ABCMdb

ABCC8 p.Asn10Gln

Predicted by SNAP2:	A: D (59%), C: D (71%), D: N (53%), E: D (53%), F: D (71%), G: N (61%), H: N (61%), I: D (75%), K: D (59%), L: D (75%), M: D (71%), P: D (63%), Q: N (57%), R: N (53%), S: N (66%), T: N (57%), V: D (71%), W: D (80%), Y: N (53%),
Predicted by PROVEAN:	A: D, C: D, D: N, E: D, F: D, G: D, H: N, I: N, K: N, L: D, M: D, P: D, Q: D, R: N, S: N, T: N, V: N, W: D, Y: D,

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[hide] Molecular biology of adenosine triphosphate-sensit... Endocr Rev. 1999 Apr;20(2):101-35. Aguilar-Bryan L, Bryan J
Molecular biology of adenosine triphosphate-sensitive potassium channels.
Endocr Rev. 1999 Apr;20(2):101-35., [PMID:10204114]

Abstract [show]
KATP channels are a newly defined class of potassium channels based on the physical association of an ABC protein, the sulfonylurea receptor, and a K+ inward rectifier subunit. The beta-cell KATP channel is composed of SUR1, the high-affinity sulfonylurea receptor with multiple TMDs and two NBFs, and KIR6.2, a weak inward rectifier, in a 1:1 stoichiometry. The pore of the channel is formed by KIR6.2 in a tetrameric arrangement; the overall stoichiometry of active channels is (SUR1/KIR6.2)4. The two subunits form a tightly integrated whole. KIR6.2 can be expressed in the plasma membrane either by deletion of an ER retention signal at its C-terminal end or by high-level expression to overwhelm the retention mechanism. The single-channel conductance of the homomeric KIR6.2 channels is equivalent to SUR/KIR6.2 channels, but they differ in all other respects, including bursting behavior, pharmacological properties, sensitivity to ATP and ADP, and trafficking to the plasma membrane. Coexpression with SUR restores the normal channel properties. The key role KATP channel play in the regulation of insulin secretion in response to changes in glucose metabolism is underscored by the finding that a recessive form of persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is caused by mutations in KATP channel subunits that result in the loss of channel activity. KATP channels set the resting membrane potential of beta-cells, and their loss results in a constitutive depolarization that allows voltage-gated Ca2+ channels to open spontaneously, increasing the cytosolic Ca2+ levels enough to trigger continuous release of insulin. The loss of KATP channels, in effect, uncouples the electrical activity of beta-cells from their metabolic activity. PHHI mutations have been informative on the function of SUR1 and regulation of KATP channels by adenine nucleotides. The results indicate that SUR1 is important in sensing nucleotide changes, as implied by its sequence similarity to other ABC proteins, in addition to being the drug sensor. An unexpected finding is that the inhibitory action of ATP appears to be through a site located on KIR6.2, whose affinity for ATP is modified by SUR1. A PHHI mutation, G1479R, in the second NBF of SUR1 forms active KATP channels that respond normally to ATP, but fail to activate with MgADP. The result implies that ATP tonically inhibits KATP channels, but that the ADP level in a fasting beta-cell antagonizes this inhibition. Decreases in the ADP level as glucose is metabolized result in KATP channel closure. Although KATP channels are the target for sulfonylureas used in the treatment of NIDDM, the available data suggest that the identified KATP channel mutations do not play a major role in diabetes. Understanding how KATP channels fit into the overall scheme of glucose homeostasis, on the other hand, promises insight into diabetes and other disorders of glucose metabolism, while understanding the structure and regulation of these channels offers potential for development of novel compounds to regulate cellular electrical activity.

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

[hide] Membrane topology of the amino-terminal region of ... J Biol Chem. 1999 Oct 8;274(41):29122-9. Raab-Graham KF, Cirilo LJ, Boettcher AA, Radeke CM, Vandenberg CA
Membrane topology of the amino-terminal region of the sulfonylurea receptor.
J Biol Chem. 1999 Oct 8;274(41):29122-9., [PMID:10506167]

Abstract [show]
The sulfonylurea receptor (SUR) is a member of the ATP-binding cassette family that is associated with Kir 6.x to form ATP-sensitive potassium channels. SUR is involved in nucleotide regulation of the channel and is the site of pharmacological interaction with sulfonylurea drugs and potassium channel openers. SUR contains three hydrophobic domains, TM(0), TM(1), and TM(2), with nucleotide binding folds following TM(1) and TM(2). Two topological models of SUR have been proposed containing either 13 transmembrane segments (in a 4+5+4 arrangement) or 17 transmembrane segments (in a 5+6+6 arrangement) (Aguilar-Bryan, L., Nichols, C. G., Wechsler, S. W., Clement, J. P. t., Boyd, A. E., III, Gonzalez, G., Herrera-Sosa, H., Nguy, K., Bryan, J., and Nelson, D. A. (1995) Science 268, 423-426; Tusnady, G. E., Bakos, E., Varadi, A., and Sarkadi, B. (1997) FEBS Lett. 402, 1-3; Aguilar-Bryan, L., Clement, J. P., IV, Gonzalez, G., Kunjilwar, K., Babenko, A., and Bryan, J. (1998) Physiol. Rev. 78, 227-245). We analyzed the topology of the amino-terminal TM(0) region of SUR1 using glycosylation and protease protection studies. Deglycosylation using peptide-N-glycosidase F and site-directed mutagenesis established that Asn(10), near the amino terminus, and Asn(1050) are the only sites of N-linked glycosylation, thus placing these sites on the extracellular side of the membrane. To study in detail the topology of SUR1, we constructed and expressed in vitro fusion proteins containing 1-5 hydrophobic segments of the TM(0) region fused to the reporter prolactin. The fusion proteins were subjected to a protease protection assay that reported the accessibility of the prolactin epitope. Our results indicate that the TM(0) region is comprised of 5 transmembrane segments. These data support the 5+6+6 model of SUR1 topology.

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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

[hide] Membrane targeting of ATP-sensitive potassium chan... J Biol Chem. 2002 Jul 12;277(28):25416-22. Epub 2002 May 6. 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., [PMID:11994306]

Abstract [show]
Oligosaccharides play significant roles in trafficking, folding, and sorting of membrane proteins. Sulfonylurea receptors (SURx), members of the ATP binding cassette family of proteins, associate with the inward rectifier Kir6.x to form ATP-sensitive potassium channels (K(ATP)). These channels are found on the plasma membrane in many tissues and play a pivotal role in synchronizing electrical excitability with cell metabolic state. Trafficking defects resulting from three independent SUR1 mutations involved in the disease persistent hyperinsulinemic hypoglycemia of infancy have been described. Two of these mutations displayed notable decreases in glycosylation. Here we have investigated the relationship between the two N-linked glycosylation sites (Asn(10) and Asn(1050)) and SUR1 trafficking. 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. Additionally, we show prominent retention within the ER of the SUR1 double glycosylation mutant under the same conditions. Further investigation revealed that mutation of the ER retention signal was able to partially restore surface expression of the SUR1 double glycosylation mutant. These studies suggest that SUR1 glycosylation is a key element for the proper trafficking and surface expression of K(ATP) channels.

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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