ABCMdb

PMID: 11994306

Conti LR, Radeke CM, Vandenberg CA
Membrane targeting of ATP-sensitive potassium channel. Effects of glycosylation on surface expression.
J Biol Chem. 2002 Jul 12;277(28):25416-22. Epub 2002 May 6., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC8 p.Asn10Gln Using patch clamp analysis, surface biotinylation, and immunofluorescence microscopy, we demonstrate a significant decrease in surface expression of SUR1 single or double glycosylation site mutants (N10Q,N1050Q) when co-expressed with Kir6.2. Login to comment 59 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln N-Linked glycosylation sites were removed by replacing asparagine 10 and/or asparagine 1050 with glutamines (SUR1 N10Q, SUR1 N1050Q, and SUR1 N10Q,N1050Q) (27). Login to comment 60 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln N-Linked glycosylation sites were removed by replacing asparagine 10 and/or asparagine 1050 with glutamines (SUR1 N10Q, SUR1 N1050Q, and SUR1 N10Q,N1050Q) (27). Login to comment 101 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln RESULTS Glycosylation of SUR1 and SUR1 Glycosylation Mutants-To examine the effect of glycosylation in SUR1 trafficking and surface expression, the two N-linked glycosylation sites on SUR1 were replaced with glutamines to form single glycosylation mutants (SUR1 N10Q, SUR1 N1050Q) and double site glycosylation mutant (SUR1 N10Q,N1050Q). Login to comment 102 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln RESULTS Glycosylation of SUR1 and SUR1 Glycosylation Mutants-To examine the effect of glycosylation in SUR1 trafficking and surface expression, the two N-linked glycosylation sites on SUR1 were replaced with glutamines to form single glycosylation mutants (SUR1 N10Q, SUR1 N1050Q) and double site glycosylation mutant (SUR1 N10Q,N1050Q). Login to comment 104 ABCC8 p.Asn10Gln While the SUR1 N10Q complex and core glycosylated bands showed distinct separation from one another, the SUR1 N1050Q bands consistently migrated as a compact doublet. Login to comment 105 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln While the SUR1 N10Q complex and core glycosylated bands showed distinct separation from one another, the SUR1 N1050Q bands consistently migrated as a compact doublet. Login to comment 106 ABCC8 p.Asn10Gln The SUR1 N10Q,N1050Q double mutant produced only a single band with a mobility similar to that of core glycosylated SUR1. Login to comment 108 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln All glycosylation mutants (SUR1 N10Q, SUR1 N1050Q, and SUR1 N10Q,N1050Q) yielded functional channels when co-expressed with Kir6.2 in transiently transfected COS-1 cells. Login to comment 109 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln All glycosylation mutants (SUR1 N10Q, SUR1 N1050Q, and SUR1 N10Q,N1050Q) yielded functional channels when co-expressed with Kir6.2 in transiently transfected COS-1 cells. Login to comment 110 ABCC8 p.Asn10Gln To quantitatively examine KATP channel expression, the current magnitude of the double mutant SUR1 N10Q,N1050Q was compared with WT SUR1. Login to comment 111 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln To quantitatively examine KATP channel expression, the current magnitude of the double mutant SUR1 N10Q,N1050Q was compared with WT SUR1. Login to comment 112 ABCC8 p.Asn10Gln SUR1 N10Q,N1050Q displayed a striking and significant decrease in channel activity compared with WT SUR1 when co-expressed with Kir6.2 (Fig. 2, A and B). Login to comment 117 ABCC8 p.Asn10Gln Consistent with patch clamp analysis, SUR1 N10Q,N1050Q with Kir6.2 showed reduced labeling when compared with WT SUR1 with Kir6.2 (Fig. 3). Login to comment 118 ABCC8 p.Asn10Gln Consistent with patch clamp analysis, SUR1 N10Q,N1050Q with Kir6.2 showed reduced labeling when compared with WT SUR1 with Kir6.2 (Fig. 3). Login to comment 123 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln Glycosylation of SUR1 and SUR1 glycosylation mutants (SUR1 N10Q, SUR1 N1050Q, and SUR1 N10Q,N1050Q) in the presence of Kir6.2. Login to comment 124 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln Glycosylation of SUR1 and SUR1 glycosylation mutants (SUR1 N10Q, SUR1 N1050Q, and SUR1 N10Q,N1050Q) in the presence of Kir6.2. Login to comment 127 ABCC8 p.Asn10Gln Patch clamp current recordings of SUR1 and SUR1 N10Q,N1050Q constructs. Login to comment 128 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln Patch clamp current recordings of SUR1 and SUR1 N10Q,N1050Q constructs. Login to comment 129 ABCC8 p.Asn10Gln COS-1 cells were co-transfected with SUR1 or SUR1 N10Q,N1050Q together with Kir6.2 and pEGFP. Login to comment 132 ABCC8 p.Asn10Gln B, quantitation of patch clamp recordings was performed by averaging the mean current acquired at afa;60 mV for excised patches from cells expressing SUR1 af9; Kir6.2 and SUR1 N10Q,N1050Q af9; Kir6.2. Login to comment 133 ABCC8 p.Asn10Gln B, quantitation of patch clamp recordings was performed by averaging the mean current acquired at -60 mV for excised patches from cells expressing SUR1 ϩ Kir6.2 and SUR1 N10Q,N1050Q ϩ Kir6.2. Login to comment 137 ABCC8 p.Asn10Gln COS-1 cells were transfected with SUR1 together with Kir6.2, SUR1 N10Q,N1050Q together with Kir6.2, or SUR1 èc;F1388 alone. Login to comment 138 ABCC8 p.Asn10Gln COS-1 cells were transfected with SUR1 together with Kir6.2, SUR1 N10Q,N1050Q together with Kir6.2, or SUR1 ⌬F1388 alone. Login to comment 145 ABCC8 p.Asn10Gln Consistent with patch clamp and surface immunofluorescence data, biotin maleimide labeling of SUR1 N10Q,N1050Q, when co-expressed with Kir6.2, was reduced compared with WT SUR1 (Fig. 4A). Login to comment 146 ABCC8 p.Asn10Gln Consistent with patch clamp and surface immunofluorescence data, biotin maleimide labeling of SUR1 N10Q,N1050Q, when co-expressed with Kir6.2, was reduced compared with WT SUR1 (Fig. 4A). Login to comment 153 ABCC8 p.Asn10Gln The single glycosylation mutants (SUR1 N10Q, and SUR1 N1050Q) were also investigated and similarly showed a significant decrease in surface expression (Fig. 4B). Login to comment 154 ABCC8 p.Asn10Gln The single glycosylation mutants (SUR1 N10Q, and SUR1 N1050Q) were also investigated and similarly showed a significant decrease in surface expression (Fig. 4B). Login to comment 158 ABCC8 p.Asn10Gln This trend also was observed for both single glycosylation mutants, SUR1 N10Q and SUR1 N1050Q. Login to comment 159 ABCC8 p.Asn10Gln This trend also was observed for both single glycosylation mutants, SUR1 N10Q and SUR1 N1050Q. Login to comment 163 ABCC8 p.Asn10Gln To determine subcellular distribution of SUR1 or SUR1 N10Q,N1050Q, immunofluorescent labeling of permeabilized COS-1 cells expressing SUR1 constructs, in the presence of Kir6.2, was investigated. Login to comment 164 ABCC8 p.Asn10Gln To determine subcellular distribution of SUR1 or SUR1 N10Q,N1050Q, immunofluorescent labeling of permeabilized COS-1 cells expressing SUR1 constructs, in the presence of Kir6.2, was investigated. Login to comment 167 ABCC8 p.Asn10Gln WT SUR1 af9; Kir6.2 showed distinct plasma membrane staining, which was only rarely observed with SUR1 N10Q,N1050Q af9; Kir6.2, and almost never with SUR1 èc;F1388 (Fig. 5, A and B, arrowheads). Login to comment 168 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln WT SUR1 ϩ Kir6.2 showed distinct plasma membrane staining, which was only rarely observed with SUR1 N10Q,N1050Q ϩ Kir6.2, and almost never with SUR1 ⌬F1388 (Fig. 5, A and B, arrowheads). Login to comment 169 ABCC8 p.Asn10Gln In addition, labeling of SUR1 ϩ Kir6.2, SUR1 ⌬F1388, and SUR1 N10Q,N1050Q ϩ Kir6.2 all displayed prominent ER co-localization with pECFP-ER (Fig. 5A). Login to comment 171 ABCC8 p.Asn10Gln While WT SUR1 af9; Kir6.2 tended to overlap with the Golgi at low intensity, SUR1 èc;F1388 or SUR1 N10Q,N1050Q af9; Kir6.2 did not generally overlap with the pECFP-Golgi marker. Login to comment 172 ABCC8 p.Asn10Gln While WT SUR1 ϩ Kir6.2 tended to overlap with the Golgi at low intensity, SUR1 ⌬F1388 or SUR1 N10Q,N1050Q ϩ Kir6.2 did not generally overlap with the pECFP-Golgi marker. Login to comment 181 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln B, quantitation of surface biotinylation data: SUR1 af9; Kir6.2, 5.09% af9; 0.45 (n afd; 5); SUR1 èc;F1388, 0.52% af9; 0.05 (n afd; 4); SUR1 N10Q af9; Kir6.2, 1.65% af9; 0.70 (n afd; 5); SUR1 N1050Q af9; Kir6.2, 2.24% af9; 1.47 (n afd; 3); SUR1 N10Q,N1050Q af9; Kir6.2, 1.3% af9; 0.85 (n afd; 4). Login to comment 182 ABCC8 p.Asn10Gln B, quantitation of surface biotinylation data: SUR1 ϩ Kir6.2, 5.09% ϩ 0.45 (n ϭ 5); SUR1 ⌬F1388, 0.52% ϩ 0.05 (n ϭ 4); SUR1 N10Q ϩ Kir6.2, 1.65% ϩ 0.70 (n ϭ 5); SUR1 N1050Q ϩ Kir6.2, 2.24% ϩ 1.47 (n ϭ 3); SUR1 N10Q,N1050Q ϩ Kir6.2, 1.3% ϩ 0.85 (n ϭ 4). Login to comment 194 ABCC8 p.Asn10Gln Additionally, mutation of the ER retention signal sustained partial recovery of SUR1AAA N10Q,N1050Q surface expression in the presence or absence of Kir6.2 (Fig. 7). Login to comment 195 ABCC8 p.Asn10Gln Additionally, mutation of the ER retention signal sustained partial recovery of SUR1AAA N10Q,N1050Q surface expression in the presence or absence of Kir6.2 (Fig. 7). Login to comment 202 ABCC8 p.Asn10Gln The increase in apparent molecular mass of SUR1AAA and SUR1AAA glycosylation mutants (SUR1AAA N10Q, and SUR1AAA N1050Q) was demonstrated to be a result of oligosaccharide modification. Login to comment 203 ABCC8 p.Asn10Gln The increase in apparent molecular mass of SUR1AAA and SUR1AAA glycosylation mutants (SUR1AAA N10Q, and SUR1AAA N1050Q) was demonstrated to be a result of oligosaccharide modification. Login to comment 204 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln Both SUR1AAA N10Q and SUR1AAA N1050Q show increased apparent molecular mass in the absence of Kir6.2 (Fig. 7, Input, right panel), which decreased toward the size of SUR1 N10Q and SUR1 N1050Q, respectively, in the presence of Kir6.2 (Fig. 7, Input, left panel). Login to comment 205 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln Both SUR1AAA N10Q and SUR1AAA N1050Q show increased apparent molecular mass in the absence of Kir6.2 (Fig. 7, Input, right panel), which decreased toward the size of SUR1 N10Q and SUR1 N1050Q, respectively, in the presence of Kir6.2 (Fig. 7, Input, left panel). Login to comment 208 ABCC8 p.Asn10Gln Immunolocalization of SUR1 d19; Kir6.2, SUR1 èc;F1388, and SUR1 N10Q,N1050Q d19; Kir6.2. Login to comment 209 ABCC8 p.Asn10Gln Immunolocalization of SUR1 ؉ Kir6.2, SUR1 ⌬F1388, and SUR1 N10Q,N1050Q ؉ Kir6.2. Login to comment 216 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln SUR1 af9; Kir6.2, 3.73% af9; 0.59 (n afd; 4); SUR1 èc;F1388, 0.75% af9; 0.26 (n afd; 4); SUR1, 1.77% af9; 0.61 (n afd; 5); SUR1 N10Q, 0.82% af9; 0.68 (n afd; 2); SUR1 N1050Q, 0.43% af9; 0.27 (n afd; 2); SUR1 N10Q,N1050Q, 0.23% af9; 0.15 (n afd; 2). Login to comment 217 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln SUR1 ϩ Kir6.2, 3.73% ϩ 0.59 (n ϭ 4); SUR1 ⌬F1388, 0.75% ϩ 0.26 (n ϭ 4); SUR1, 1.77% ϩ 0.61 (n ϭ 5); SUR1 N10Q, 0.82% ϩ 0.68 (n ϭ 2); SUR1 N1050Q, 0.43% ϩ 0.27 (n ϭ 2); SUR1 N10Q,N1050Q, 0.23% ϩ 0.15 (n ϭ 2). Login to comment 220 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln The study presented here utilized single and double SUR1 glycosylation mutants (SUR1 N10Q, SUR1 N1050Q, and SUR1 N10Q,N1050Q) to demonstrate the significance of SUR1 oligosaccharide modification in the surface membrane expression of KATP channels. Login to comment 221 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln The study presented here utilized single and double SUR1 glycosylation mutants (SUR1 N10Q, SUR1 N1050Q, and SUR1 N10Q,N1050Q) to demonstrate the significance of SUR1 oligosaccharide modification in the surface membrane expression of KATP channels. Login to comment 222 ABCC8 p.Asn10Gln We demonstrate a significant reduction in the surface expression of SUR1 N10Q, SUR1 N1050Q, and SUR1N10Q,N1050Q compared with WT SUR1 when co-expressed with Kir6.2. Login to comment 261 ABCC8 p.Asn10Gln To address whether drugs could increase the stability of SUR1 N10Q,1050Q and consequently increase surface expression, surface biotinylation was carried out following 24-h incubation with sulfonylureas or potassium channel openers. Login to comment 262 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln To address whether drugs could increase the stability of SUR1 N10Q,1050Q and consequently increase surface expression, surface biotinylation was carried out following 24-h incubation with sulfonylureas or potassium channel openers. Login to comment 263 ABCC8 p.Asn10Gln The addition of diazoxide or glibenclamide had no apparent impact on the surface expression of SUR1 N10Q,N1050Q (data not shown). Login to comment 273 ABCC8 p.Asn10Gln The greater apparent molecular mass of the SUR1 N10Q upper band compared with the SUR1 N1050Q upper band suggests that the Asn1050 site is glycosylated more extensively. Login to comment 274 ABCC8 p.Asn10Gln The greater apparent molecular mass of the SUR1 N10Q upper band compared with the SUR1 N1050Q upper band suggests that the Asn1050 site is glycosylated more extensively. Login to comment