Figure 2. Deletion of RA domain in SNX27b affects localization of GIRK2c/3 channels monitored with BiFC.

A, Left, Schematic shows placement of split YFP on GIRK2c and GIRK3. Note the C-terminal domains are free to interact with other proteins. Right, BiFC-tagged GIRK2c/3 channels are functional. Current-voltage plot is shown for ^CYGIRK2c/^NYGIRK3 channels. Baclofen (100 µM) activates and Ba²⁺ (1 mM) inhibits inwardly rectifying current. HEK293T cells were transfected with cDNA encoding GABA_B1a, GABA_B2 and ^CYGIRK2c/^NYGIRK3. Average baclofen-induced current densities were –13.2±6.0 pA⋅pF⁻¹ (n = 3) for ^CYGIRK2c/^NYGIRK3. B, HEK293 cells were co-transfected with ^CYGIRK2c, ^NYGIRK3 and either control cDNA (i), wild-type SNX27b (ii), SNX27b-ΔRA (deletion of Asp272-Trp358) (iii) or SNX27b-Y51L (a PDZ mutation) (iv). Green fluorescence in images represents molecular recombination of ^CYGIRK2c/^NYGIRK3 heterotetramers. Coexpression of wild-type SNX27b induced formation of puncta. By contrast, ^CYGIRK2c/^NYGIRK3 fluorescence was diffuse in the cytoplasm for SNX27b-ΔRA and for SNX27b-Y51L, similar to control. Inset shows zoom of boxed area. C, SNX27b-ΔRA exhibited a pattern of punctate expression similar that of wild-type SNX27b. YFP was fused to the C-terminus of SNX27b or SNX27b-ΔRA to directly visualize expression. HEK293T cells were transfected with cDNA for SNX27b-YFP and SNX27bΔRA-YFP. Scale bar: 10 µm.