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.