Palmitoylation of Gephyrin Controls Receptor Clustering and Plasticity of GABAergic Synapses
Figure 5
Gephyrin is palmitoylated by DHHC-12.
(a) Individual HA-tagged DHHC proteins or GST-HA as control were co-expressed with gephyrin-GFP for 24 h in HEK293 cells and analyzed with the ABE assay. Omitting hydroxylamine demonstrated specify of the assay. (b) Gephyrin-GFP was co-expressed with individual HA-tagged DHHC enzymes or GST-HA as control in primary hippocampal neurons, and gephyrin cluster size was quantified by confocal laser scanning microscopy. In the histogram, DHHC enzymes are labeled according to [16]. (c) Representative images show phenotypical differences of gephyrin-GFP clusters in DHHC-12–expressing neurons compared to GST-expressing controls. Scale bar, 10 µm. (d) Gephyrin-GFP cluster density after 24 h expression of DHHC-12 compared to control neurons. (e–g) Quantification of gephyrin-GFP cluster size and density and representative image upon expression of dominant-negative DHHS-12–HA compared to mCherry-expressing control neurons. Scale bar, 5 µm. (h) Quantitative analysis of 212,284Geph cluster size in control mCherry and DHHC-12-HA–expressing neurons. (i) Expression of dhhc12 mRNA in cultured hippocampal neurons (h. neurons) was validated by PCR using cDNA prepared from neurons. A plasmid encoding the respective dhhc gene was used as control. (j) Primary hippocampal neurons were grown in medium containing cell-penetrating siRNAs to knock down expression of DHHC-12 or DHHC-16. Lysates were subjected to ABE, and the palmitoylation level of gephyrin, GABAAR γ2, and PSD-95 was analyzed by immunoblotting. We used 10% of the lysate to detect total levels of the respective proteins; scr., scrambled. (k) Quantification of three independent experiments shows gephyrin palmitoylation levels normalized to total protein (***p<0.001 using Student's t test).