ABCMdb

PMID: 10993895

Matsuo M, Trapp S, Tanizawa Y, Kioka N, Amachi T, Oka Y, Ashcroft FM, Ueda K
Functional analysis of a mutant sulfonylurea receptor, SUR1-R1420C, that is responsible for persistent hyperinsulinemic hypoglycemia of infancy.
J Biol Chem. 2000 Dec 29;275(52):41184-91., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC8 p.Arg1420Cys We analyzed the functional consequences of the PHHI missense mutation R1420C, which lies in the second nucleotide-binding fold (NBF2) of SUR1. Login to comment 3 ABCC8 p.Arg1420Cys Labeling of NBF1 with 8-azido-[␣-32 P]ATP was inhibited by MgATP and MgADP with similar Ki for wild-type SUR1 and SUR1-R1420C. Login to comment 4 ABCC8 p.Arg1420Cys However, the MgATP and MgADP affinities of NBF2 of SUR1-R1420C were about 5-fold lower than those of wild-type SUR1. Login to comment 5 ABCC8 p.Arg1420Cys MgATP and MgADP stabilized 8-azido-ATP binding at NBF1 of wild-type SUR1 by interacting with NBF2, but this cooperative nucleotide binding was not observed for SUR1-R1420C. Login to comment 6 ABCC8 p.Arg1420Cys Studies on macroscopic currents recorded in inside-out membrane patches revealed that the SUR1-R1420C mutation exhibits reduced expression but does not affect inhibition by ATP or tolbutamide or activation by diazoxide. Login to comment 7 ABCC8 p.Arg1420Cys However, co-expression with Kir6.2-R50G, which renders the channel less sensitive to ATP inhibition, revealed that the SUR1-R1420C mutation increases the EC50 for MgADP activation from 74 to 197 ␮M. Login to comment 8 ABCC8 p.Arg1420Cys We suggest that the lower expression of the mutant channel and the reduced affinity of NBF2 for MgADP may lead to a smaller KATP ؉ current in R1420C-PHHI beta-cells and thereby to the enhanced insulin secretion. Login to comment 27 ABCC8 p.Arg1420Cys Recently, we identified a missense SUR1 mutation (R1420C) in Japanese PHHI patients (31). Login to comment 42 ABCC8 p.Arg1420Cys An 86 Rbϩ efflux study revealed that KATP ϩ channels composed of SUR1-R1420C and Kir6.2 are not activated by metabolic inhibition as much as wild-type channels. Login to comment 43 ABCC8 p.Arg1420Cys This may be related to the fact that the expression level of SUR1-R1420C was only about half that of the wild-type channel when it was transiently expressed in COS-7 cells (31). Login to comment 44 ABCC8 p.Arg1420Cys We also reported that MgADP, either by direct binding to NBF2 or by hydrolysis of bound MgATP at NBF2, stabilizes the binding of 8-azido-ATP at NBF1 of wild-type SUR1 (19), and that the R1420C mutation impairs this effect without altering high affinity 8-azido-ATP binding to NBF1 (31). Login to comment 46 ABCC8 p.Arg1420Cys ABCC8 p.Arg1420Cys In this study, we examined the nucleotide-binding properties of the two NBFs of SUR1-R1420C by photoaffinity labeling experiments and studied the properties of Kir6.2/SUR1-R1420C channels by electrophysiological analysis. Login to comment 51 ABCC8 p.Arg1420Cys Crude membranes, containing similar amounts of wild-type SUR1 and SUR1-R1420C as determined by Western blotting, were incubated with 50 ␮M 8-azido-[32 P]ATP in the presence or absence of 0.1-1000 ␮M ATP or ADP in 3 ␮l of TEM buffer (40 mM Tris-Cl (pH 7.5), 0.1 mM EGTA, 1 mM MgSO4) containing 2 mM ouabain. Login to comment 78 ABCC8 p.Arg1420Cys ABCC8 p.Arg1420Cys RESULTS Photoaffinity Labeling of SUR1-R1420C with 8-Azido-[␣- 32 P]ATP and 8-Azido-[␥-32 P]ATP-To examine the biochemical properties of SUR1-R1420C, we investigated 8-azido-[32 P]ATP photoaffinity labeling of NBF1 and NBF2 (20). Login to comment 79 ABCC8 p.Arg1420Cys Crude membranes from COS-7 cells expressing SUR1 or SUR1-R1420C were incubated with 50 ␮M 8-azido-[␣-32 P]ATP followed by mild trypsin digestion. Login to comment 81 ABCC8 p.Arg1420Cys This indicates that both NBFs of SUR1-R1420C can bind 8-azido-ATP, as is found for wild-type SUR1. Login to comment 83 ABCC8 p.Arg1420Cys We therefore examined whether SUR1-R1420C is photoaffinity-labeled with 8-azido-[␥-32 P]ATP. Login to comment 84 ABCC8 p.Arg1420Cys Both NBF1 of SUR1 and of SUR1-R1420C were photoaffinity-labeled with 8-azido-[␥- 32 P]ATP (Fig. 1, lanes 5 and 7), indicating that NBF1 either does not have ATPase activity or has little ATPase activity under our experimental conditions. Login to comment 85 ABCC8 p.Arg1420Cys In contrast, NBF2s of SUR1 and SUR1-R1420C were not photoaffinity-labeled with 8-azido-[␥-32 P]ATP (Fig. 1, lanes 6 and 8), although they were photoaffinity-labeled by 8-azido-[␣-32 P]ATP (Fig. 1, lanes 2 and 4). Login to comment 86 ABCC8 p.Arg1420Cys These results suggest that the ␥-phosphate dissociates from 8-azido-[32 P]ATP bound at NBF2 and thus that NBF2 of SUR1- R1420C might have ATPase activity as is found for wild-type SUR1. Login to comment 87 ABCC8 p.Arg1420Cys ABCC8 p.Arg1420Cys Affinity of NBFs of SUR1-R1420C for ATP and ADP-To characterize the biochemical properties of SUR1-R1420C further, we investigated the affinities of both NBF1 and NBF2 for ATP and ADP. Login to comment 88 ABCC8 p.Arg1420Cys When crude membranes from COS-7 cells expressing SUR1 or SUR1-R1420C were incubated with 50 ␮M 8-azido-[␣-32 P]ATP in the presence of ATP or ADP followed by mild trypsin digestion, photoaffinity labeling of tryptic fragments containing either NBF1 or NBF2 was inhibited in a concentration-dependent manner (Fig. 2). Login to comment 89 ABCC8 p.Arg1420Cys This indicates that SUR1 and SUR1-R1420C can bind ATP and ADP. Login to comment 91 ABCC8 p.Arg1420Cys The Ki values of NBF1 of SUR1 for ATP and ADP were 1.6 Ϯ 0.64 (n ϭ 3) and 17 Ϯ 7.9 ␮M (n ϭ 3) respectively, and those of SUR1-R1420C were 1.5 Ϯ 0.26 (n ϭ 3) and 13 Ϯ 6.8 ␮M (n ϭ 3), respectively. Login to comment 92 ABCC8 p.Arg1420Cys This indicates that the affinity of NBF1 for nucleotides is not significantly different between wild-type SUR1 and SUR1-R1420C and that the affinity for ATP is significantly higher than that for ADP. Login to comment 95 ABCC8 p.Arg1420Cys However, the Ki values of NBF2 of SUR1-R1420C for ATP and ADP (350 Ϯ 36 (n ϭ 3) and 290 Ϯ 66 ␮M (n ϭ 3), respectively) were significantly higher than those of wild-type SUR1 (64 Ϯ 4.7 (n ϭ 3) and 65 Ϯ 16 ␮M (n ϭ 3), respectively). Login to comment 96 ABCC8 p.Arg1420Cys These results demonstrate that the R1420C mutation decreases the affinity of NBF2 for both ATP and ADP. Login to comment 97 ABCC8 p.Arg1420Cys ABCC8 p.Arg1420Cys Cooperative Nucleotide Binding of SUR1-R1420C-To explore the possibility that the impaired cooperative nucleotide binding observed for SUR1-R1420C is due to the low nucleotide-binding affinity of NBF2, we examined the dependence of cooperative binding on nucleotide concentration. Login to comment 102 ABCC8 p.Arg1420Cys In contrast, neither ATP nor ADP (1 mM) stabilized 8-azido-[32 P]ATP binding at NBF1 of SUR1-R1420C despite the fact that 1 mM MgATP and MgADP inhibited photoaffinity labeling of NBF2 (68 Ϯ 3.2 (n ϭ 3) and 76 Ϯ 2.9% (n ϭ 3), respectively) as efficiently as that of wild-type SUR1 (74 Ϯ 10 (n ϭ 3) and 73 Ϯ 1.5% (n ϭ 3), FIG. 1. Login to comment 103 ABCC8 p.Arg1420Cys Photoaffinity labeling of the NBFs of SUR1 and SUR1-R1420C with 8-azido-[␣-32 P]ATP and 8-azido-[␥-32 P]ATP. Login to comment 104 ABCC8 p.Arg1420Cys Membrane proteins (10-20 ␮g) from COS-7 cells expressing wild-type SUR1 (lanes 1, 2, 5, and 6) or SUR1-R1420C (lanes 3, 4, 7, and 8) were incubated with 50 ␮M 8-azido-[␣-32 P]ATP (lanes 1-4) or 8-azido-[␥- 32 P]ATP (lanes 5-8) for 10 min at 37 °C and then UV-irradiated. Login to comment 110 ABCC8 p.Arg1420Cys Inhibition of photoaffinity labeling of the NBFs of SUR1 and SUR1-R1420C by nucleotides. Login to comment 111 ABCC8 p.Arg1420Cys Membrane proteins (10-20 ␮g) from COS-7 cells expressing wild-type SUR1 or SUR1-R1420C were incubated with 50 ␮M 8-azido-[␣-32 P]ATP in the presence of 0.1-1000 ␮M ATP or ADP for 10 min at 0 °C and then UV-irradiated. Login to comment 113 ABCC8 p.Arg1420Cys Relative photoaffinity labeling of wild-type SUR1 in the presence of ATP (open squares) or ADP (closed squares) and of SUR1-R1420C in the presence of ATP (open circles) or ADP (closed circles) are expressed as percentages of that obtained in the absence of cold nucleotides. Login to comment 115 ABCC8 p.Arg1420Cys The symbols show the mean values and the S.E. TABLE I Ki values for interaction of ATP and ADP with the NBFs of SUR1 and SUR1-R1420C The data of Fig. 2 were fit by the Hill equation, and Ki values were obtained. Login to comment 116 ABCC8 p.Arg1420Cys Ki Wild-type R1420C ␮M ␮M NBF1-ATP 1.6 Ϯ 0.64 1.5 Ϯ 0.26 NBF1-ADP 17 Ϯ 7.9 13 Ϯ 6.8 NBF2-ATP 64 Ϯ 4.7 350 Ϯ 36 NBF2-ADP 65 Ϯ 16 290 Ϯ 66 respectively) (Fig. 2). Login to comment 117 ABCC8 p.Arg1420Cys These results suggest that neither MgATP nor MgADP bound at NBF2 can stabilize 8-azido-ATP binding at NBF1 of SUR1-R1420C. Login to comment 118 ABCC8 p.Arg1420Cys ABCC8 p.Arg1420Cys Electrophysiological Analysis-To examine the functional properties of the mutant KATP ϩ channels, we coexpressed SUR1-R1420C with Kir6.2 in Xenopus oocytes. As observed for the wild-type channel (Kir6.2/SUR1), no significant current was detected for Kir6.2/SUR1-R1420C channels in the cell-attached configuration, but large currents developed following patch excision into nucleotide-free solution. Login to comment 119 ABCC8 p.Arg1420Cys The mean current amplitude at -100 mV was 3.0 Ϯ 1.0 nA (n ϭ 10) for Kir6.2/SUR1 and 1.5 Ϯ 0.6 nA (n ϭ 15) for Kir6.2/SUR1-R1420C channels. Login to comment 121 ABCC8 p.Arg1420Cys A quantitatively similar reduction in SUR1-R1420C protein expression was observed in COS-7 cells, indicating that the lower expression of mutant SUR1 is confined to the oocyte expression system. Login to comment 122 ABCC8 p.Arg1420Cys Pharmacological Regulation-We first examined the effect of the R1420C mutation on the activation of the channel by the potassium channel opener diazoxide (Fig. 4) and its inhibition by the sulfonylurea tolbutamide. Login to comment 125 ABCC8 p.Arg1420Cys Diazoxide increased Kir6.2/SUR1 currents by 694 Ϯ 40% (n ϭ 4) and Kir6.2/SUR1-R1420C currents by 499 Ϯ 126% (n ϭ 7). Login to comment 126 ABCC8 p.Arg1420Cys The sulfonylurea tolbutamide blocked Kir6.2/SUR1 currents by 53 Ϯ 2% (n ϭ 3) and Kir6.2/SUR1-R1420C currents by 68 Ϯ 7% (n ϭ 3). Login to comment 127 ABCC8 p.Arg1420Cys Thus the R1420C mutation does not affect the pharmacological properties of the channel. Login to comment 128 ABCC8 p.Arg1420Cys Nucleotide Regulation-Inhibition of the channel by ATP was not affected by the R1420C mutation. Login to comment 129 ABCC8 p.Arg1420Cys Kir6.2/SUR1 currents were inhibited with an IC50 of 13 Ϯ 2 ␮M and a Hill coefficient of 0.99 Ϯ 0.18 (n ϭ 5), whereas Kir6.2/SUR1-R1420C currents were blocked with an IC50 of 12 Ϯ 2 ␮M and a Hill coefficient of 0.90 Ϯ 0.14 (n ϭ 6; Fig. 5). Login to comment 130 ABCC8 p.Arg1420Cys ADP produced a mean current increase of 402 Ϯ 82% (n ϭ 3) and 347 Ϯ 52% (n ϭ 6) for Kir6.2/SUR1 and Kir6.2/SUR1-R1420C, respectively, when tested in the presence of 100 ␮M MgATP. Login to comment 132 ABCC8 p.Arg1420Cys Cooperative binding of nucleotides and 8-azido-[␣- 32 P]ATP to SUR1 and SUR1-R1420C. Login to comment 133 ABCC8 p.Arg1420Cys Membrane proteins (10-20 ␮g) from COS-7 cells expressing wild-type SUR1 or SUR1-R1420C were preincubated with 10 ␮M 8-azido-[␣-32 P]ATP for 3 min at 37 °C. Free 8-azido-[␣-32 P]ATP was then removed by washing the membranes with excess cold buffer. Login to comment 134 ABCC8 p.Arg1420Cys ABCC8 p.Arg1420Cys ABCC8 p.Arg1420Cys Proteins were UV-irradiated after a postincubation for 15 min at 37 °C in the absence of nucleotide (SUR1, square with cross; SUR1-R1420C, circle with cross), in the presence of ATP (SUR1, open squares; SUR1-R1420C, open circles), or in the presence of ADP (SUR1, closed squares; SUR1-R1420C, closed circles). Login to comment 137 ABCC8 p.Arg1420Cys Diazoxide and nucleotide modulation of Kir6.2/SUR1-R1420C. Login to comment 138 ABCC8 p.Arg1420Cys A, macroscopic currents recorded from a giant patch on an oocyte coinjected with Kir6.2 and SUR1-R1420C mRNA. Login to comment 141 ABCC8 p.Arg1420Cys B, mean macroscopic currents recorded from oocytes coexpressing Kir6.2 and either wild-type SUR1 or SUR1-R1420C in the presence of the nucleotides indicated. The mean conductance in the presence of nucleotide (G) is expressed relative to the mean of that measured in control solution before and after removal of nucleotide (Gc; indicated by the dashed line). Login to comment 144 ABCC8 p.Arg1420Cys ATP sensitivity of Kir6.2/SUR1-R1420C. Login to comment 145 ABCC8 p.Arg1420Cys Mean MgATP concentration-response relationships for Kir6.2/SUR1 (filled squares, n ϭ 5) and Kir6.2-SUR1-R1420C (open squares, n ϭ 6) currents are shown. Login to comment 151 ABCC8 p.Arg1420Cys Therefore, to quantify the difference in the extent of nucleotide activation of wild-type and mutant KATP ϩ channels, we coexpressed either SUR1 or SUR1-R1420C with a mutant form of Kir6.2, Kir6.2-R50G, which exhibits a greatly reduced ATP sensitivity (18, 36). Login to comment 152 ABCC8 p.Arg1420Cys ABCC8 p.Arg1420Cys Both Kir6.2-R50G/SUR1 and Kir6.2-R50G/SUR1-R1420C currents were activated by 100 ␮M MgATP and by 100 ␮M MgGTP (Fig. 6A), and in both cases, the extent of activation was greater for Kir6.2-R50G/SUR1-R1420C channels. Login to comment 154 ABCC8 p.Lys719Ala ABCC8 p.Arg1420Cys As Fig. 6A also shows, Kir6.2-R50G/SUR1-K719A currents were neither activated nor blocked by 100 ␮M MgATP, demonstrating that, at this ATP concentration, activation of Kir6.2-R50G/SUR1 and Kir6.2-R50G/SUR1-R1420C currents is not partially masked by an inhibitory effect of ATP. Login to comment 155 ABCC8 p.Lys719Ala At ATP concentrations of 1 mM and above, however, Kir6.2-R50G/SUR1-K719A currents are inhibited. Login to comment 157 ABCC8 p.Arg1420Cys Fig. 6B shows the relationship between MgADP concentration and activation of Kir6.2-R50G/SUR1 and Kir6.2-R50G/ SUR1-R1420C channels. Login to comment 159 ABCC8 p.Arg1420Cys ABCC8 p.Arg1420Cys The EC50 for ADP activation was also increased by the SUR1-R1420C mutation from 74 Ϯ 30 ␮M (n ϭ 5) for Kir6.2-R50G/SUR1 channels to 197 Ϯ 20 ␮M (n ϭ 6) for Kir6.2-R50G/SUR1-R1420C channels. Login to comment 160 ABCC8 p.Arg1420Cys These values are comparable with those found for ADP displacement of 8-azido-ATP binding to NBF2 of SUR1 and SUR1-R1420C, respectively (Table I). Login to comment 161 ABCC8 p.Arg1420Cys One possible explanation for the fact that ADP causes greater activation of Kir6.2-R50G/SUR1-R1420C channels is that their open probability (Po) is less than that of Kir6.2-R50G/SUR1 channels. Login to comment 164 ABCC8 p.Arg1420Cys Kir6.2/SUR1 and Kir6.2/SUR1-R1420C channels had Po of 0.29 Ϯ 0.09 (n ϭ 4) and 0.32 Ϯ 0.03 (n ϭ 5), respectively. Login to comment 165 ABCC8 p.Arg1420Cys When SUR1 or SUR1-R1420C was coexpressed with Kir6.2-R50G, the Po was 0.35 Ϯ 0.07 (n ϭ 6) and 0.34 Ϯ 0.07 (n ϭ 7), respectively. Login to comment 167 ABCC8 p.Arg1420Cys Whole-cell Studies-To explore the effect of metabolic inhibition on Kir6.2/SUR1 and Kir6.2/SUR1-R1420C channels, we recorded whole-cell KATP ϩ currents from intact oocytes and used 3 mM azide as a metabolic poison. Login to comment 168 ABCC8 p.Arg1420Cys In control solution, oocytes expressing both types of channels exhibited very small current amplitudes, similar to those of water-injected oocytes. As shown in Fig. 8, metabolic poisoning produced a large increase in both Kir6.2/SUR1 and Kir6.2/SUR1-R1420C currents. Login to comment 170 ABCC8 p.Arg1420Cys The fact that Kir6.2/SUR1-R1420C currents induced by metabolic inhibition are smaller than those of the wild-type channel may reflect, at least in part, a lower level of expression of the mutant channel because in inside-out membrane patches the currents activated on patch excision were also smaller (Fig. 8B). Login to comment 171 ABCC8 p.Arg1420Cys DISCUSSION We analyzed the functional properties of SUR1 containing a PHHI missense mutation, R1420C, and demonstrated that the mutation lowers the affinities of NBF2 for ATP and ADP and impairs the ability of MgATP and MgADP to stabilize ATP binding at NBF1. Login to comment 172 ABCC8 p.Arg1420Cys ABCC8 p.Arg1420Cys This cooperative nucleotide binding was not observed for SUR1-R1420C even with 1 mM MgATP or MgADP, although 1 mM MgATP and MgADP inhibited the photoaffinity labeling of NBF2 of wild-type SUR1 and SUR1-R1420C with similar efficiency. Login to comment 173 ABCC8 p.Arg1420Cys The affinities of NBF1s for ATP and ADP are similar for wild-type SUR1 and SUR1-R1420C. Login to comment 174 ABCC8 p.Arg1420Cys These results suggest that the R1420C mutation not only decreases the nucleotide-binding affinity of NBF2 but also impedes the transduction of a conformational change at NBF2 and thereby its ability to stabilize ATP binding at NBF1. Login to comment 176 ABCC8 p.Arg1420Cys Nucleotide activation of Kir6.2/SUR1-R1420C. Login to comment 177 ABCC8 p.Lys719Ala ABCC8 p.Arg1420Cys A, mean macroscopic currents recorded from oocytes coexpressing Kir6.2-R50G and either wild-type SUR1, SUR1-R1420C, or SUR1-K719A in the presence of 100 ␮M of the nucleotides indicated. The mean conductance in the presence of nucleotide (G) is expressed relative to the mean of that measured in control solution before and after removal of nucleotide (Gc indicated by the dashed line). Login to comment 178 ABCC8 p.Arg1420Cys Numbers of oocytes are given above the bars. B, relationship between the MgADP concentration and the KATP ϩ current for Kir6.2-R50G/SUR1 (circles, n ϭ 4) and Kir6.2-R50G/ SUR1-R1420C (squares, n ϭ 5) channels. Login to comment 184 ABCC8 p.Arg1420Cys ABCC8 p.Arg1420Cys As found for binding studies, the EC50 for channel activation by ADP increased to 197 Ϯ 20 ␮M when arginine 1420 was mutated to cysteine, which compares favorably with the Ki of 290 Ϯ 66 ␮M found for ADP binding to NBF2 of SUR1-R1420C. Login to comment 190 ABCC8 p.Arg1420Cys Second, the R1420C mutation may reduce the affinity of MgADP binding yet at the same time enhance the transduction of the conformational change in response to MgADP. Login to comment 192 ABCC8 p.Arg1420Cys This might occur if, for example, channel activation is greater when MgADP binds to both NBFs than when MgATP binds to NBF1 and MgADP to NBF2. Our results suggest that the level of expression of Kir6.2/ SUR1-R1420C KATP ϩ channels in Xenopus oocytes is about half that of wild-type KATP ϩ channels because mutant currents in excised patches were about half those of wild-type currents. Login to comment 193 ABCC8 p.Arg1420Cys We have previously reported that Rbϩ efflux from COS-7 cells expressing Kir6.2/SUR1-R1420C KATP ϩ channels is about half that of cells expressing wild-type KATP ϩ channels when intracellular ATP is depleted by metabolic inhibition with 2-deoxyglucose and oligomycin. Login to comment 195 ABCC8 p.Arg1420Cys Thus the R1420C mutation results in a reduced KATP ϩ current whether expressed in oocytes or in mammalian cells. Login to comment 196 ABCC8 p.Arg1420Cys However, this cannot be the only explanation for the ability of the R1420C mutation to cause PHHI because heterozygotes carrying other PHHI mutations that result in a total loss of protein, which would also be expected to produce KATP ϩ currents of about half the normal amplitude, do not develop PHHI. Login to comment 201 ABCC8 p.Arg1420Cys We have proposed that the greater ability of SUR1 to stimulate opening of KATP ϩ channels when metabolism falls is linked to the higher nucleotide-binding affinities of NBF1 and/or NBF2. Our finding that the PHHI missense mutation R1420C lowers the affinity of NBF2 for ATP and ADP and increases the EC50 for ADP activation of channel activity adds further support to the idea that ADP binding to NBF2 of SUR1 is important for KATP ϩ channel stimulation. Login to comment 208 ABCC8 p.Arg1420Cys Single Kir6.2/SUR1 and Kir6.2/ SUR1-R1420C channel currents. Login to comment 209 ABCC8 p.Arg1420Cys Single channel currents recorded at -60 mV from patches excised from oocytes expressing Kir6.2/SUR1 and Kir6.2/SUR1-R1420C channels are shown. Login to comment 211 ABCC8 p.Arg1420Cys Metabolic regulation of Kir6.2/SUR1-R1420C. Login to comment 213 ABCC8 p.Arg1420Cys Oocytes were coinjected with mRNA encoding Kir6.2 and SUR1 or SUR1-R1420C. Login to comment 215 ABCC8 p.Arg1420Cys Oocytes were coinjected with mRNA encoding Kir6.2 and SUR1 or SUR1-R1420C. Login to comment 225 ABCC8 p.Arg1420Cys In this study, we have demonstrated that the R1420C PHHI mutation strongly affects the biochemical properties of SUR1; it lowers the affinity of NBF2 for ATP and ADP and abolishes the ability of nucleotide binding at NBF2 to stabilize 8-azido-ATP binding at NBF1. Login to comment