ABCMdb

PMID: 16956886

Yan FF, Casey J, Shyng SL
Sulfonylureas correct trafficking defects of disease-causing ATP-sensitive potassium channels by binding to the channel complex.
J Biol Chem. 2006 Nov 3;281(44):33403-13. Epub 2006 Sep 6., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Previously, we reported that sulfonylureas, oral hypoglycemic drugs widely used to treat type II diabetes, correct the endoplasmic reticulum to the plasma membrane trafficking defect caused by two SUR1 mutations, A116P and V187D. Login to comment 5 ABCC8 p.Tyr230Ala Attenuating or abolishing the ability of mutant SUR1 to bind sulfonylureas or glinides by the following mutations: Y230A, S1238Y, or both, accordingly diminished the rescuing effects of the drugs. Login to comment 38 ABCC8 p.Tyr230Ala Deletion of this cytoplasmic loop leads to loss of [3 H]glibenclamide binding in recombinant SUR1 expressed in insect cells (21) and mutation of tyrosine 230 in L0 to alanine (Y230A) abolishes photoaffinity labeling of SUR1 by [125 I]azidoglibenclamide (14). Login to comment 40 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro We have previously reported that sulfonylureas rescue surface expression defects of KATP channels caused by two CHI-associated SUR1 mutations, A116P and V187D (23). Login to comment 42 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro In this work, we investigated the mechanism by which sulfonylureas correct the channel surface expression defects caused by the A116P or V187D mutations and by the PNDM-associated Kir6.2 mutations. Login to comment 44 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Interestingly and somewhat unexpectedly, we found that Kir6.2 is required for sulfonylureas to rescue the A116P and V187D mutant SUR1 at the cell surface. Login to comment 58 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Tyr230Ala A, topology model of SUR1 showing the three transmembrane domains, the location of the A116P and V187D trafficking mutations, the location of the -RKR-ER retention motif, and the location of the S1238Y and Y230A mutations that have been proposed to disrupt the A- and B- sulfonyl- urea binding sites (as shown in B), respectively. Login to comment 59 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Tyr230Ala A, topology model of SUR1 showing the three transmembrane domains, the location of the A116P and V187D trafficking mutations, the location of the -RKR-ER retention motif, and the location of the S1238Y and Y230A mutations that have been proposed to disrupt the Aand B- sulfonyl- urea binding sites (as shown in B), respectively. Login to comment 88 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Of these, two mutations, A116P and V187D, were rescued by the pharmacological agent sulfonylurea (23). Login to comment 89 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Of these, two mutations, A116P and V187D, were rescued by the pharmacological agent sulfonylurea (23). Login to comment 90 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Because both A116P and V187D are located in TMD0, we first tested if sulfonylureas bind directly to TMD0 to facilitate mutant protein biogenesis and trafficking, even though TMD0 has not been implicated in sulfonylurea binding in prior studies (21). Login to comment 91 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Because both A116P and V187D are located in TMD0, we first tested if sulfonylureas bind directly to TMD0 to facilitate mutant protein biogenesis and trafficking, even though TMD0 has not been implicated in sulfonylurea binding in prior studies (21). Login to comment 92 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro We compared surface expression of recombinant SUR1-TMD0 (amino acids 1-197) containing either the A116P or the V187D mutation in the presence or absence of 5 ␮M glibenclamide. Login to comment 94 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro By con- FIGURE2.ThefirsttransmembranedomainofSUR1(TMD0)doesnotconfer the sulfonylurea rescue effect on the A116P or V187D mutations. Login to comment 95 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro By con- FIGURE2.ThefirsttransmembranedomainofSUR1(TMD0)doesnotconfer the sulfonylurea rescue effect on the A116P or V187D mutations. Login to comment 96 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro A, schematic showing the fSUR1-TMD0 (amino acids 1-197) and the Kir6.2⌬C25 constructs used in experiments shown in B. B, surface expression of fSUR1-TMD0 harboring mutations A116P or V187D in cells treated with or without glibenclamide (5 ␮M for 24 h). Login to comment 99 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Each bar represents the mean Ϯ S.E. of three to four independent experiments. Sulfonylureas and KATP Channel Trafficking trast, when the A116P or V187D mutations were introduced into fSUR1-TMD0, surface expression was greatly reduced (by Ͼ70%), even though the total mutant recombinant proteins were abundantly expressed as assessed by Western blots and immunofluorescent staining of permeabilized cells (not shown). Login to comment 102 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Restoration of A116P- and V187D-mutant Channel Expression by Sulfonylureas Is Dependent on Intact Sulfonylurea Binding Sites in SUR1-Several studies indicate that the high affinity tolbutamide binding site in SUR1 resides in transmembrane segments 13-16 (19, 20). Login to comment 105 ABCC8 p.Ser1237Tyr ABCC8 p.Val187Asp ABCC8 p.Ala116Pro If sulfonylureas rescue the A116P and V187D trafficking mutants by binding to the channel protein, then introducing the S1237Y mutation should also reduce or abolish the ability of sulfonylureas to correct the trafficking defect. Login to comment 106 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro We made the equivalent sulfonylurea binding site mutation (S1238Y) in hamster SUR1 (16) and examined how it affects the response of the A116P- or V187D-mutant channels to sulfonylureas. Login to comment 107 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Initial assessment by immunofluorescent staining indicates that the S1238Y mutation by itself does not affect fSUR1 surface expression when coexpressed with Kir6.2; however, when combined with the A116P or V187D mutation, it indeed reduced or prevented the ability of glibenclamide to rescue the surface expression defect caused by the A116P and V187D mutations (Fig. 3). Login to comment 109 ABCC8 p.Ala116Pro Consistent results were obtained using Western blot analysis. A representative blot of A116P fSUR1 in the WT or S1238Y background from cells treated with or without 5 ␮M glibenclamide for 24 h is shown in Fig. 4A. Login to comment 115 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro However, when combined with the A116P- or V187D-SUR1 trafficking mutations, S1238Y completely abolished the rescue effect of tolbutamide at 300 (Fig. 4B) and 600 òe;M (not shown). Login to comment 116 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro However, when combined with the A116P- or V187D-SUR1 trafficking mutations, S1238Y completely abolished the rescue effect of tolbutamide at 300 (Fig. 4B) and 600 ␮M (not shown). Login to comment 117 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro In the WT background (no sulfonylurea binding mutation), 24-h treatment with 1 òe;M glibenclamide increased surface expression of the A116P mutant from 3.1 afe; 0.7 to 34.5 afe; 7.2% and the V187D mutant from 12.8 afe; 1.6 to 49.2 afe; 6.8% of WT channels. Login to comment 118 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro In the WT background (no sulfonylurea binding mutation), 24-h treatment with 1 ␮M glibenclamide increased surface expression of the A116P mutant from 3.1 Ϯ 0.7 to 34.5 Ϯ 7.2% and the V187D mutant from 12.8 Ϯ 1.6 to 49.2 Ϯ 6.8% of WT channels. Login to comment 119 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro But in the S1238Y background, the same treatment only slightly increased surface expression of the A116P and V187D mutants, from 0.7 Ϯ 1.0 to 6.6 Ϯ 2.1% and 9.8 Ϯ 1.4 to 15.6 Ϯ 2.3% of WT, respectively. Login to comment 120 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Increasing the concentration of glibenclamide to 5 ␮M led to a much greater effect on surface expression of the mutants (to 21.8 and 23.9% of WT for A116P and V187D, respectively; Fig. 4C). Login to comment 124 ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala Bryan et al. (14) have shown that mutation of Tyr230 located in the intracellular loop between TMD0 and TMD1 to alanine (Y230A) results in loss of [125 I]azidoglibenclamide photoaffinity labeling, suggesting that the benzamido group lies in close proximity to Tyr230 during binding, although it is possible that Y230A abolishes binding indirectly by affecting a distant site. Login to comment 125 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala Bryan et al. (14) have shown that mutation of Tyr230 located in the intracellular loop between TMD0 and TMD1 to alanine (Y230A) results in loss of [125 I]azidoglibenclamide photoaffinity labeling, suggesting that the benzamido group lies in close proximity to Tyr230 during binding, although it is possible that Y230A abolishes binding indirectly by affecting a distant site. Login to comment 126 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Tyr230Ala We therefore tested whether this mutation also interferes with the ability of sulfonylureas to rescue the A116P and V187D trafficking mutants. Login to comment 127 ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala Indeed, the Y230A mutation reduced the rescue effect of glibenclamide, as assessed by immunostaining (Fig. 3) as well as chemiluminescence assays (Fig. 5A). Login to comment 128 ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala We then asked whether this reduction is attributable to loss of binding of FIGURE 3. Impact of the sulfonylurea binding mutations on the effectiveness of glibenclamide to rescue KATP channel trafficking defects in the presence of Kir6.2, using immunocytochemistry analysis. A, top panel, surface staining of COSm6 cells transiently transfected with Kir6.2 and WT-, Y230A-, S1238Y-, or Y230A/S1238Y-fSUR1, using the M2 anti-FLAG mouse monoclonal antibodies followed by Cy3-conjugated anti-mouse secondary antibody. Login to comment 132 ABCC8 p.Ala116Pro B, addition of the A116P mutation in WT or the various sulfonylurea binding mutation backgrounds abolished surface staining (top panel), although the mutant proteins were detected inside the cell (middle panel). Login to comment 133 ABCC8 p.Ala116Pro B, addition of the A116P mutation in WT or the various sulfonylurea binding mutation backgrounds abolished surface staining (top panel), although the mutant proteins were detected inside the cell (middle panel). Login to comment 134 ABCC8 p.Ala116Pro Treatment of cells with 5 òe;M glibenclamide significantly increased surface expression of the A116P mutant as reported previously. Login to comment 135 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala Treatment of cells with 5 ␮M glibenclamide significantly increased surface expression of the A116P mutant as reported previously. Login to comment 136 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala The Y230A or S1238Y mutations reduced the ability of glibenclamide to restore surface expression of the A116P mutant and simultaneous mutation of Y230A and S1238Y completely abolished the ability of glibenclamide to rescue A116P to the cell surface. Login to comment 137 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro C, same as B except that the V187D trafficking mutation was introduced into WT or sulfonylurea binding mutation fSUR1 background. Login to comment 138 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Note as previously reported, the V187D mutation showed slightly higher surface expression compared with the A116P mutation, with faint surface staining barely visible (23). Login to comment 139 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Tyr230Ala Accordingly, we examined how repaglinide, a glinide that contains the benzamido moiety but not the sulfonylurea moiety, affects surface expression of A116P and V187D channels in the presence or absence of the Y230A mutation. Login to comment 140 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Tyr230Ala Accordingly, we examined how repaglinide, a glinide that contains the benzamido moiety but not the sulfonylurea moiety, affects surface expression of A116P and V187D channels in the presence or absence of the Y230A mutation. Login to comment 141 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Tyr230Ala Fig. 5B shows that repaglinide at 10 ␮M effectively rescued surface expression of the A116P and V187D mutants, whereas the Tyr230 mutation abolished this rescue effect; in contrast, the S1238Y mutation had little effect on the ability of repaglinide to rescue the trafficking mutants (not shown). Login to comment 142 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala Surprisingly, we found that the Y230A mutation also renders tolbutamide unable to rescue the A116P and V187D trafficking mutants (Fig. 5C), suggesting a role of Tyr230 in tolbutamide binding or in conferring tolbutamide sensitivity toward trafficking rescue through an allosteric effect. Login to comment 143 ABCC8 p.Tyr230Ala Finally, introducing the Y230A and S1238Y mutations simultaneously completely abolished the rescue effect by tolbutamide, repaglinide, or glibenclamide (Figs. Login to comment 148 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro For the A116P-fSUR1, however, only the immature band was detected in untreated cells (A116P); upon treatment with 1 òe;M glibenclamide for 24 h (A116Paf9;Glib),theupperA116P-fSUR1bandbecameapparent,indicatingres- cue of the mutant protein out of the ER. Login to comment 149 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro For the A116P-fSUR1, however, only the immature band was detected in untreated cells (A116P); upon treatment with 1 ␮M glibenclamide for 24 h (A116PϩGlib),theupperA116P-fSUR1bandbecameapparent,indicatingres- cue of the mutant protein out of the ER. Login to comment 150 ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro When the S1238Y mutation was introduced into A116P-fSUR1 (A116P/S1238Y), the same glibenclamide treatment was less effective in promoting expression of the upper band (A116P/ S1238YϩGlib). Login to comment 152 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Surface expression of tolbutamide-treated cells was significantly higher than untreated for A116P and V187D (p b0d; 0.001) but not A116P/S1238Y and V187D/S1238Y. Login to comment 153 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Surface expression of tolbutamide-treated cells was significantly higher than untreated for A116P and V187D (p Ͻ 0.001) but not A116P/S1238Y and V187D/S1238Y. Login to comment 154 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Without the S1238Y mutation, both 1 and 5 òe;M glibenclamide significantly increased surface expression of the A116P and V187D trafficking mutants (p b0d; 0.001). Login to comment 155 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Without the S1238Y mutation, both 1 and 5 ␮M glibenclamide significantly increased surface expression of the A116P and V187D trafficking mutants (p Ͻ 0.001). Login to comment 156 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro However, for the A116P/ S1238Y and V187D/S1238Y mutants, 1 ␮M glibenclamide did not lead to a statistically significant increase in surface expression, whereas 5 ␮M glibenclamide did (p Ͻ 0.01). Login to comment 157 ABCC8 p.Tyr230Ala FIGURE 5. Impact of the Y230A mutation on the effectiveness of sulfonylureas and glinides to rescue KATP channel trafficking defects in the presence of Kir6.2. Login to comment 158 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala FIGURE 5. Impact of the Y230A mutation on the effectiveness of sulfonylureas and glinides to rescue KATP channel trafficking defects in the presence of Kir6.2. Login to comment 159 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Tyr230Ala The A116P or V187D trafficking mutation was introduced onto the WTor Y230A-fSUR1 background. Login to comment 162 ABCC8 p.Tyr230Ala On the Y230A mutation background, however, the glibenclamide rescue effect was attenuated although still significant (p b0d; 0.01), whereas the effects of repaglinide and tolbutamide were both abolished. Login to comment 163 ABCC8 p.Tyr230Ala On the Y230A mutation background, however, the glibenclamide rescue effect was attenuated although still significant (p Ͻ 0.01), whereas the effects of repaglinide and tolbutamide were both abolished. Login to comment 164 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Each bar is the mean afe; S.E. of three to five independent experiments. Sulfonylureas and KATP Channel Trafficking 33408 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281ߦNUMBER 44ߦNOVEMBER 3, 2006 the trafficking defects of the A116P and V187D mutants via direct interactions with the mutant channel proteins. Login to comment 165 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala Each bar is the mean Ϯ S.E. of three to five independent experiments. Sulfonylureas and KATP Channel Trafficking 33408 the trafficking defects of the A116P and V187D mutants via direct interactions with the mutant channel proteins. Login to comment 166 ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala Comparison of the Effects of Sulfonylurea Binding Site Mutations on Trafficking Rescue Versus Channel Activity Block by Sulfonylureas-Given the somewhat unexpected result that Y230A not only attenuated and abolished channel trafficking rescue by glibenclamide and repaglinide, respectively, but also abrogated the rescue effect of tolbutamide, we sought to further characterize the role of Tyr230 in channel response to sulfonylureas by examining how Y230A affects the ability of glibenclamide and tolbutamide to inhibit KATP channel activity, the best characterized effect of these drugs. Login to comment 167 ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala Inside-out patch clamp recording was used to monitor tolbutamide or glibenclamide block of channel activity in cells expressing Y230A, S1238Y, or Y230A/Y1238Y mutant channels. Login to comment 168 ABCC8 p.Tyr230Ala In the Y230A mutant, we observed reduced tolbutamide block at 10 and 300 òe;M, indicating a role of Tyr230 in mediating tolbutamide response both in the context of channel trafficking rescue and channel activity block. Login to comment 169 ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala In the Y230A mutant, we observed reduced tolbutamide block at 10 and 300 ␮M, indicating a role of Tyr230 in mediating tolbutamide response both in the context of channel trafficking rescue and channel activity block. Login to comment 170 ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala As expected, Y230A also reduced glibenclamide block at 10 nM and 1 ␮M. Login to comment 171 ABCC8 p.Tyr230Ala However, Y230A did not render glibenclamide inhibition reversible; this is in contrast to the S1238Y mutation that caused reduced block by 10 nM glibenclamide and rendered the block reversible upon washout (Fig. 7A) as reported previously by others (20). Login to comment 172 ABCC8 p.Tyr230Ala Finally, combining Y230A and S1238Y had a greater effect on preventing tolbutamide and glibenclamide block than either mutation alone (Fig. 7). Login to comment 173 ABCC8 p.Tyr230Ala Finally, combining Y230A and S1238Y had a greater effect on preventing tolbutamide and glibenclamide block than either mutation alone (Fig. 7). Login to comment 174 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The Role of Kir6.2 in Sulfonylurea Rescue of Channel Trafficking Defects-The data we presented so far demonstrate that intact sulfonylurea binding sites in SUR1 are necessary for effective rescue of the A116P- or V187D-SUR1 trafficking mutants by sulfonylureas. Login to comment 175 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The Role of Kir6.2 in Sulfonylurea Rescue of Channel Trafficking Defects-The data we presented so far demonstrate that intact sulfonylurea binding sites in SUR1 are necessary for effective rescue of the A116P- or V187D-SUR1 trafficking mutants by sulfonylureas. Login to comment 176 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro To investigate the role of Kir6.2 in sulfonylurea rescue of the A116P- or V187D-SUR1 trafficking mutants, we took advantage of the fact that inactivation of the -RKR-ER retention/retrieval motif by mutation to AAA (referred to as WTAAA in Fig. 8A) in SUR1 allows SUR1 to traffic to the cell surface without co-expression of Kir6.2 (29). Login to comment 177 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro To investigate the role of Kir6.2 in sulfonylurea rescue of the A116P- or V187D-SUR1 trafficking mutants, we took advantage of the fact that inactivation of the -RKR-ER retention/retrieval motif by mutation to AAA (referred to as WTAAA in Fig. 8A) in SUR1 allows SUR1 to traffic to the cell surface without co-expression of Kir6.2 (29). Login to comment 178 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro If sulfonylurea binding to SUR1 is sufficient to correct the defect caused by the A116P or V187D mutations, we expect sulfonylureas to improve the surface expression of A116P- or V187D-SUR1 in which the -RKR- motif has been mutated to -AAA- (referred to as A116PAAA and V187DAAA) in the absence of Kir6.2. Login to comment 179 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro These results are consistent with the A116P and V187D mutations causing defects in the SUR1 protein itself. Login to comment 180 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro These results are consistent with the A116P and V187D mutations causing defects in the SUR1 protein itself. Login to comment 184 ABCC8 p.Tyr230Ala Effect of the Y230A/S1238Y double mutation on sulfonylurea and glinide rescue of KATP channel trafficking mutants in the presence of Kir6.2. Login to comment 185 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala Effect of the Y230A/S1238Y double mutation on sulfonylurea and glinide rescue of KATP channel trafficking mutants in the presence of Kir6.2. Login to comment 186 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala The experiments were as described in the legend to Fig. 5 except that A116P and V187D were each introduced onto the WTor Y230A/S1238Y-fSUR1 backgrounds. Login to comment 187 ABCC8 p.Tyr230Ala The Y230A/S1238Y double mutation completely abolished the rescue effects by glibenclamide (A), repaglinide (B), and tolbutamide (C). Login to comment 191 ABCC8 p.Tyr230Ala Effect of the Y230A and S1238Y mutations on channel block by sulfonylureas. Login to comment 192 ABCC8 p.Tyr230Ala Effect of the Y230A and S1238Y mutations on channel block by sulfonylureas. Login to comment 200 ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala The relative current amplitudes of Y230A or S1238Y in 10 or 300 òe;M tolbutamide are not significantly different from WT (p b0e; 0.05), whereas that of Y230A/S1238Y in either 10 or 300 òe;M tolbutamide is significantly different from WT (p b0d; 0.05). Login to comment 201 ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala The relative current amplitudes of Y230A or S1238Y in 10 or 300 ␮M tolbutamide are not significantly different from WT (p Ͼ 0.05), whereas that of Y230A/S1238Y in either 10 or 300 ␮M tolbutamide is significantly different from WT (p Ͻ 0.05). Login to comment 202 ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala For glibenclamide inhibition, the response of Y230A, S1238Y, and Y230A/S1238Y are all significantly different from WT in either 10 nM or 1 ␮M (p Ͻ 0.05). Login to comment 211 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Our results demonstrate that mutations in SUR1 previously reported to abolish or reduce sulfonylurea binding also abolish or reduce the ability of sulfonylureas to rescue channel trafficking defects caused by the A116P or V187D SUR1 mutations and that both the sulfonylurea and benzoamido moieties of glibenclamide contribute to the rescue effect. Login to comment 212 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Our results demonstrate that mutations in SUR1 previously reported to abolish or reduce sulfonylurea binding also abolish or reduce the ability of sulfonylureas to rescue channel trafficking defects caused by the A116P or V187D SUR1 mutations and that both the sulfonylurea and benzoamido moieties of glibenclamide contribute to the rescue effect. Login to comment 217 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro A reasonable hypothesis for how sulfonylureas improve surface expression of the A116P and V187D mutants is that A116P or V187D cause SUR1 misfolding and sulfonylureas, upon binding to the mutant SUR1, help it to adopt the correct conformation. Login to comment 218 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro A reasonable hypothesis for how sulfonylureas improve surface expression of the A116P and V187D mutants is that A116P or V187D cause SUR1 misfolding and sulfonylureas, upon binding to the mutant SUR1, help it to adopt the correct conformation. Login to comment 219 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Our results that the A116P or V187D mutations are sufficient to prevent trafficking of TMD0 as well as SUR1AAA to the cell surface support the idea that the A116P or V187D mutations have an effect on the folding/processing of SUR1 protein itself. Login to comment 226 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The A116P or V187D trafficking mutation was engineered into WT-fSUR1 or fSUR1 in which the -RKR-ER retention motif has been inactivated by mutation to AAA. Login to comment 227 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The A116P or V187D trafficking mutation was engineered into WT-fSUR1 or fSUR1 in which the -RKR-ER retention motif has been inactivated by mutation to AAA. Login to comment 236 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro It is also worth pointing out that TMD0 harboring the A116P and V187D mutations have been shown to not coimmunoprecipitate with Kir6.2 (27), suggesting weakened association between mutant SUR1 and Kir6.2. Login to comment 238 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro It is also worth pointing out that TMD0 harboring the A116P and V187D mutations have been shown to not coimmunoprecipitate with Kir6.2 (27), suggesting weakened association between mutant SUR1 and Kir6.2. Login to comment 243 ABCC8 p.Tyr230Ala Consistent with this picture, we found that mutation S1238Y in the proposed A site abolished the effect of tolbutamide and attenuated the effect of glibenclamide on the trafficking mutants, whereas mutation Y230A in the proposed B site rendered repaglinide ineffective and glibenclamide less effective in rescuing the trafficking mutants. Login to comment 244 ABCC8 p.Tyr230Ala To our surprise, mutation Y230A in the proposed B site also prevented tolbutamide from rescuing the trafficking mutants; whereas this may suggest a role of Tyr230 in tolbutamide binding, an alternative possibility is that the residue is involved in post-binding events that are important for the rescue effect of the drug. Login to comment 245 ABCC8 p.Tyr230Ala Consistent with this picture, we found that mutation S1238Y in the proposed A site abolished the effect of tolbutamide and attenuated the effect of glibenclamide on the trafficking mutants, whereas mutation Y230A in the proposed B site rendered repaglinide ineffective and glibenclamide less effective in rescuing the trafficking mutants. Login to comment 246 ABCC8 p.Tyr230Ala ABCC8 p.Tyr230Ala To our surprise, mutation Y230A in the proposed B site also prevented tolbutamide from rescuing the trafficking mutants; whereas this may suggest a role of Tyr230 in tolbutamide binding, an alternative possibility is that the residue is involved in post-binding events that are important for the rescue effect of the drug. Login to comment 247 ABCC8 p.Tyr230Ala Interestingly, we observed parallel effects of S1238Y and/or Y230A on the ability of sulfonylureas to rescue trafficking mutants and block channel activity, suggesting the two processes likely share similar transduction mechanisms. Login to comment 248 ABCC8 p.Tyr230Ala The Y230A mutation might abolish the tolbutamide rescue effect on the trafficking mutants by disrupting the cross-talk between SUR1 and Kir6.2, which we have shown to be necessary for the sulfonylurea rescue effects. Login to comment 249 ABCC8 p.Tyr230Ala Interestingly, we observed parallel effects of S1238Y and/or Y230A on the ability of sulfonylureas to rescue trafficking mutants and block channel activity, suggesting the two processes likely share similar transduction mechanisms. Login to comment 252 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Although we used the A116P and V187D-SUR1 mutations as two examples for probing the mechanism by which sulfonylureas rescue channel trafficking defect, we have found many more CHI-causing KATP mutants with trafficking defects to respond to sulfonylurea rescue.3 Our findings are therefore applicable to a growing number of naturally occurring channel mutations whose trafficking defects could be targeted for therapy. Login to comment 254 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Although we used the A116P and V187D-SUR1 mutations as two examples for probing the mechanism by which sulfonylureas rescue channel trafficking defect, we have found many more CHI-causing KATP mutants with trafficking defects to respond to sulfonylurea rescue.3 Our findings are therefore applicable to a growing number of naturally occurring channel mutations whose trafficking defects could be targeted for therapy. Login to comment