ABCMdb

PMID: 19151370

Pratt EB, Yan FF, Gay JW, Stanley CA, Shyng SL
Sulfonylurea receptor 1 mutations that cause opposite insulin secretion defects with chemical chaperone exposure.
J Biol Chem. 2009 Mar 20;284(12):7951-9. Epub 2009 Jan 16., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Here we report that two hyperinsulinism-associated SUR1 missense mutations, R74W and E128K, surprisingly reduce channel inhibition by intracellular ATP, a gating defect expected to yield the opposite disease phenotype neonatal diabetes. Login to comment 5 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp The R74W and E128K mutants thus rescued to the cell surface paradoxically exhibited ATP sensitivity 6and 12-fold lower than wild-type channels, respectively. Login to comment 42 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Here, we report two mutants, R74W and E128K, which, upon rescue to the cell surface, surprisingly revealed reduced ATP sensitivity-gating defects. Login to comment 45 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Interestingly, unlike previously reported ATP-insensitive mutants, which tend to have increased intrinsic Po, the R74W and E128K mutants showed reduced intrinsic Po. Login to comment 46 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp The finding suggests R74W and E128K diminish channel ATP sensitivity by a distinct mechanism that likely involves functional uncoupling between SUR1 and Kir6.2. Login to comment 106 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp RESULTS The R74W and E128K Mutations Reduce Channel Sensitivity to ATP-Previously, we reported several CHI-associated SUR1 mutations that reduce surface expression of KATP channels could be rescued to the cell surface efficiently by sulfonylureas such as glibenclamide and tolbutamide (13, 15). Login to comment 107 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Asn24Lys ABCC8 p.Phe27Ser These mutations are all in the TMD0 of SUR1 (amino acids 1-196) and include G7R, N24K, F27S, R74W, A116P, E128K, and V187D. Login to comment 108 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The functional properties of rescued A116P and V187D mutant channels had been characterized in detail and shown to be normal (13). Login to comment 112 ABCC8 p.Asn24Lys ABCC8 p.Phe27Ser Of the five mutants, G7R, N24K, and F27S had WT-like or slightly increased ATP sensitivity, and either normal or reduced MgADP response that is commonly associated with CHI mutations (Fig. 1) (23). Login to comment 113 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp To our surprise, however, two of the mutants, R74W and E128K, exhibited significantly reduced ATP sensitivities (Fig. 2, A and B). Login to comment 114 ABCC8 p.Arg74Trp The R74W mutation has been reported in two focal cases (disease causing a paternally derived mutation that was expressed due to loss of heterozygosity for the maternal allele) (24). Login to comment 116 ABCC8 p.Arg74Trp R74W was also reported as a compound heterozygous mutation with another mutation, R1215Q, in a patient with diffuse HI who failed medical therapy with diazoxide and required a near-total pancreatectomy (15, 25). Login to comment 117 ABCC8 p.Glu128Lys The E128K mutation occurred in homozygous form in a child with diffuse hyperinsulinism, severe neonatal onset hypoglycemia, failed diazoxide therapy, and required near-total pancreatectomy for control of hypoglycemia (15). Login to comment 120 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Although expression levels of R74W and E128K mutants were very low, sufficient currents in a small fraction of transfected cells (identified by co-transfected GFP) were detected. Login to comment 122 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Arg74Trp SUR1 Mutations, Hyperinsulinism, and Diabetes MARCH 20, 2009•VOLUME 284•NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 7953 Mechanisms of Reduced ATP Inhibition in R74W and E128K Channels-Several studies have shown that PNDM-causing SUR1 or Kir6.2 mutations can reduce channel ATP sensitivity by enhancing channel response to Mg-nucleotide stimulation (26, 27). Login to comment 123 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp In inside-out patches, MgADP stimulated channel activity in both R74W and E128K mutants (Fig. 3A); however, because the mutants were much less sensitive to nucleotide inhibition, it is difficult to directly compare their MgADP sensitivities to WT channels. Login to comment 124 ABCC8 p.Lys719Met ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp We therefore tested the effect of R74W or E128K on channel ATP sensitivity in the background of SUR1-NBD mutations such as G1479D and G1479R in NBD2 and K719M in NBD1, which are known to abolish channel response to MgADP stimulation (28). Login to comment 125 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Arg74Trp In the E128K/G1479R double mutants, ATP sensitivity was as reduced as the E128K mutant; the R74W/G1479D double mutant also showed significantly reduced ATP sensitivity, although to a lesser degree than the R74W single mutant (Fig. 3, B and C). Login to comment 126 ABCC8 p.Lys719Met ABCC8 p.Lys719Met ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys We also combined E128K with NBD1 mutation K719M (E128K/K719M), and the resulting channels were as insensitive to ATP as E128K (not shown). Login to comment 127 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp These results indicate that, even if the mutations increased Mg-nucleotide stimulation, this effect alone could not explain the reduced ATP sensitivity in R74W and E128K. Login to comment 129 ABCC8 p.Phe132Leu The recently identified PNDM-causing SUR1 F132L mutation is an example, so are many Kir6.2 mutations reported earlier (8, 29). Login to comment 130 ABCC8 p.Glu128Lys To examinethispossibility,wefirstmeasuredthePo oftheR74Wand E128K channels expressed in COSm6 cells by single channel recording. Login to comment 132 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Unexpectedly, however, the average Po values for R74W and E128K (0.35 Ϯ 0.08 and 0.18 Ϯ 0.06, respectively) were significantly lower than that of WT channels (0.63 Ϯ 0.06; Fig. 4B). Login to comment 133 ABCC8 p.Glu128Lys Of note, 10 out of 11 patches of the E128K mutant had consistently lower Po with only one outlier showing a Po of 0.69 (Fig. 4B). Login to comment 134 ABCC8 p.Arg74Trp ABCC8 p.Asn24Lys The R74W mutant exhibited more variable Po FIGURE1.NucleotidesensitivitiesofTMD0mutantsG7R,N24K,andF27S. Login to comment 141 ABCC8 p.Asn24Lys ABCC8 p.Phe27Ser Scale bars: WT: 200 pA, 10 s; G7R: 200 pA, 10 s; N24K: 20 pA, 10 s; F27S: 50 pA, 10 s. B, quantification of channel response to ATP and MgADP. Login to comment 143 ABCC8 p.Asn24Lys ABCC8 p.Asn24Lys ABCC8 p.Phe27Ser The ATP sensitivity of N24K is significantly higher than WT while the MgADP sensitivity of both N24K and F27S are significantly lower than WT channels (*, p Ͻ 0.05; Student`s t test). Login to comment 146 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp R74W and E128K decrease channel sensitivity to ATP inhibition. Login to comment 148 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Scale bars: WT: 500 pA, 5 s; R74W and E128K: 50 pA, 5 s. B, ATP dose-response relationships. Parameters describing best-fit curves to the Hill equation (Irel ϭ 1/(1 ϩ ([ATP]/ IC50)H )), including the [ATP] necessary for half-maximal inhibition (IC50) and Hill coefficient (H), are shown. Login to comment 152 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Scale bars: WT: 500 pA, 10 s; R74W and E128K: 20 pA, 10 s. SUR1 Mutations, Hyperinsulinism, and Diabetes 7954 ranging from 0.01 to 0.88 (Fig. 4B). Login to comment 153 ABCC8 p.Arg74Trp Two recordings representing both ends of the R74W Po spectrum are shown in Fig. 4A. Login to comment 154 ABCC8 p.Phe132Leu As a control, we also analyzed the Po of the F132L mutant. Login to comment 155 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu As a control, we also analyzed the Po of the F132L mutant. Login to comment 156 ABCC8 p.Phe132Leu Consistent with that reported previously (30), the Po of F132L (0.71 Ϯ 0.05) tends to be higher than that of WT (Fig. 4), although the difference did not reach statistical significance. Login to comment 157 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp The Po values thus derived are 0.84 afe; 0.02 (n afd; 10), 0.71 afe; 0.06 (n afd; 9), and 0.35 afe; 0.05 (n afd; 9) for WT, R74W, and E128K, respectively. Login to comment 158 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Arg74Trp The Po values thus derived are 0.84 Ϯ 0.02 (n ϭ 10), 0.71 Ϯ 0.06 (n ϭ 9), and 0.35 Ϯ 0.05 (n ϭ 9) for WT, R74W, and E128K, respectively. Login to comment 159 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp These numbers, while all consid- erablyhigher,areneverthelessingeneralagreementwiththetrend observed in single channel recording experiments, with the Po of R74W and E128K significantly lower than that of WT (p Ͻ 0.05). Login to comment 160 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Together, our data point to reduced intrinsic channel open probabilities for the R74W and the E128K mutant. Login to comment 161 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Together, our data point to reduced intrinsic channel open probabilities for the R74W and the E128K mutant. Login to comment 163 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Mutant Channel Biogenesis Defects and Correction by Sulfonylureas in Insulin-secreting Cells-The reduced ATP sensitivity of the R74W and E128K mutant channels predicts that, were the channels able to overcome their trafficking defect, they would be insensitive to metabolic stimuli and would cause beta-cell dysfunction resembling neonatal diabetes. Login to comment 164 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Mutant Channel Biogenesis Defects and Correction by Sulfonylureas in Insulin-secreting Cells-The reduced ATP sensitivity of the R74W and E128K mutant channels predicts that, were the channels able to overcome their trafficking defect, they would be insensitive to metabolic stimuli and would cause beta-cell dysfunction resembling neonatal diabetes. Login to comment 165 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Reduced ATP sensitivity in R74W and E128K is independent of MgADP stimulation. Login to comment 166 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Reduced ATP sensitivity in R74W and E128K is independent of MgADP stimulation. Login to comment 169 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Arg74Trp Scale bars: WT: 50 pA, 10 s; R74W: 50 pA, 10 s; E128K: 500 pA, 10 s. B and C, ATP sensitivity is still reduced in R74W and E128K containing SUR1-NBD mutations G1479D or G1479R. Login to comment 170 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Arg74Trp Scale bars: WT: 50 pA, 10 s; R74W: 50 pA, 10 s; E128K: 500 pA, 10 s. B and C, ATP sensitivity is still reduced in R74W and E128K containing SUR1-NBD mutations G1479D or G1479R. Login to comment 171 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Scale bars: WT: 300 pA, 5 s; RW/GD (R74W/G1479D): 100 pA, 5 s; EK/GR (E128K/G1479R): 100 pA, 5 s. C, ATP dose-response relationships. Parameters describing best-fit curves are given as in Fig. 2B. All cells were pretreated with 300 òe;M tolbutamide to increase surface expression. Login to comment 172 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Scale bars: WT: 300 pA, 5 s; RW/GD (R74W/G1479D): 100 pA, 5 s; EK/GR (E128K/G1479R): 100 pA, 5 s. C, ATP dose-response relationships. Parameters describing best-fit curves are given as in Fig. 2B. Login to comment 173 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp R74W and E128K reduce channel intrinsic open probability. Login to comment 175 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp R74W and E128K reduce channel intrinsic open probability. Login to comment 183 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Western blots showed that, without glibenclamide treatment, the R74W and E128K mutant fSUR1 was seen only as a lower band corresponding to the core-glycosylated form in the ER, in contrast to WT fSUR1 seen as both a lower band and an upper band corresponding to the mature complex-glycosylated form found post medial-Golgi (35). Login to comment 185 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Western blots showed that, without glibenclamide treatment, the R74W and E128K mutant fSUR1 was seen only as a lower band corresponding to the core-glycosylated form in the ER, in contrast to WT fSUR1 seen as both a lower band and an upper band corresponding to the mature complex-glycosylated form found post medial-Golgi (35). Login to comment 186 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp By contrast, surface R74W- and E128K-fSUR1 was only easily detectable following overnight tolbutamide treatment. Login to comment 188 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Arg74Trp By contrast, surface R74W- and E128K-fSUR1 was only easily detectable following overnight tolbutamide treatment. Login to comment 189 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp The trafficking and rescue characteristics of R74W and E128K were also confirmed in rat islets. Login to comment 190 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Following 24-h treatment with 5 ␮M glibenclamide, the expression level of R74W and E128K mutants increased from 9% to 90% and 9% to 80% that of WT, respectively (Fig. 5C). Login to comment 191 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp The trafficking and rescue characteristics of R74W and E128K were also confirmed in rat islets. Login to comment 193 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Expression of R74W or E128K Mutant Alters INS-1 Cell Responses to Glucose Stimulation-Having established the sulfonylurea-dependent expression of mutant channels in INS-1 cells, we next determined if expression of the mutants at the cell surface alters membrane electrical properties. Login to comment 195 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Expression of R74W or E128K Mutant Alters INS-1 Cell Responses to Glucose Stimulation-Having established the sulfonylurea-dependent expression of mutant channels in INS-1 cells, we next determined if expression of the mutants at the cell surface alters membrane electrical properties. Login to comment 199 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp R74W and E128K have inappropriate channel openings in intact cells following high glucose stimulation. Login to comment 201 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp R74W and E128K have inappropriate channel openings in intact cells following high glucose stimulation. Login to comment 202 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Representative current traces showing that both uninfected control and WT-infected cells had little or no channel activity; however, both R74W and E128K had robust channel openings. Login to comment 204 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Representative current traces showing that both uninfected control and WT-infected cells had little or no channel activity; however, both R74W and E128K had robust channel openings. Login to comment 205 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Scale bars: WT: 50 pA, 10 s; R74W: 100 pA, 10 s; E128K: 50 pA, 10 s. FIGURE 5. Login to comment 206 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp R74W and E128K surface expression was rescued by sulfonylurea treatment in insulin-secreting cells. Login to comment 207 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Scale bars: WT: 50 pA, 10 s; R74W: 100 pA, 10 s; E128K: 50 pA, 10 s. FIGURE 5. Login to comment 208 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp R74W and E128K surface expression was rescued by sulfonylurea treatment in insulin-secreting cells. Login to comment 210 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp The upper band is undetectable in untreated R74W- and E128K-infectedcells,indicatingdefectivechannelprocessingandtrafficking.Sulfonylurea treatment, however, restores upper band expression. Login to comment 212 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Arg74Trp The upper band is undetectable in untreated R74W- and E128K-infectedcells,indicatingdefectivechannelprocessingandtrafficking.Sulfonylurea treatment, however, restores upper band expression. Login to comment 213 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Under control conditions, R74W and E128K both express at 9% of WT. Login to comment 214 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Arg74Trp B, surface immunostaining with FLAG-antibody of fSUR1 showed that R74W and E128K mutant channels are only detected at the cell surface following tolbutamide treatment.C,KATP surfaceexpressioninINS-1cellswasquantifiedusingchemilu- minescence assays. Login to comment 215 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Under control conditions, R74W and E128K both express at 9% of WT. Login to comment 216 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Sulfonylurea treatment greatly improves R74W and E128K expression to90and80%,respectively.ErrorbarsrepresentϮS.E.ofthreeexperiments.D, is- letsisolatedfromratpancreaswereculturedfor48handtheninfectedwithKATP subunit-encoding adenoviruses. Login to comment 217 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp In contrast, all cells infected with the R74W or E128K channel subunits and pretreated with tolbutamide had high on-cell activities (Fig. 6A). Login to comment 219 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Arg74Trp In contrast, all cells infected with the R74W or E128K channel subunits and pretreated with tolbutamide had high on-cell activities (Fig. 6A). Login to comment 221 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Cells that were infected with R74W or E128K but not rescued by tolbutamide were also tested; a small fraction of each mutant displayed some on-cell channel activity but less than that observed in tolbutamide-treated cells and decreased channel ATP sensitivity upon patch excision (supplemental Fig. S1), indicating some mutant channels were able to traffic to the plasma membrane without pharmacologic chaperone. Login to comment 228 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp In contrast, both R74W- and E128K-expressing cells receiving tolbutamide pretreatment were significantly more hyperpolarized at 12 mM glucose. Login to comment 229 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp The initial RMP was afa;33 afe; 3.8 mV for R74W and afa;54 afe; 4.0 mV for E128K. Login to comment 230 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp In contrast, both R74W- and E128K-expressing cells receiving tolbutamide pretreatment were significantly more hyperpolarized at 12 mM glucose. Login to comment 231 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp The initial RMP was -33 Ϯ 3.8 mV for R74W and -54 Ϯ 4.0 mV for E128K. Login to comment 233 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Taken together, these results are in line with the idea that expression of the R74W or E128K mutant channels at the INS-1 cell surface render the cell membrane potentials unable to depolarize in response to glucose stimulation. Login to comment 234 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Lastly, we determined if rescue of the R74W or E128K mutant channels to the cell surface would cause defective insulin secretion in response to glucose stimulation. Login to comment 235 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Taken together, these results are in line with the idea that expression of the R74W or E128K mutant channels at the INS-1 cell surface render the cell membrane potentials unable to depolarize in response to glucose stimulation. Login to comment 236 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Lastly, we determined if rescue of the R74W or E128K mutant channels to the cell surface would cause defective insulin secretion in response to glucose stimulation. Login to comment 244 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp C, insulin secretion at basal (3 mM) and 12 mM glucose in uninfected controls and WT-, R74W-, or E128K-infected INS-1 cells. Login to comment 245 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Arg74Trp C, insulin secretion at basal (3 mM) and 12 mM glucose in uninfected controls and WT-, R74W-, or E128K-infected INS-1 cells. Login to comment 246 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Arg74Trp R74W- and E128K-infected cells pretreated with 300 ␮M tolbutamide for 4 h to rescue surface expression had significantly less insulin secretion relative to control or WT-infected cells. Login to comment 247 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp In R74W- or E128K-infected cells without tolbutamide rescue, insulin secretion was also reduced likely due to some leak expression of the mutants, although the extent of reduction was less than tolbutamide-rescued cells. Login to comment 253 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp These data led us to conclude that rescue of the CHI-causing R74W or E128K mutant KATP channels by sulfonylureas inverses the beta-cell dysfunction phenotype to diabetic. Login to comment 254 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp These data led us to conclude that rescue of the CHI-causing R74W or E128K mutant KATP channels by sulfonylureas inverses the beta-cell dysfunction phenotype to diabetic. Login to comment 258 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Mechanisms of Reduced ATP Sensitivities in the Mutant Channels-Our results indicate that the decreased ATP sensitivities of R74W and E128K are not due to enhanced MgADP stimulation, at least alone, because elimination of channel MgADP response by mutations in the nucleotide binding folds did not restore their ATP sensitivity to the level of WT channels (Fig. 3). Login to comment 259 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Arg74Trp Mechanisms of Reduced ATP Sensitivities in the Mutant Channels-Our results indicate that the decreased ATP sensitivities of R74W and E128K are not due to enhanced MgADP stimulation, at least alone, because elimination of channel MgADP response by mutations in the nucleotide binding folds did not restore their ATP sensitivity to the level of WT channels (Fig. 3). Login to comment 260 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Arg74Trp Furthermore, we found that, rather than increasing channel intrinsic Po, the R74W and E128K mutations significantly lowered the average intrinsic Po (Fig. 4). Login to comment 261 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp These properties are unlike the previously reported ATP-insensitive mutants and place the R74W and E128K mutants in a distinct category in terms of the underlying mechanisms for loss of ATP sensitivity. Login to comment 263 ABCC8 p.Glu128Lys That the E128K mutant has poor surface expression, lower Po, and reduced ATP sensitivity closer to those seen in Kir6.2èc;C channels suggests the mutation likely disrupts the functional coupling between TMD0-SUR1 and Kir6.2. Login to comment 264 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp That the E128K mutant has poor surface expression, lower Po, and reduced ATP sensitivity closer to those seen in Kir6.2⌬C channels suggests the mutation likely disrupts the functional coupling between TMD0-SUR1 and Kir6.2. Login to comment 265 ABCC8 p.Arg74Trp The R74W mutation might also disrupt functional interactions between TMD0-SUR1 and Kir6.2; however, this disruption would be predicted to be less pronounced as the mutation only caused mild reduction in ATP sensitivity and Po. Login to comment 269 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp In fact, the reduced ATP sensitivities observed in the R74W and E128K mutants indicate TMD0 is necessary for normal channel ATP sensitivity. Login to comment 270 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp In fact, the reduced ATP sensitivities observed in the R74W and E128K mutants indicate TMD0 is necessary for normal channel ATP sensitivity. Login to comment 271 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp If R74W and E128K cause functional uncoupling between TMD0-SUR1 and Kir6.2, one might ask if the mutations also result in reduced physical association between the two subunits. Login to comment 272 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Phe132Leu If R74W and E128K cause functional uncoupling between TMD0-SUR1 and Kir6.2, one might ask if the mutations also result in reduced physical association between the two subunits. Login to comment 273 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu Several SUR1-TMD0 mutations have been reported to reduce physical association between TMD0 and Kir6.2 in co-immunoprecipitation experiments, including CHI-causing A116P and V187D mutations and PNDM-causing F132L mutation (10, 30). Login to comment 274 ABCC8 p.Phe132Leu The former two do not affect the gating properties of the channel (13), whereas the F132L mutation reduces ATP sensitivity by increasing channel intrinsic Po (30). Login to comment 275 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp It would not be surprising if R74W and E128K do not affect the extent of co-immunoprecipitation between SUR1 and Kir6.2, because there are likely multiple chemical interactions retained (such as those mediated by Ala-116, Val-187, and Phe-132) to allow association of the two subunits. Login to comment 276 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp It would not be surprising if R74W and E128K do not affect the extent of co-immunoprecipitation between SUR1 and Kir6.2, because there are likely multiple chemical interactions retained (such as those mediated by Ala-116, Val-187, and Phe-132) to allow association of the two subunits. Login to comment 277 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp R74W and E128K, like all other neonatal diabetes-causing mutations, render KATP channels less sensitive to ATP inhibition during glucose stimulation, and yet they were identified in patients with severe hyperinsulinism, because they also prevent channels from being expressed at the cell membrane. Login to comment 278 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp R74W and E128K, like all other neonatal diabetes-causing mutations, render KATP channels less sensitive to ATP inhibition during glucose stimulation, and yet they were identified in patients with severe hyperinsulinism, because they also prevent channels from being expressed at the cell membrane. Login to comment 280 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp The R74W was identified in one patient with diffuse disease who also carries an R1215Q mutation that reduces channel sensitivity to MgADP and two patients with focal disease (23-25), SUR1 Mutations, Hyperinsulinism, and Diabetes 7958 and E128K was identified as a disease-causing homozygous mutation in a patient with diffuse disease (15). Login to comment 281 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp The R74W was identified in one patient with diffuse disease who also carries an R1215Q mutation that reduces channel sensitivity to MgADP and two patients with focal disease (23-25), SUR1 Mutations, Hyperinsulinism, and Diabetes 7958 and E128K was identified as a disease-causing homozygous mutation in a patient with diffuse disease (15). Login to comment 286 ABCC8 p.Phe132Leu In this regard, it is important to note that we have found the neonatal diabetes-causing mutation F132L also significantly reduces channel expression at the cell surface (57.05 afe; 1.75% of WT; n afd; 3), and that sulfonylureas restore mutant channel surface expression to the same level as WT (109.2 afe; 7.05; n afd; 3). Login to comment 287 ABCC8 p.Phe132Leu In this regard, it is important to note that we have found the neonatal diabetes-causing mutation F132L also significantly reduces channel expression at the cell surface (57.05 Ϯ 1.75% of WT; n ϭ 3), and that sulfonylureas restore mutant channel surface expression to the same level as WT (109.2 Ϯ 7.05; n ϭ 3). Login to comment