ABCMdb

ABCC8 p.Asn10Gln

None has been submitted yet.

Publications

PMID: 10204114 [PubMed] Aguilar-Bryan L et al: "Molecular biology of adenosine triphosphate-sensitive potassium channels."

No. Sentence Comment

277 The N10Q or N1050Q mutations partially eliminate glycosylation, while the double mutant is not glycosylated. Login to comment

PMID: 10506167 [PubMed] Raab-Graham KF et al: "Membrane topology of the amino-terminal region of the sulfonylurea receptor."

No. Sentence Comment

65 Deletion of Glycosylation Sites-Candidate N-linked glycosylation sites at Asn10 and/or Asn1050 were mutated to create proteins lacking one (SUR-N10Q and SUR-N1050Q) or both (SUR-N10Q,N1050Q) putative glycosylation sites. Login to comment
69 SUR1, SUR-N10Q, SUR-N1050Q, and SUR-N10Q,N1050Q (20-30 ␮g/100-mm plate) were cotransfected with Kir 6.2 (1-4 ␮g/ 100-mm plate) using pFx-2 (Invitrogen). Login to comment
118 Full-length SUR1, SUR-N10Q, SUR-N1050Q, and SUR-N10Q,N1050Q were coexpressed with Kir 6.2 in COS-1 cells and identified by immunoblotting with an antibody to a carboxyl-terminal V5 epitope tag. Login to comment
123 To determine which residues were glycosylated, the predicted glycosylation acceptor sites Asn10 and Asn1050 were mutated either individually (SUR-N10Q, and SUR-N1050Q) or together (SUR-N10Q,1050Q). Login to comment
124 Expression of SUR-N10Q (Fig. 2, third and fourth lanes) and SUR-N1050Q (Fig. 2, fifth and sixth lanes) demonstrated that elimination of either of the Asn10 or Asn1050 glycosylation consensus sites resulted in a significant decrease of mature glycosylated receptor compared with wild type SUR1. Login to comment
125 Furthermore, simultaneous removal of both the Asn10 and Asn1050 sites in SUR-N10Q,N1050Q resulted only in a band that comigrated with the unglycosylated form of the protein (Fig. 2, seventh and eighth lanes), indicating that both Asn10 and Asn1050 were glycosylated in wild type SUR1. Login to comment
126 A reduction in glycosylation on SUR-N10Q relative to wild type SUR1 is in agreement with previous studies suggesting that Asn10 on the SUR1 amino terminus is glycosylated (14, 15). Login to comment
144 Mutation of that site in SUR285PL-N10Q produced a single band at the same apparent molecular mass as the unglycosylated SUR285PL protein, and its mobility was not shifted by PNGase F treatment (Fig. 4, third and fourth lanes), showing that Asn10 is glycosylated in the SUR285PL fusion protein. Login to comment
156 Glycosylation of SUR285PL and SUR285PL-N10Q fusion proteins. Login to comment
157 Autoradiograms are shown of SUR285PL and SUR285PL-N10Q fusion proteins that were translated in vitro in the presence of [35 S]Met and canine pancreatic microsomes. Login to comment
158 Asn10 in SUR285PL-N10Q was mutated to remove the N-glycosylation acceptor site. Login to comment

PMID: 11994306 [PubMed] Conti LR et al: "Membrane targeting of ATP-sensitive potassium channel. Effects of glycosylation on surface expression."

No. Sentence Comment

6 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
60 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
102 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
105 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 The SUR1 N10Q,N1050Q double mutant produced only a single band with a mobility similar to that of core glycosylated SUR1. Login to comment
109 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
111 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 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
118 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
124 Glycosylation of SUR1 and SUR1 glycosylation mutants (SUR1 N10Q, SUR1 N1050Q, and SUR1 N10Q,N1050Q) in the presence of Kir6.2. Login to comment
128 Patch clamp current recordings of SUR1 and SUR1 N10Q,N1050Q constructs. Login to comment
129 COS-1 cells were co-transfected with SUR1 or SUR1 N10Q,N1050Q together with Kir6.2 and pEGFP. Login to comment
133 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
138 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
146 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
154 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
159 This trend also was observed for both single glycosylation mutants, SUR1 N10Q and SUR1 N1050Q. Login to comment
164 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
168 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 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
172 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
182 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
195 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
203 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
205 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
209 Immunolocalization of SUR1 ؉ Kir6.2, SUR1 ⌬F1388, and SUR1 N10Q,N1050Q ؉ Kir6.2. Login to comment
217 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
221 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 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
262 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 The addition of diazoxide or glibenclamide had no apparent impact on the surface expression of SUR1 N10Q,N1050Q (data not shown). Login to comment
274 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
59 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 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 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
108 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 To quantitatively examine KATP channel expression, the current magnitude of the double mutant SUR1 N10Q,N1050Q was compared with WT SUR1. Login to comment
117 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 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 Patch clamp current recordings of SUR1 and SUR1 N10Q,N1050Q constructs. Login to comment
132 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
137 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
145 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 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 This trend also was observed for both single glycosylation mutants, SUR1 N10Q and SUR1 N1050Q. Login to comment
163 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 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
171 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
181 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
194 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 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 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 Immunolocalization of SUR1 d19; Kir6.2, SUR1 èc;F1388, and SUR1 N10Q,N1050Q d19; Kir6.2. Login to comment
216 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
220 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
261 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
273 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