ABCMdb

PMID: 14707124

Yan F, Lin CW, Weisiger E, Cartier EA, Taschenberger G, Shyng SL
Sulfonylureas correct trafficking defects of ATP-sensitive potassium channels caused by mutations in the sulfonylurea receptor.
J Biol Chem. 2004 Mar 19;279(12):11096-105. Epub 2004 Jan 5., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro We report here that sulfonylureas also function as chemical chaperones to rescue KATP channel trafficking defects caused by two SUR1 mutations, A116P and V187D, identified in patients with congenital hyperinsulinism. Login to comment 3 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Sulfonylureas markedly increased cell surface expression of the A116P and V187D mutants by stabilizing the mutant SUR1 proteins and promoting their maturation. Login to comment 5 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Importantly, both mutant channels rescued to the cell surface have normal ATP, MgADP, and diazoxide sensitivities, demonstrating that SUR1 harboring either the A116P or the V187D mutation is capable of associating with Kir6.2 to form functional KATP channels. Thus, sulfonylureas may be used to treat congenital hyperinsulinism caused by certain KATP channel trafficking mutations. Login to comment 42 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Here, we report that two PHHI-associated SUR1 mutations, A116P and V187D (2, 21, 29, 37), located in the first transmembrane domain (TM0), prevent trafficking of KATP channels from the ER to the plasma membrane. Login to comment 43 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Here, we report that two PHHI-associated SUR1 mutations, A116P and V187D (2, 21, 29, 37), located in the first transmembrane domain (TM0), prevent trafficking of KATP channels from the ER to the plasma membrane. Login to comment 90 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The data were presented as the means afe; S.E. RESULTS Both the A116P and V187D Mutations in SUR1 Prevent Normal Cell Surface Expression of KATP Channels-Several recent studies have shown that defective KATP channel trafficking is an underlying mechanism of congenital hyperinsulinism. Login to comment 92 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro To examine whether mutations located in other parts of the molecule also affect channel trafficking, we focused our attention to two mutations, A116P and V187D, that are located in the first transmembrane domain, or TM0, of SUR1 (Fig. 1), and that have previously been reported to not form functional channels when co-expressed with Kir6.2 (6, 21, 37). Login to comment 93 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro RESULTS Both the A116P and V187D Mutations in SUR1 Prevent Normal Cell Surface Expression of KATP Channels-Several recent studies have shown that defective KATP channel trafficking is an underlying mechanism of congenital hyperinsulinism. Login to comment 95 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro To examine whether mutations located in other parts of the molecule also affect channel trafficking, we focused our attention to two mutations, A116P and V187D, that are located in the first transmembrane domain, or TM0, of SUR1 (Fig. 1), and that have previously been reported to not form functional channels when co-expressed with Kir6.2 (6, 21, 37). Login to comment 96 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro To investigate how the A116P and V187D mutations lead to loss of functional KATP channels, we first performed Western blot analysis. Login to comment 98 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Fig. 2A shows that, although both the immature and mature forms were seen with cells co-expressing WT-fSUR1 and Kir6.2, only the immature form was evident in cells co-expressing Kir6.2 and the A116P- or the V187D-fSUR1 mutants. Login to comment 101 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Fig. 2A shows that, although both the immature and mature forms were seen with cells co-expressing WT-fSUR1 and Kir6.2, only the immature form was evident in cells co-expressing Kir6.2 and the A116P- or the V187D-fSUR1 mutants. Login to comment 103 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro These results led us to conclude that the A116P and V187D mutations cause loss of functional KATP channels by preventing channels from trafficking to the cell surface. Login to comment 104 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro In contrast to the abundant surface staining observed in cells transfected with Kir6.2 and WT-fSUR1, surface staining in cells transfected with Kir6.2 and A116P-fSUR1 or V187D-fSUR1 was barely detectable (Fig. 2B, top panels). Login to comment 106 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro These results led us to conclude that the A116P and V187D mutations cause loss of functional KATP channels by preventing channels from trafficking to the cell surface. Login to comment 107 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro The Trafficking Defects of the A116P and V187D Mutants Are Intrinsic to SUR1-One potential explanation for the trafficking defects seen with the A116P- and V187D-fSUR1 mutations is that the SUR1 mutants are unable to associate with Kir6.2. Login to comment 108 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Although WT fSUR1/Kir6.2 fusion protein was expressed at a level comparable with that observed in cells transfected with WT-fSUR1 and Kir6.2 as individual subunits, fusion proteins carrying the A116P- or V187D-SUR1 mutation had poor surface expression, b03;10 and 20% that of WT fSUR1/ Kir6.2 fusion, respectively (Fig. 3A). Login to comment 109 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro To address this possibility, we used a heterotandem dimer construct in which the C terminus of the mutant fSUR1 has been fused to the N terminus of Kir6.2 (referred to as A116P- or V187D-fSUR1/Kir6.2 fusion) to achieve obligatory physical association between the two subunits; similar SUR1/ Kir6.2 fusion constructs have been used previously by a number of groups for structure-function and trafficking studies (7, 9, 10, 28, 34). Login to comment 111 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Although WT fSUR1/Kir6.2 fusion protein was expressed at a level comparable with that observed in cells transfected with WT-fSUR1 and Kir6.2 as individual subunits, fusion proteins carrying the A116P- or V187D-SUR1 mutation had poor surface expression, ϳ10 and 20% that of WT fSUR1/ Kir6.2 fusion, respectively (Fig. 3A). Login to comment 114 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Analysis of the A116- and V187D-SUR1 mutants by immunoblotting and immunofluorescent staining experiments. Login to comment 115 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The total steady-state protein level of A116P- and V187D-fSUR1 also appears less than that of WT-fSUR1. Login to comment 117 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro In contrast, only the immature band is observed in cells expressing Kir6.2 and A116P- or V187D-fSUR1. Login to comment 118 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The total steady-state protein level of A116P- and V187D-fSUR1 also appears less than that of WT-fSUR1. Login to comment 119 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Whereas cells expressing WT-fSUR1 channels have abundant surface staining, those expressing A116P- or V187D-fSUR1 channels have barely detectable staining. Login to comment 120 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro B, top panels, surface staining of COSm6 cells transiently transfected with Kir6.2 and either WT-, A116P-, or V187D-fSUR1 using the M2 anti-FLAG mouse monoclonal antibodies followed by Cy-3-conjugated anti-mouse secondary antibody. Login to comment 122 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Whereas cells expressing WT-fSUR1 channels have abundant surface staining, those expressing A116P- or V187D-fSUR1 channels have barely detectable staining. Login to comment 123 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1. Login to comment 125 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Both A116P- and V187D-fSUR1 were detected inside the cell, with a perinuclear staining pattern. Login to comment 126 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1. Login to comment 128 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The A116P and V187D mutations in SUR1. Login to comment 129 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The locations of the A116P and V187D mutations in SUR1 are shown. Login to comment 130 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The surface expression levels of A116P-fSUR1AAA and V187D-fSUR1AAA are 10 and 20% that of WT-fSUR1AAA, respectively (Fig. 3B). Login to comment 132 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro In addition, although the surface expression levels of the A116P and V187D mutant channels were very low (7 and 19% of WT; see Figs. Login to comment 133 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The surface expression levels of A116P-fSUR1AAA and V187D-fSUR1AAA are 10 and 20% that of WT-fSUR1AAA, respectively (Fig. 3B). Login to comment 135 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro In addition, although the surface expression levels of the A116P and V187D mutant channels were very low (7 and 19% of WT; see Figs. Login to comment 136 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Taken together, the results led us to propose that the A116P and V187D mutations cause trafficking defects in SUR1, possibly by promoting protein misfolding, but that the mutant proteins retain the ability to associate with Kir6.2 to form functional channels. Login to comment 137 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Another potential mechanism for the deficient surface expression of KATP channels is that the A116P and V187D mutations interfere with proper shielding of the RKR signals in the channel complex. Login to comment 139 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Taken together, the results led us to propose that the A116P and V187D mutations cause trafficking defects in SUR1, possibly by promoting protein misfolding, but that the mutant proteins retain the ability to associate with Kir6.2 to form functional channels. Login to comment 140 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Another potential mechanism for the deficient surface expression of KATP channels is that the A116P and V187D mutations interfere with proper shielding of the RKR signals in the channel complex. Login to comment 141 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Glibenclamide Corrects the Channel Trafficking Defects Caused by the A116P and V187D Mutations in SUR1-The data presented so far suggest that the two mutations likely cause defective channel trafficking by promoting misfolding of SUR1. Login to comment 142 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro We found that inactivation of the RKR signal in SUR1 slightly increased surface expression of the A116P mutant channels (from 6 to 18% of normal expression level) but not the V187D mutant, and removal of the RKR signal in Kir6.2 (Kir6.2⌬C25) also had very little effect on the surface expression of either mutant (Fig. 3C). Login to comment 144 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Glibenclamide Corrects the Channel Trafficking Defects Caused by the A116P and V187D Mutations in SUR1-The data presented so far suggest that the two mutations likely cause defective channel trafficking by promoting misfolding of SUR1. Login to comment 148 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro We found that treating cells with 5% glycerol for 24 h slightly improved surface expression of both the A116P and V187D mutants as well as the WT channel (Fig. 4A). Login to comment 149 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Treating cells with 5 òe;M glibenclamide, however, dramatically increased surface expression of A116P-fSUR1, from 5 to 55%, and of V187D-fSUR1, from 19% to 70% of normal WT channel expression level (Fig. 4A). Login to comment 151 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro We found that treating cells with 5% glycerol for 24 h slightly improved surface expression of both the A116P and V187D mutants as well as the WT channel (Fig. 4A). Login to comment 152 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Treating cells with 5 ␮M glibenclamide, however, dramatically increased surface expression of A116P-fSUR1, from 5 to 55%, and of V187D-fSUR1, from 19% to 70% of normal WT channel expression level (Fig. 4A). Login to comment 153 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro A, obligatory association between SUR1 and Kir6.2 does not overcome trafficking defects caused by A116P or V187D. Login to comment 154 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro The effect of glibenclamide on the A116P and V187D mu- FIG. 3. Login to comment 155 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The trafficking defects of the A116P- and V187D-fSUR1 mutants are intrinsic to SUR1. Login to comment 156 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro A, obligatory association between SUR1 and Kir6.2 does not overcome trafficking defects caused by A116P or V187D. Login to comment 157 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Fusion fSUR1/Kir6.2 constructs containing either A116P or V187D mutation still exhibit poor surface expression compared with the WT fusion construct. Login to comment 159 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro B, the A116P and V187D mutations cause trafficking defects in SUR1. Login to comment 161 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro However, introducing A116P or V187D to fSUR1AAA (fA116PAAA and fV187DAAA) abolishes the ability of the proteins to traffic to the cell surface. Login to comment 162 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro B, the A116P and V187D mutations cause trafficking defects in SUR1. Login to comment 163 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Inactivation of the RKR signal in SUR1 (SUR1RKR/AAA) only slightly improved surface expression of A116P but not V187D, whereas removal of RKR in Kir6.2 (Kir6.2èc;C25) slightly improved surface expression of V187D but not A116P. Login to comment 164 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro However, introducing A116P or V187D to fSUR1AAA (fA116PAAA and fV187DAAA) abolishes the ability of the proteins to traffic to the cell surface. Login to comment 165 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro C, trafficking defects caused by A116P and V187D do not involve improper shielding of RKR signals in the channel complex. Login to comment 166 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Inactivation of the RKR signal in SUR1 (SUR1RKR/AAA) only slightly improved surface expression of A116P but not V187D, whereas removal of RKR in Kir6.2 (Kir6.2⌬C25) slightly improved surface expression of V187D but not A116P. Login to comment 167 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1. Login to comment 168 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Glibenclamide rescues surface expression of the A116P and V187D mutant KATPchannels. A, cells co-expressing Kir6.2 and WT-, A116P-, or V187D-fSUR1 were subjected to the different drug treatments indicated for 24 h, and surface expression of fSUR1 was quantified using the chemiluminescence assay as described in Fig. 3. Login to comment 169 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Without any drug treatment, expression levels of the A116P and V187D mutants are 6.4 afe; 1.2 and 19.1 afe; 4.8% that of WT, respectively. Login to comment 170 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The response of the A116P- and the V187D-fSUR1 mutants to glibenclamide was specific; another SUR1 ligand, FIG. 4. Login to comment 171 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Glibenclamide rescues surface expression of the A116P and V187D mutant KATPchannels. A, cells co-expressing Kir6.2 and WT-, A116P-, or V187D-fSUR1 were subjected to the different drug treatments indicated for 24 h, and surface expression of fSUR1 was quantified using the chemiluminescence assay as described in Fig. 3. Login to comment 172 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Without any drug treatment, expression levels of the A116P and V187D mutants are 6.4 Ϯ 1.2 and 19.1 Ϯ 4.8% that of WT, respectively. Login to comment 173 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro B, Western blots showing that in cells expressing Kir6.2 and the A116P or V187D mutant fSUR1, treatment with 5 òe;M glibenclamide for 24 h led to appearance of the mature band, which was not detected in untreated cells (Fig. 2A). Login to comment 174 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Glibenclamide treatment at 5 ␮M for 24 h dramatically improved surface expression of both A116P- and V187D-fSUR1 (to 55.4 Ϯ 4.8 and 70.4 Ϯ 14.5% of WT, respectively) but only slightly increased WT expression (by 6.6 Ϯ 2.8%). Login to comment 175 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Top panels, surface staining of COSm6 cells transfected with Kir6.2 and either WT-, A116P-, or V187D-fSUR1, as described for Fig. 2B. Login to comment 176 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro B, Western blots showing that in cells expressing Kir6.2 and the A116P or V187D mutant fSUR1, treatment with 5 ␮M glibenclamide for 24 h led to appearance of the mature band, which was not detected in untreated cells (Fig. 2A). Login to comment 177 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro In contrast to the data shown in Fig. 2B, cells expressing A116P- or V187D-fSUR1 mutant channels had strong surface staining that is nearly comparable with cells expressing WT-fSUR1 channels. Login to comment 178 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Top panels, surface staining of COSm6 cells transfected with Kir6.2 and either WT-, A116P-, or V187D-fSUR1, as described for Fig. 2B. Login to comment 180 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro In contrast to the data shown in Fig. 2B, cells expressing A116P- or V187D-fSUR1 mutant channels had strong surface staining that is nearly comparable with cells expressing WT-fSUR1 channels. Login to comment 182 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro In fact, diazoxide slightly decreased the surface expression of both A116P and V187D mutants. Login to comment 183 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1; Glib, glibenclamide. Login to comment 185 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro In fact, diazoxide slightly decreased the surface expression of both A116P and V187D mutants. Login to comment 186 ABCC8 p.Ala116Pro To test this, we examined the effect of glibenclamide on metabolically labeled A116P-fSUR1 in cells co-expressing Kir6.2. Login to comment 188 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Glibenclamide Rescues Surface Expression of A116P Mutant Channels by Slowing the Degradation of the Mutant SUR1 Protein-We presume that glibenclamide acts as a chemical chaperone to facilitate folding of the mutant fSUR1 in the ER, thereby increasing maturation and cell surface expression of the channel complex. Login to comment 189 ABCC8 p.Ala116Pro To test this, we examined the effect of glibenclamide on metabolically labeled A116P-fSUR1 in cells co-expressing Kir6.2. Login to comment 190 ABCC8 p.Ala116Pro We quantified the degradation rate of A116P-fSUR1 co-expressed with Kir6.2 in control or in glibenclamide-treated cells (the sum of both immature and mature forms) and compared it with that of WT-fSUR1 (coexpressed with Kir6.2) in control cells (the sum of both immature and mature forms). Login to comment 191 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro In the absence of glibenclamide, A116P-fSUR1 was detected as a core glycosylated immature band following a 30-min pulse-labeling period and remained as the immature form throughout the chase period of up to 24 h (Fig. 6A). Login to comment 192 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro To further determine whether glibenclamide stabilizes the A116P-fSUR1 mutant protein by stabilizing the mutant SUR1 itself, we measured the degradation rate of A116P-fSUR1 in the absence of Kir6.2. Login to comment 193 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro We quantified the degradation rate of A116P-fSUR1 co-expressed with Kir6.2 in control or in glibenclamide-treated cells (the sum of both immature and mature forms) and compared it with that of WT-fSUR1 (coexpressed with Kir6.2) in control cells (the sum of both immature and mature forms). Login to comment 194 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro As shown in Fig. 6B, whereas the overall degradation rate of the A116P-fSUR1 mutant protein in glibenclamide-treated cells is similar to that of WT-fSUR1 in control cells, it is markedly slower than that of A116P-fSUR1 in control cells. Login to comment 195 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro To further determine whether glibenclamide stabilizes the A116P-fSUR1 mutant protein by stabilizing the mutant SUR1 itself, we measured the degradation rate of A116P-fSUR1 in the absence of Kir6.2. Login to comment 196 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Cells expressing A116P-fSUR1 alone were pulse-labeled for 30 min and chased for up to 18 h in the presence or absence of 5 ␮M glibenclamide. Login to comment 197 ABCC8 p.Ala116Pro We found that glibenclamide indeed slowed the degradation of A116P-fSUR1, although the rate is still faster than that of WT-fSUR1 in control cells (Fig. 6C). Login to comment 199 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Tolbutamide Also Rescues SUR1 A116P Mutant Channels to the Cell Surface, and the Expressed Channels Are Fully Functional after Tolbutamide Washout-Following our observation that glibenclamide significantly improves cell surface expression of the A116P and V187D mutants, the question arises as to whether the rescued channels are still glibenclamide-bound and whether they are physiologically functional. Login to comment 201 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Concentration and time dependence of the effect of glibenclamide on surface expression of A116P- and V187D-fSUR1 mutant KATPchannels. A, cells transfected with Kir6.2 and WT-, A116P-, or V187D-fSUR1 were treated with different concentrations of glibenclamide for 24 h, and the surface expression of fSUR1 was quantified by the chemiluminescence assay. Login to comment 204 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Concentration and time dependence of the effect of glibenclamide on surface expression of A116P- and V187D-fSUR1 mutant KATPchannels. A, cells transfected with Kir6.2 and WT-, A116P-, or V187D-fSUR1 were treated with different concentrations of glibenclamide for 24 h, and the surface expression of fSUR1 was quantified by the chemiluminescence assay. Login to comment 207 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The cells expressing A116P- or V187D-fSUR1 mutant channels were treated with 5 òe;M for different periods of time as indicated, and surface expression of the mutant channel was quantified by chemiluminescence assays. Login to comment 209 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Each data point represents the average from 2-3 experiments, and the error bar is the deviation from the average or the S.E. fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1. Login to comment 210 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The cells expressing A116P- or V187D-fSUR1 mutant channels were treated with 5 ␮M for different periods of time as indicated, and surface expression of the mutant channel was quantified by chemiluminescence assays. Login to comment 212 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Each data point represents the average from 2-3 experiments, and the error bar is the deviation from the average or the S.E. fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1. Login to comment 213 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Fig. 7A shows that in chemiluminescence assays, tolbutamide also increases surface expression of both A116P- and V187D-fSUR1 mutant channels, at concentrations of 100 and 300 òe;M that we tested (only 300 òe;M is shown). Login to comment 214 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Consistently, in inside-out patch clamp recording experiments, tolbutamide treatment led to parallel increases in the current size of both mutant channels; the average patch current amplitudes in K-INT for A116P- and V187D-fSUR1 channels are 3.24 afe; 0.75 nA (n afd; 12) and 5.40 afe; 1.19 nA (n afd; 13), respectively, compared with 7.05 afe; 1.02 nA (n afd; 17) for control WT-fSUR1 channels. Login to comment 216 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Fig. 7A shows that in chemiluminescence assays, tolbutamide also increases surface expression of both A116P- and V187D-fSUR1 mutant channels, at concentrations of 100 and 300 ␮M that we tested (only 300 ␮M is shown). Login to comment 217 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Consistently, in inside-out patch clamp recording experiments, tolbutamide treatment led to parallel increases in the current size of both mutant channels; the average patch current amplitudes in K-INT for A116P- and V187D-fSUR1 channels are 3.24 Ϯ 0.75 nA (n ϭ 12) and 5.40 Ϯ 1.19 nA (n ϭ 13), respectively, compared with 7.05 Ϯ 1.02 nA (n ϭ 17) for control WT-fSUR1 channels. Login to comment 220 ABCC8 p.Ala116Pro The ability of the rescued mutant channels to respond to metabolic changes was further examined by 86 Rbaf9; efflux experiments using the A116P mutant as an example. Login to comment 221 ABCC8 p.Ala116Pro Without tolbutamide treatment, the cells transfected with A116P exhibited very low KATP channel activities upon metabolic inhibition (11% efflux in 40 min; compare with 9% in untransfected cells) in contrast to cells transfected with WT channels (81% efflux). Login to comment 222 ABCC8 p.Ala116Pro Following tolbutamide treatment (300 òe;M for 24 h) and subsequent washout of tolbutamide (for 12 h), cells transfected with Kir6.2 and A116P exhibited a substantial increase in channel activities upon metabolic inhibition (b03;40% efflux; compare with b03;80% in cells transfected with Kir6.2 and WT-fSUR1 and 10% in untransfected cells). Login to comment 223 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro The ability of the rescued mutant channels to respond to metabolic changes was further examined by 86 Rbϩ efflux experiments using the A116P mutant as an example. Login to comment 224 ABCC8 p.Ala116Pro Without tolbutamide treatment, the cells transfected with A116P exhibited very low KATP channel activities upon metabolic inhibition (11% efflux in 40 min; compare with 9% in untransfected cells) in contrast to cells transfected with WT channels (81% efflux). Login to comment 225 ABCC8 p.Ala116Pro Following tolbutamide treatment (300 ␮M for 24 h) and subsequent washout of tolbutamide (for 12 h), cells transfected with Kir6.2 and A116P exhibited a substantial increase in channel activities upon metabolic inhibition (ϳ40% efflux; compare with ϳ80% in cells transfected with Kir6.2 and WT-fSUR1 and 10% in untransfected cells). Login to comment 226 ABCC8 p.Ala116Pro Thus, tolbutamide can be used as a pharmacological chaperone to recruit A116P mutant channels to the cell surface. Login to comment 228 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro In this study, we show that two SUR1 point mutations, A116P and V187D, identified in patients with congenital hyperinsulinism (2, 37) cause defective trafficking and a lack of cell surface expression of KATP channels. Login to comment 230 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Mechanisms of Trafficking Defects Caused by the A116P and V187D Mutations-Multiple steps are involved in the proper expression of KATP channels on the cell surface. Login to comment 231 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro In this study, we show that two SUR1 point mutations, A116P and V187D, identified in patients with congenital hyperinsulinism (2, 37) cause defective trafficking and a lack of cell surface expression of KATP channels. Login to comment 233 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Mechanisms of Trafficking Defects Caused by the A116P and V187D Mutations-Multiple steps are involved in the proper expression of KATP channels on the cell surface. Login to comment 236 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro In our pulse-chase labeling experiments, the mutant A116P-fSUR1 never became complex-glycosylated, arguing that the lack of surface expression was a result of ER retention rather than increased degradation of surface channels. Login to comment 237 ABCC8 p.Ala116Pro A, metabolic pulse-chase of A116P-fSUR1 co-expressed with Kir6.2 (labeled as fA116P-SUR1 in the figure). Login to comment 239 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Glibenclamide slows the degradation of A116P-fSUR1. Login to comment 240 ABCC8 p.Ala116Pro A, metabolic pulse-chase of A116P-fSUR1 co-expressed with Kir6.2 (labeled as fA116P-SUR1 in the figure). Login to comment 241 ABCC8 p.Ala116Pro B, degradation of A116P-fSUR1 in cells co-expressing Kir6.2. Login to comment 242 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Although A116P-fSUR1 in cells not treated with glibenclamide appeared as a single immature band throughout the chase, A116P-fSUR1 in cells treated with 5 ␮M glibenclamide following the pulse label was converted to the mature form with time. Login to comment 243 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro The overall degradation rate of A116P-fSUR1 in glibenclamide-treated cells (filled squares) is comparable to that of WT-fSUR1 in control cells (open circles) but obviously slower than that of A116P-fSUR1 in control (without glibenclamide treatment) cells (open squares). Login to comment 244 ABCC8 p.Ala116Pro B, degradation of A116P-fSUR1 in cells co-expressing Kir6.2. Login to comment 245 ABCC8 p.Ala116Pro In glibenclamide-treated cells expressing A116P-fSUR1 and Kir6.2 and in control cells expressing WT-fSUR1 and Kir6.2, both the mature band and immature band were included for the quantification of residual label. Login to comment 246 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro The overall degradation rate of A116P-fSUR1 in glibenclamide-treated cells (filled squares) is comparable to that of WT-fSUR1 in control cells (open circles) but obviously slower than that of A116P-fSUR1 in control (without glibenclamide treatment) cells (open squares). Login to comment 247 ABCC8 p.Ala116Pro The degradation rate of A116P-fSUR1 in glibenclamide-treated cells (filled squares) is apparently slower than that in control untreated cells (open squares), but it is still faster than that of WT-fSUR1 in control cells (open circles). Login to comment 249 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro C, degradation of A116P-fSUR1 in the absence of Kir6.2. Login to comment 250 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro The degradation rate of A116P-fSUR1 in glibenclamide-treated cells (filled squares) is apparently slower than that in control untreated cells (open squares), but it is still faster than that of WT-fSUR1 in control cells (open circles). Login to comment 252 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro fWT, WT-fSUR1; fA116P, A116P-fSUR1. Login to comment 253 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ported by our metabolic pulse-chase labeling experiments, which showed that glibenclamide slowed the degradation rate of the A116P mutant SUR1 and, in the presence of Kir6.2, promoted maturation of the mutant protein. Login to comment 254 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro A, cells transfected with Kir6.2 and A116P- or V187D-fSUR1 were treated with 300 òe;M tolbutamide for 24 h, and surface expression of channels was measured by chemiluminescence assays. Login to comment 255 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro This conclusion differs somewhat from that reached by Chan et al. (29), who proposed that the A116P and V187D mutations cause PHHI by preventing association between SUR1 and FIG. 7. Login to comment 256 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Tolbutamide rescues functional A116P and V187D mutant channels to the cell surface. Login to comment 257 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro A, cells transfected with Kir6.2 and A116P- or V187D-fSUR1 were treated with 300 ␮M tolbutamide for 24 h, and surface expression of channels was measured by chemiluminescence assays. Login to comment 258 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro At 300 ␮M, tolbutamide was nearly as effective as 5 ␮M glibenclamide and restored surface expression of A116P- and V187D-fSUR1 channels from 6.4 Ϯ 1.2 to 49.9 Ϯ 7.6% and from 19.1 Ϯ 4.8 to 48.9 Ϯ 6.0% of normal levels, respectively. Login to comment 264 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro C, representative KATP current traces recorded from inside-out membrane patches containing WT-fSUR1, A116P-fSUR1, or V187D-fSUR1 channels 2 h after tolbutamide removal. Login to comment 267 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro C, representative KATP current traces recorded from inside-out membrane patches containing WT-fSUR1, A116P-fSUR1, or V187D-fSUR1 channels 2 h after tolbutamide removal. Login to comment 269 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1. Login to comment 272 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1. Login to comment 279 ABCC8 p.Ala116Pro It is expected that mutation of alanine 116 to a proline and valine 187 to a charged aspartate would be disruptive to the transmembrane ॷ-helix structure and affect the normal protein folding process. Login to comment 281 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro However, the first transmembrane domain (TM0) where the A116P and V187D mutations are located has not been implicated in glibenclamide binding. Login to comment 282 ABCC8 p.Ala116Pro It is expected that mutation of alanine 116 to a proline and valine 187 to a charged aspartate would be disruptive to the transmembrane ␣-helix structure and affect the normal protein folding process. Login to comment 284 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro However, the first transmembrane domain (TM0) where the A116P and V187D mutations are located has not been implicated in glibenclamide binding. Login to comment 289 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro By contrast, diazoxide, which too binds SUR1 but is structurally quite different from sulfonylureas and results in channel stimulation, does not correct the trafficking defect of either A116P- or V187D-fSUR1. Login to comment 290 ABCC8 p.Val1550Asp ABCC8 p.Ala1457Thr ABCC8 p.Leu1551Val Comparison with Other Trafficking Mutants-A number of missense or point deletion mutations in SUR1 have been reported to reduce or prevent cell surface expression of KATP channels, including èc;F1388, R1394H, L1544P, A1457T, V1550D, and L1551V (34-36, 50). Login to comment 292 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro By contrast, diazoxide, which too binds SUR1 but is structurally quite different from sulfonylureas and results in channel stimulation, does not correct the trafficking defect of either A116P- or V187D-fSUR1. Login to comment 293 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Val1550Asp ABCC8 p.Ala1457Thr ABCC8 p.Leu1551Val Comparison with Other Trafficking Mutants-A number of missense or point deletion mutations in SUR1 have been reported to reduce or prevent cell surface expression of KATP channels, including ⌬F1388, R1394H, L1544P, A1457T, V1550D, and L1551V (34-36, 50). Login to comment 296 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Although like A116P and V187D they all result in a lack of surface channel expression phenotype, the mechanisms leading to this phenotype differ, as revealed by their responses to the different rescuing strategies. Login to comment 297 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Although sulfonylureas rescue the surface expression of mutant channels bearing the A116P or the V187D mutation, they do not rescue surface expression of either èc;F1388 or L1544P mutant channels (36). Login to comment 300 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Although sulfonylureas rescue the surface expression of mutant channels bearing the A116P or the V187D mutation, they do not rescue surface expression of either ⌬F1388 or L1544P mutant channels (36). Login to comment 304 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Although genetic and clinical data on the A116P mutation have not been published, the V187D mutation has been shown to account for the majority of PHHI cases in Finland (37). Login to comment 306 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The results presented in this study show that sulfonylureas have rapid, potent, and long lasting (for at least 12 h after drug removal) effects on rescuing the A116P and V187D mutant channels to the cell surface. Login to comment 307 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Although genetic and clinical data on the A116P mutation have not been published, the V187D mutation has been shown to account for the majority of PHHI cases in Finland (37). Login to comment 309 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The results presented in this study show that sulfonylureas have rapid, potent, and long lasting (for at least 12 h after drug removal) effects on rescuing the A116P and V187D mutant channels to the cell surface. Login to comment 310 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Most importantly, both A116P- and V187D-SUR1 mutant channels rescued to the cell surface by tolbutamide are fully functional upon drug removal and respond to MgADP and diazoxide stimulation like WT channels. Thus, mutant channels rescued to the surface will be able to respond to metabolic signals and to diazoxide treatment. Login to comment