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

<p><b>A</b>, <i>Left</i>, Schematic shows placement of split YFP on GIRK2c and GIRK3. Note the C-terminal domains are free to interact with other proteins. <i>Right</i>, 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>B</b>, HEK293 cells were co-transfected with ^CYGIRK2c, ^NYGIRK3 and either control cDNA (<i>i</i>), wild-type SNX27b (<i>ii</i>), SNX27b-ΔRA (deletion of Asp272-Trp358) (<i>iii</i>) or SNX27b-Y51L (a PDZ mutation) (<i>iv</i>). 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. <b>C,</b> 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.</p

Deletion of RA domain in SNX27b impairs functional regulation of GIRK channels.

<p><b>A</b>, Cartoon shows model of GIRK channels regulation by SNX27b. GIRK channels recycle through endosomal compartments. SNX27b associating with GIRK2c/3 channels in the early endosome (EE) reduces plasma membrane expression (PM) by targeting some channels to the late endosome (LE). <b>B</b>, SNX27 contains three functional domains; PDZ, PX and RA. GIRK2c and GIRK3 contain a C-terminal PDZ binding motif (-E(S/N)ESKV). <b>C,</b> Examples of baclofen-induced (100 µM) and Ba²⁺-sensitive (1 mM Ba²⁺) currents in HEK293T cells transfected with cDNA for GABA_B1a/B2 receptors, GIRK2c/GIRK3 and either control vector, SNX27b or SNX27b-ΔRA. Agonist-independent basal currents are revealed by inhibition with 1 mM Ba²⁺. <b>D</b>, Average baclofen-induced current densities (I_Baclofen) for control (–41.3±5.2 pA⋅pF⁻¹, n = 8), SNX27b (–11.0±3.6 pA⋅pF⁻¹, n = 8) and SNX27b- ΔRA (–55.9±8.2 pA⋅pF⁻¹, n = 13) with GIRK2c/GIRK3. <b>E</b>, Average I_Baclofen for control (–15.7±3.6 pA⋅pF⁻¹, n = 6) and SNX27b-ΔRA (–18.6±4.9 pA⋅pF⁻¹, n = 5) with GIRK1/GIRK3 (**P<0.05, one way ANOVA followed by Bonferroni <i>post hoc</i> test; n.s. – not significant).</p

Dominant-negative H-Ras (H-Ras_S17NDN) prevents SNX27b-dependent down-regulation of GIRK channels.

<p><b>A</b>, Schematic shows GIRK2c and GIRK3 with PDZ-binding motif and GIRK2a and GIRK3-RR, which lack motifs that interact with SNX27-PDZ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Balana1" target="_blank">[7]</a>. <b>B</b>, Examples of baclofen-induced (100 µM) and Ba²⁺-sensitive (1 mM Ba²⁺) currents in HEK293 cells transfected with cDNA for GABA_B1a/B2, GIRK2c/GIRK3 and either empty vector (Control), H-Ras_S17NDN or H-Ras_S17NDN plus SNX27b. <b>C</b>, Bar graph shows average I_Baclofen for GIRK2c/GIRK3 alone (–47.7±8.5 pA⋅pF⁻¹, n = 6), GIRK2c/GIRK3 and H-Ras_S17NDN (–22.0±3.8 pA⋅pF⁻¹, n = 19) or GIRK2c/GIRK3, SNX27b and H-Ras_S17NDN (–23.7±4.7 pA⋅pF⁻¹, n = 16). <b>D</b>, Bar graph shows I_Baclofen for control (GIRK2a/GIRK3-RR alone) (–18.8±3.0 pA⋅pF⁻¹, n = 10) and GIRK2a/GIRK3-RR plus H-Ras_S17NDN (–18.6±2.0 pA⋅pF⁻¹, n = 11). <b>E</b>, Bar graph shows average I_Barium for GIRK2c/GIRK3 alone (–27.6±9.8 pA⋅pF⁻¹, n = 6), GIRK2c/GIRK3 and H-Ras_S17NDN (–7.3±2.2 pA⋅pF⁻¹, n = 18) or GIRK2c/GIRK3, SNX27b and H-Ras_S17NDN (–7.1±1.5 pA⋅pF⁻¹, n = 14). <b>F</b>, Bar graph shows average I_Barium for GIRK2a/GIRK3-RR alone (−2.49±0.9 pA⋅pF⁻¹, n = 10), GIRK2c/GIRK3 and H-Ras_S17NDN (−1.84±0.4 pA⋅pF⁻¹, n = 11). **P<0.05, one way ANOVA followed by Bonferroni post hoc test; n.s. – not significant.</p

K305A point mutation in SNX27b RA domain disrupts interaction with constitutively active H-Ras (H-Ras_G12VCA).

<p><b>A</b>, Cartoons of SNX27b, GST fused to SNX27b-RA_WT or SNX27b-RA_K305A. <b>B</b>, Coomassie stain shows purified GST, His₈-Ras_G12VCA, GST-RA_WT and GST-RA_K305A. <b>C,</b> Western blot using anti-His₆ antibody shows His₈-Ras_G12VCA binding to GST-RA_WT but not to GST-RA_K305A. <b>C</b>, Quantification of pull-down shows consistent decrease in association of GST-RA_K305A mutant with His₈-Ras_G12VCA (assay repeated 4 times).</p

K305A point mutation in SNX27b RA domain disrupts functional regulation of GIRK2c/GIRK3 channels.

<p><b>A</b>, Alignment of two different RA domains: a RID (Ras interacting domain) from RalGDS and a RA domain of RGL, with RA domain of SNX27. Residues implicated in Ras binding in Raf and RalGDS domains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Block1" target="_blank">[28]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Vetter1" target="_blank">[30]</a> are highlighted in red. <b>B</b>, High-resolution structure shows R20, K32 and K52 at the binding interface of RID and Ras <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Huang1" target="_blank">[26]</a>. <b>C,</b> RA domain mutations impair the ability of SNX27b to induce formation of GIRK2c/3 puncta using BiFC. HEK293T cells were co-transfected with cDNA for ^CYGIRK2c/^NYGIRK3 and either wild-type SNX27b (<b>i</b>), SNX27b-R276A (<b>ii</b>), SNX27b-R288A/K291A (<b>iii</b>) or SNX27b-K305A (<b>iv</b>). Green fluorescence indicates molecular complementation of ^CYGIRK2c/^NYGIRK3. Scale bar: 10 µm. <b>D</b>, Colocalization of wild-type SNX27b (SNX27b-YFP; green) and SNX27b-K305A (anti-SNX27; green) with an early endosomal marker (anti-EEA1, green). Scale bar: 5 µm. <b>E</b>, Average I_Baclofen currents for control (–53.4±9.4 pA⋅pF⁻¹, n = 5), SNX27b (–8.05±3.44 pA⋅pF⁻¹, n = 6) and SNX27b-K305A (–52.9±8.8 pA⋅pF⁻¹, n = 10) with GIRK2c/GIRK3 channels.</p