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    • Besides the described changes in protein expression and

    2023-01-24

    Besides the described changes in protein expression and thus in current amplitudes, Ki8751 of PORCN also leads to accelerated decay kinetics of evoked and spontaneous AMPAR currents. These changes in channel kinetics are most likely a secondary effect due to the selective depletion of γ-8 but not γ-2 from AMPAR complexes in the absence of PORCN, as γ-8 has greater effects on AMPAR desensitization (Cho et al., 2007; Milstein et al., 2007). However, decay kinetics could not be rescued by overexpression of γ-8. Changes in the synaptic current decay kinetics could also be explained by the selective reduction of GluA2/3 protein levels in the PSD fraction of PORCN KO mice, leaving faster desensitizing GluA2-lacking receptors at the synapse. Experiments in dissociated neuronal cultures, however, did not show significant changes in rectification properties or block by philanthotoxin demonstrating that most neuronal AMPARs are still GluA2 containing. A direct effect of PORCN on AMPAR kinetics is alternatively possible, but unlikely as PORCN levels in the PSD are minimal. Both ABHD6 and PORCN have transmembrane domains, and both have well-characterized enzyme activities. ABHD6 serves as the rate-limiting enzyme in degrading the endocannabinoid 2-AG and localizes postsynaptically (Marrs et al., 2010). Knockdown of ABHD6 or pharmacological inhibition augments endocannabinoid signaling and thereby modulates synaptic plasticity (Zhong et al., 2011). However, these properties are not essential for actions described here, as a catalytically inactive ABHD6 mutant (Navia-Paldanius et al., 2012) continued to modulate AMPARs. Porcupine was discovered genetically as acting upstream of the segment polarity gene wingless (Wg), a Drosophila Wnt family member (Kadowaki et al., 1996). Elegant genetic and biochemical studies later showed that PORCN mediates palmitoylation of a specific serine on Wg and mammalian Wnt isoforms (Galli et al., 2007). These Wg/Wnt palmitoylations occur in the lumen of the ER and are therefore categorically distinct from cytoplasmic palmitoylations catalyzed by DHHC enzymes that modify the C-terminal tail of AMPARs, PSD-95, and many other synaptic proteins (El-Husseini and Bredt, 2002; Hayashi et al., 2005). Palmitoyl-transferase activity of PORCN does not mediate its effects on AMPARs, as a catalytically dead mutant (Galli et al., 2007) showed full activity to control GluA1 stability. Additionally, a highly specific and potent PORCN inhibitor did not abolish the PORCN-mediated effects on AMPARs in HEK cells or neurons. Importantly, recent genetic studies found that PORCN effects on a subset of cancer cell lines are independent of its enzyme activity. Indeed, PORCN mutated at the same His used here was fully effective in controlling cell growth in multiple breast cancer cell lines (Covey et al., 2012). Thus, PORCN’s functional interaction with AMPARs occurs in a “moonlighting” role independent of Wnt signaling. Another multi-functional ion channel modulator is gephyrin, which both clusters glycine/GABA receptors in neurons and catalyzes the last step in molybdenum cofactor biosynthesis throughout the body (Feng et al., 1998). Like gephyrin, PORCN is a single gene subject to complex alternative splicing. It will be important to understand how these alternative forms may specify PORCN actions on Wnt and non-Wnt pathways. PORCN has differential effects on AMPARs in heterologous cells and neurons. Whereas PORCN expression dramatically increases levels of AMPARs in both systems, PORCN reduces receptor functionality in non-neuronal cells. This reduced functionality is due to PORCN-dependent retention of GluA subunits in intracellular compartments as demonstrated by biotinylation experiments, and it is not due to an increased cytoplasmic polyamine block as shown by rectification experiments (data not shown). As AMPARs are expressed almost exclusively in neurons and glial cells, this intracellular trapping in HEK293T cells may be artificial. Alternatively, PORCN may physiologically retain GluA subunits in the ER and release them following proper assembly of the AMPAR complex. Co-expression of TARPs did not overcome PORCN-mediated intracellular retention of GluA1 (data not shown), so other neuronal factors or other AMPAR auxiliary subunits must contribute. Cellular context is also critical in function of CNIH-2, which promotes ER export of AMPARs in mammalian neurons (Harmel et al., 2012) but reduces AMPAR surface levels in oocytes (Brockie et al., 2013). Furthermore, in C. elegans muscle cells, CNIH-2 or its worm homolog CNI-1 blocks ER export and CNIH-2 reduces surface AMPAR levels (Brockie et al., 2013).