP show binding at the subunit interface, but with an orientation or binding pose that differs from classical nicotinic agonists and antagonists and from the previously analyzed set of 2-aminopyrimidines that displayed distinct cooperative interactions with AChBP. Orientations of aromatic side chains of these complexes are distinctive, suggesting new modes of binding at the agonist-antagonist site and perhaps an allosteric action for heteromeric nAChRs.
Stromal interaction molecules (STIM1, 2) are acting as sensors for Ca 2+ in intracellular stores and activate Orai channels at the plasma membrane for store-operated Ca 2+ entry (SOCE), while classical transient receptor potential (TRPC) channel mediate receptor-operated Ca 2+ entry (Roce). Several reports, however, indicate a role for TRPC in SOCE in certain cell types. Here, we analyzed Ca 2+ influx and cell function in TRPC1/6-deficient (TRPC1/6 −/− ) and STIM1/2-deficient (STIM1/2 ΔpmLf ) primary murine lung fibroblasts (pmLF). As expected, SOCE was decreased in STIM1/2-deficient pmLF and ROCE was decreased in TRPC1/6 −/− pmLF compared to control cells. By contrast, SOCE was not significantly different in TRPC1/6 −/− pmLF and ROCE was similar in STIM1/2-deficient pmLF compared to Wt cells. Most interestingly, cell proliferation, migration and nuclear localization of nuclear factor of activated T-cells (NFATc1 and c3) were decreased after ablation of STIM1/2 proteins in pmLF. In conclusion, TRPC1/6 channels are not involved in SOCE and STIM1/2 deficiency resulted in decreased cell proliferation and migration in pmLf.Store-operated Ca 2+ entry (SOCE) also named capacitive Ca 2+ entry (CCE) was first described by J.W. Putney Jr. more than 30 years ago as depletion of intracellular Ca 2+ stores which induces the opening of plasma membrane (PM) Ca 2+ channels 1 . Since then, candidate proteins like classical transient receptor potential (TRPC) channels 2 and mechanisms, e.g. coupling of TRPC proteins to inositol 1-4-5 trisphosphate (IP3) receptor channels in the endoplasmic reticulum 3,4 for SOCE, were intensively discussed in the scientific community. In 2005 however, stromal interaction molecules (Stim in Drosophila and STIM1, STIM2 in humans) were identified as Ca 2+ sensors in the ER directly regulating SOCE in two different large-scale screening approaches 5,6 . One year later, Ca 2+ selective channels at the plasma membrane (Orai) were discovered 7-9 , which were responsible for Ca 2+ release activated Ca 2+ (CRAC) currents originally described in mast cells 10 . A molecular model was developed to support the concept that upon ER Ca 2+ depletion STIM proteins homo-multimerize and translocate to ER-PM junctions 11,12 , where they recruit and gate Orai channels via direct interaction 13 . Ca 2+ influx through Orai channels is important for cellular remodeling, e.g. in cardiovascular diseases 14 , and mutations in these channels are responsible for multiple channelopathies 15 . Irrespective of these events, TRP channels trigger Ca 2+ influx in response to extracellular stimuli or receptor activation (receptor-operated Ca 2+ influx, ROCE) independently of STIM and Orai 16 . Some labs, however, reported that TRPC channels also interact with STIM proteins 17 and/or Orai channels 18 . Along these lines, TRPC channels like TRPC1 were invoked in SOCE in certain cells of salivary glands 19 and pancreatic acini 20 , while in vascular smooth muscle cells TRPC1 channels work independently of SOCE 21 . The role of TRPC1 i...
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