Iquitylation could play a role within this method as Ub has been discovered to regulate surface expression and degradation of other members of the Kir household (25). Thus, we evaluated the background ubiquitylation levels of recombinant WT and K346T proteins by performing WB analysis with anti-polyubiquitin and anti-Kir2.1 antibodies and compared with that of K346T. Equal amounts of His-tagged WT and K346T protein eluates have been resolved by SDS Page and ubiquitylation levels were evaluated by WB (Supplementary Material, Fig. S4A). These experiments initially revealed that Kir2.1 is ubiquitylated; in addition they showed that the ubiquitylation levels for K346T channels have been reduced than the WT (Supplementary Material, Fig. S4A and B). We confirmed that these data by using an in vitro ubiquitylation assay. Cells expressing WT or K346T channels had been transfected withHuman Molecular Genetics, 2014, Vol. 23, No.Figure 5. The K346T mutation impacts the distribution of Kir2.1 channels in membrane lipid rafts. (A) WB analysis of cholesterol-rich (triton insoluble fractions: three ) and cholesterol-poor membrane 858474-14-3 site fractions (triton soluble fractions: 102) of WT or K346T Kir2.1-expressing cells. WT channels are mostly distributed in triton insoluble fractions (gray box), whereas K346T is also abundantly localized in cholesterol-poor fractions (black boxes). Cav-1 and flotillin-1 recognize the caveolar raft fractions. Molecular weight markers are around the left (kDa). (B E) Standard distributions of total protein (indicated on top) in membrane fractions isolated by sucrose density gradient. The levels of protein in each fraction are normalized towards the total protein amount recovered from all of the fractions with each other.simulations of cholesterol revealed that K346T is situated 1014 A away in the known and newly identified cholesterolbinding sites (Supplementary Material, Fig. S5). Kir2.1 interacts with Cav-1 and Cav-2 proteins The details that (i) the K346T mutation also resides within the proximity of a putative caveolin-binding motif and (ii) caveolins influence cell surface expression, raft compartmentalization and trafficking of various type of K+ channels (31 33), prompted us to investigate no matter if Kir2.1 interacts with caveolin proteins which are expressed in cultured astrocytes (34), along with the feasible effects of K346T mutation. By performing the His-affinity co-purification assay described above, we discovered that Cav-1, the key structural element of caveolar rafts, similarly interacted with WT and K346T channels (Fig. 6A and B). In contrast, K346T mutation drastically reduced the association of Kir2.1 with Cav-2 (Fig. 6A and B), a protein directly involved in the regulation of cell signaling at raft levels (35). Cav-3, the 55028-72-3 Autophagy musclespecific caveolin isoform, could not be detected in U251 cells (M.S. Brignone, unpublished observation), confirming prior findings (34). Since Cav-1 and Cav-2 can modulate channel endocytosis major to channel degradation or inactivation (3133,36) and Cav-2 can also regulate membrane protein trafficking independently from Cav-1 (37), the outcomes obtained here recommend that the differences in the associations with Cav-2 could influence K346T channels’ membrane compartmentalization, stability and trafficking.DISCUSSIONIn this study, we offer new gain-of-function mechanisms relevant to understand SQT3S pathogenesis, recommend the potential association of SQT3S with neurological issues and uncover a multifunctional domain in Kir2.1 that controls pivotalproperties of W.
