ABCC8 p.Ile60Gly
Predicted by SNAP2: | A: N (53%), C: N (72%), D: D (80%), E: D (59%), F: D (59%), G: D (71%), H: D (71%), K: D (71%), L: N (87%), M: N (93%), N: D (71%), P: D (71%), Q: D (66%), R: D (59%), S: D (53%), T: N (66%), V: N (93%), W: D (66%), Y: D (66%), |
Predicted by PROVEAN: | A: N, C: N, D: D, E: D, F: N, G: D, H: D, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: N, T: N, V: N, W: D, Y: N, |
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[hide] Analysis of two KCNJ11 neonatal diabetes mutations... J Biol Chem. 2009 Mar 13;284(11):6752-62. Epub 2009 Jan 12. Winkler M, Lutz R, Russ U, Quast U, Bryan J
Analysis of two KCNJ11 neonatal diabetes mutations, V59G and V59A, and the analogous KCNJ8 I60G substitution: differences between the channel subtypes formed with SUR1.
J Biol Chem. 2009 Mar 13;284(11):6752-62. Epub 2009 Jan 12., [PMID:19139106]
Abstract [show]
beta-Cell-type K(ATP) channels are octamers assembled from Kir6.2/KCNJ11 and SUR1/ABCC8. Adenine nucleotides play a major role in their regulation. Nucleotide binding to Kir6.2 inhibits channel activity, whereas ATP binding/hydrolysis on sulfonylurea receptor 1 (SUR1) opposes inhibition. Segments of the Kir6.2 N terminus are important for open-to-closed transitions, form part of the Kir ATP, sulfonylurea, and phosphoinositide binding sites, and interact with L0, an SUR cytoplasmic loop. Inputs from these elements link to the pore via the interfacial helix, which forms an elbow with the outer pore helix. Mutations that destabilize the interfacial helix increase channel activity, reduce sensitivity to inhibitory ATP and channel inhibitors, glibenclamide and repaglinide, and cause neonatal diabetes. We compared Kir6.x/SUR1 channels carrying the V59G substitution, a cause of the developmental delay, epilepsy, and neonatal diabetes syndrome, with a V59A substitution and the equivalent I60G mutation in the related Kir6.1 subunit from vascular smooth muscle. The substituted channels have increased P(O) values, decreased sensitivity to inhibitors, and impaired stimulation by phosphoinositides but retain sensitivity to Ba(2+)-block. The V59G and V59A channels are either not, or poorly, stimulated by phosphoinositides, respectively. Inhibition by sequestrating phosphatidylinositol 4,5-bisphosphate with neomycin and polylysine is reduced in V59A, and abolished in V59G channels. Stimulation by SUR1 is intact, and increasing the concentration of inhibitory ATP restores the sensitivity of Val-59-substituted channels to glibenclamide. The I60G channels, strongly dependent on SUR stimulation, remain sensitive to sulfonylureas. The results suggest the interfacial helix dynamically links inhibitory inputs from the Kir N terminus to the gate and that sulfonylureas stabilize an inhibitory configuration.
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No. Sentence Comment
6 We compared Kir6.x/SUR1 channels carrying the V59G substitution, a cause of the developmental delay, epilepsy, and neonatal diabetes syndrome, with a V59A substitution and the equivalent I60G mutation in the related Kir6.1 subunit from vascular smooth muscle.
X
ABCC8 p.Ile60Gly 19139106:6:187
status: NEW10 The I60G channels, strongly dependent on SUR stimulation, remain sensitive to sulfonylureas.
X
ABCC8 p.Ile60Gly 19139106:10:4
status: NEW50 To support this hypothesis, we sought to characterize the V59G mutation further, compare it with a V59A substitution and the analogous substitution, I60G, in Kir6.1 the pore-forming subunit of the vascular KATP channel.
X
ABCC8 p.Ile60Gly 19139106:50:149
status: NEW51 This channel is composed of Kir6.1 and SUR2B (40), however, for comparison we examined the I60G/SUR1 channel.
X
ABCC8 p.Ile60Gly 19139106:51:91
status: NEWX
ABCC8 p.Ile60Gly 19139106:51:149
status: NEW56 In view of these differences in gating on one hand and of the similarity in amino acid sequence around the mutation on the other it was of interest to compare the V59G and I60G/ SUR1 KATP channels.
X
ABCC8 p.Ile60Gly 19139106:56:172
status: NEW60 The V59G, V59A, and I60G mutations were introduced into Kir6.2 and Kir6.1, respectively, using the QuikChange II XL site-directed mutagenesis kit (Stratagene).
X
ABCC8 p.Ile60Gly 19139106:60:20
status: NEW61 The mutations were verified by sequencing the relevant DNA region (V59G) or the whole coding region (I60G and V59A).
X
ABCC8 p.Ile60Gly 19139106:61:20
status: NEWX
ABCC8 p.Ile60Gly 19139106:61:101
status: NEW96 The same structural predictions were obtained for the I60G substitution in Kir6.1.
X
ABCC8 p.Ile60Gly 19139106:96:54
status: NEW173 Response of Kir6.1/SUR1 Channels to Nucleotides and Sulfonylureas-Fig. 6 compares the responses of cells expressing wild-type versus I60G/SUR1 channels to stimulation with 1 mM MgATP/0.3 mM MgGDP.
X
ABCC8 p.Ile60Gly 19139106:173:133
status: NEW175 An immediate, stable current was also obtained when cells expressing the I60G channels were dialyzed with 10 mM MgATP indicating that 10 mM MgATP did not close the mutant channel (n ϭ 5).
X
ABCC8 p.Ile60Gly 19139106:175:73
status: NEW176 Dialysis with Mg2ϩ - or nucleotide-free solutions led to a steady loss of current over 2-10 min indicating that the I60G channel required activation to remain in the open state (n ϭ 12).
X
ABCC8 p.Ile60Gly 19139106:176:73
status: NEWX
ABCC8 p.Ile60Gly 19139106:176:122
status: NEW177 GBC (10 nM) inhibited both the wild-type and I60G channels Ն90% (Fig. 6).
X
ABCC8 p.Ile60Gly 19139106:177:45
status: NEWX
ABCC8 p.Ile60Gly 19139106:177:122
status: NEW178 This inhibition of the I60G channels is in sharp contrast to the V59G Kir6.2 channels (Fig. 1b), which were insensitive to GBC up to 1 M under equivalent activating conditions.
X
ABCC8 p.Ile60Gly 19139106:178:23
status: NEWX
ABCC8 p.Ile60Gly 19139106:178:45
status: NEW181 In the whole cell configuration, wild-type Kir6.1/SUR1 channels were completely and reversibly inhibited by tolbutamide (100 M), whereas the I60G channels were 89 Ϯ 6 and 91 Ϯ 4% inhibited by 100 and 300 M tolbutamide (n ϭ 4 and 8), respectively.
X
ABCC8 p.Ile60Gly 19139106:181:149
status: NEW182 Assuming complete inhibition, an IC50 value of ϳ10 M was estimated for the I60G channel.
X
ABCC8 p.Ile60Gly 19139106:182:89
status: NEWX
ABCC8 p.Ile60Gly 19139106:182:148
status: NEW183 The experiments in the whole cell configuration (Fig. 6) suggest that, in the cellular environment, wild-type Kir6.1 channels are essentially closed, whereas the I60G channels are open.
X
ABCC8 p.Ile60Gly 19139106:183:88
status: NEWX
ABCC8 p.Ile60Gly 19139106:183:162
status: NEW188 As shown in Fig. 7b, the I60G channels were spontaneously active in on-cell patches and exhibited transitions between long lived states that differed by ϳ1.8 pA.
X
ABCC8 p.Ile60Gly 19139106:188:25
status: NEW189 Tolbutamide (300 M) reversibly inhibited the I60G channels (Fig. 7b), whereas diazoxide had no effect (n ϭ 9).
X
ABCC8 p.Ile60Gly 19139106:189:25
status: NEWX
ABCC8 p.Ile60Gly 19139106:189:53
status: NEW191 DISCUSSION Structural Hypothesis-We compared Val-59-substituted Kir6.2/SUR1 and analogous I60G Kir6.1/SUR1 channels with their respective wild-type channels.
X
ABCC8 p.Ile60Gly 19139106:191:90
status: NEW234 Whole cell and cell-attached recordings show that the I60G channels are more active than wild-type in intact cells (Fig. 7).
X
ABCC8 p.Ile60Gly 19139106:234:52
status: NEW235 This suggests that under physiologic conditions the I60G channels either have a reduced sensitivity to inhibitory ATP versus wild-type or are more efficiently activated by MgATP.
X
ABCC8 p.Ile60Gly 19139106:235:19
status: NEWX
ABCC8 p.Ile60Gly 19139106:235:52
status: NEW236 We did not observe I60G channel activity in the absence of Mg2ϩ and activating nucleotides consistent with earlier studies on the Kir6.1/SUR1 wild-type (34, 40, 42, 43), whereas both the Val-59-substituted and wild-type Kir6.2 channels are open in the absence of activating nucleotides (e.g. Figs.
X
ABCC8 p.Ile60Gly 19139106:236:19
status: NEW268 Second, the I60G channels, whose activity is strongly dependent on Mg-nucleotide-dependent stimulation by SUR1, are nearly as sensitive to GBC as the wild-type (potency difference ϳ 4ϫ).
X
ABCC8 p.Ile60Gly 19139106:268:12
status: NEWX
ABCC8 p.Ile60Gly 19139106:268:75
status: NEW269 GBC limits the stimulatory input from SUR1 thereby inducing closure of the I60G channels.
X
ABCC8 p.Ile60Gly 19139106:269:75
status: NEW52 This channel is composed of Kir6.1 and SUR2B (40), however, for comparison we examined the I60G/SUR1 channel.
X
ABCC8 p.Ile60Gly 19139106:52:91
status: NEW57 In view of these differences in gating on one hand and of the similarity in amino acid sequence around the mutation on the other it was of interest to compare the V59G and I60G/ SUR1 KATP channels.
X
ABCC8 p.Ile60Gly 19139106:57:172
status: NEW62 The mutations were verified by sequencing the relevant DNA region (V59G) or the whole coding region (I60G and V59A).
X
ABCC8 p.Ile60Gly 19139106:62:101
status: NEW97 The same structural predictions were obtained for the I60G substitution in Kir6.1.
X
ABCC8 p.Ile60Gly 19139106:97:54
status: NEW174 Response of Kir6.1/SUR1 Channels to Nucleotides and Sulfonylureas-Fig. 6 compares the responses of cells expressing wild-type versus I60G/SUR1 channels to stimulation with 1 mM MgATP/0.3 mM MgGDP.
X
ABCC8 p.Ile60Gly 19139106:174:133
status: NEW179 This inhibition of the I60G channels is in sharp contrast to the V59G Kir6.2 channels (Fig. 1b), which were insensitive to GBC up to 1 òe;M under equivalent activating conditions.
X
ABCC8 p.Ile60Gly 19139106:179:23
status: NEW184 The experiments in the whole cell configuration (Fig. 6) suggest that, in the cellular environment, wild-type Kir6.1 channels are essentially closed, whereas the I60G channels are open.
X
ABCC8 p.Ile60Gly 19139106:184:162
status: NEW190 Tolbutamide (300 òe;M) reversibly inhibited the I60G channels (Fig. 7b), whereas diazoxide had no effect (n afd; 9).
X
ABCC8 p.Ile60Gly 19139106:190:52
status: NEW192 DISCUSSION Structural Hypothesis-We compared Val-59-substituted Kir6.2/SUR1 and analogous I60G Kir6.1/SUR1 channels with their respective wild-type channels.
X
ABCC8 p.Ile60Gly 19139106:192:90
status: NEW233 Whole cell and cell-attached recordings show that the I60G channels are more active than wild-type in intact cells (Fig. 7).
X
ABCC8 p.Ile60Gly 19139106:233:54
status: NEW267 Second, the I60G channels, whose activity is strongly dependent on Mg-nucleotide-dependent stimulation by SUR1, are nearly as sensitive to GBC as the wild-type (potency difference b03; 4afb;).
X
ABCC8 p.Ile60Gly 19139106:267:12
status: NEW