Skip to main content
. 2012 Sep 20;287(46):39205–39215. doi: 10.1074/jbc.M112.376053

FIGURE 7.

FIGURE 7.

Subcellular localization of independent GlyRα1 domains. A, GlyRα1 domains used in a three-domain GlyR configuration (α1-trc + Myc-iDΔ62-TM4 + TM3-4 loop) were cotransfected into HEK293 cells together with either pDsRed-ER, a vector expressing a fusion protein of the ER targeting sequence of calreticulin fused to DsRed, or pDsRed-Monomer-Mem encoding GAP-43. Overlays of two constructs are shown, with the color of each construct indicated by its letter color. Cells were fixed and permeabilized prior to staining with MAb4a to recognize the α1-trc variant (shown in green) and α-Myc antibody to detect the Myc-iDΔ62-TM4 (depicted in cyan) as well as the H18 antibody detecting an epitope within the GlyRα1 TM3-4 loop(357–418) (cyan; TM3-4 loop). Calreticulin as well as GAP-43 are shown in red. The immunoreactivity was visualized by confocal microscopy. Scale bars, 20 μm. B, whole-cell lysates prepared from HEK293 cells transfected with combinations of α1-trc, (Myc-)iDΔ62-TM4, and (Myc-)TM3-4 loop presence indicated by a plus sign below the blot. Both domains, the Myc-TM3-4 loop and Myc-iDΔ62-TM4, were detected via their Myc epitopes. The arrows point toward the observed molecular mass of 16 kDa for the TM3-4 loop as well as to 13 kDa for the Myc-iDΔ62-TM4. C, functionality of the three-domain receptor configuration. Following glycine application, no functional ion channels were observed using two domains, α1-trc + Myc-iDΔ62-TM4 or α1-trc + TM3-4 loop (top two traces). In the three-domain configuration α1-trc + Myc-iDΔ62-TM4 + TM3-4 loop (middle trace), however, receptor function was rescued (see bottom trace with α1-trc + full-length complementation construct Myc-iD-TM4 for comparison of rescue efficiency). Whole cell recordings of transfected HEK293 cells were used, and maximal Gly-gated currents were determined with 1 mm Gly. Recordings were carried out at −60 mV, [Cl−]in = [Cl−]out. Agonists were applied via U-tube for 1 s. 9 or 10 cells of each combination were measured in at least three different batches of transfected cells.