Ne name KCNMB4). To test no matter if 4-subunits are palmitoylated, we took two approaches. Firstly, the 4-subunit was transiently expressed in HEK293 cells that had been metabolically labeled with [3H]palmitate. Immunoprecipitation of the 4-subunits revealed robust incorporation of [3H]palmitate into 4-subunits (Fig. 1B). Mutation with the predicted palmitoylated cysteine residue Cys193 to alanine (C193A) abolished [3H]palmitate incorporation without having affecting protein expression of the mutated C193A 4-subunit (Fig. 1B). Secondly, applying acyl-RAC (13) that makes it possible for determination of hydroxylamine-sensitive thioester bonds that couple S-acylated cysteine residues to their cognate lipid, we identified 4-subunit S-acylation in native mouse brain (Fig. 1C). 4-Subunit Palmitoylation Controls Surface Expression and ER Exit–In many proteins, S-acylation controls trafficking and surface delivery of transmembrane proteins. To examine irrespective of whether palmitoylation of 4-subunits impacts their surface expression and trafficking per se, we undertook quantitative immunofluorescence assays. Utilizing an antibody that recognizes an extracellular epitope expression with the WT 4-subunits in HEK cells revealed no substantial surface expression (Fig. 1E) and predominant intracellular retention inside the ER in agreement with previous studies (15). The C193A palmitoylation-deficient mutant had no substantial effect on 4-subunit expression or localization (Fig. 1E). To improve the sensitivity of 4-subunit detection at the cell surface expression, we also engineered a 4-subunit having a Myc tag (Myce) inside the extracellular domain among transmembrane domains 1 and two. Probing for the Myce tag revealed low, but detectable, levels of 4-subunit surface expression, with predominant intracellular ER retention, and surface expression was abolished below the limit of detection using the C193A mutant. 4-Subunits are retained in the ER by a putative ER retention signal (KKXX) within the C terminus on the subunit (15). Hence, to enhance the signal-to-noise ratio of our assay, we engineered two trafficking-competent 4-subunits to allow characterization from the function of palmitoylation in 4-subunit trafficking. Firstly, we mutated the central Lys-206 and Arg-207 aminoVOLUME 288 ?Quantity 18 ?May 3,13138 JOURNAL OF BIOLOGICAL CHEMISTRY4-Subunit Palmitoylation Controls BK Channel TraffickingFIGURE 1. Palmitoylation controls exit with the 4-subunit from the endoplasmic reticulum. A, schematic with the 4 regulatory subunit of big conductance calcium- and voltage-activated potassium (BK) channels indicating the palmitoylated cysteine residue (Cys-193) juxtaposed towards the intracellular C terminus on the second transmembrane domain. B representative fluorographs of [3H]palmitate (3H-palm) incorporation and corresponding Western blot (anti-Myc) in the wild-type 4-subunit along with the alanine mutant C193A.Formula of 1608495-27-7 C, acyl-RAC of murine cerebellum with Western blot probed with anti-b4.Price of 351439-07-1 D, representative single confocal photos on the 4 and C193A mutant expressed in HEK293 cells and co-labeled for the ER.PMID:24059181 Scale bars are 2 m. E and F, bar graphs of membrane expression (expressed as a percentage of wild-type four) (E) and co-localization using the ER (expressed as Pearson’s correlation coefficient, R) (F) of the wild-type four and C193A mutant. Information are indicates S.E. N five, n 200. **, p 0.01 when compared with wild-type four group, ANOVA with post hoc Dunnett’s test.acids on the KKXX ER retention motif to alanine (KAAX construct), major to a significantly e.