This data was extracted from Medline abstracts and full texts (when available) in an automated manner.
The table below describes different point mutations at a given position and provides links to other documents. The sentence(s) where the point mutations in IRK11_HUMAN at position 201 were found are listed after the table.
Only one residue (R201A) simultaneously affected ATP and PIP2 sensitivity , which is consistent with the notion that these ligands , while functionally competitive , are unlikely to bind to identical sites
Nine mutations were identified as having altered sensitivity to PIP2 in this assay (R176A , R177A , R195A , R206A , K222A , R301A , and R314A [identified above] , plus R192A and R201A , which also show a nonsignificant reduction in Rb flux compared with wild-type)
Nonidentical Residues Control ATP and PIP2 Sensitivity All expressed mutants had comparable intrinsic ATP sensitivity to wild-type channels (Fig 2 D) except K185Q and R201A
Of the PIP2-sensitive residues , only R201A (Fig 4 ) also affects sensitivity to ATP
Compared with wild-type channels , R201A mutant channels actually showed only moderately increased stimulation by PIP2 (Fig 2 ) and no significant reduction of 86Rb efflux in intact cells (Fig 1 B)
However , R201A channels showed a significantly decreased ATP sensitivity (Ki ~115 µM ; Fig 4 B)
R201A mutants alter sensitivity to PIP2 and ATP
(A and C) Representative currents recorded from inside-out membrane patch containing Kir6.2[R201A] and Kir6.2[R206A] subunits coexpressed with SUR1 subunits
(B) Steady state dependence of membrane current on [ATP] (relative to current in zero ATP) for Kir6.2[R201A] or wild-type Kir6.2 subunits coexpressed with SUR1 (mean ± SEM , n = 5-7)
The data were fitted using the Hill equation , with K1 / 2 , ATP = 12 µM (WT) and 115 µM (R201A) , and H = 1.9.(WT) and 0.9 (R201A)
Only the R201A mutation affected both ATP and PIP2 interaction , reducing the sensitivity to both ligands (Fig 2 ) , which is consistent with the idea that each ligand interacts with separate , but possibly overlapping , sites on the same domain to stabilize either the closed (ATP) or open (PIP2) states
Reference #2 (Gloyn AL et al.): R201C
These partial pedigrees show families with the Q52R , V59G , V59M , R201C , R201H , and I296L mutations
NM ISPAD 55 (V59M) NN NN NN NM P ISPAD 43 (I296L) NN NN NM P ISPAD 41 (R201C) NN NN NM P ISPAD 27 (Q52R) NN NN NM NA P ISPAD 19 (R201H) NN NN NN NN NN NM P ISPAD 22 (R201H) NN NN NM P BR 1 (R201H) NN NM NM NM P ISPAD 25 (V59G) NN NN NM P ISPAD 44 (R201H) NN NN NN NM P ISPAD 54 (V59M) NN NN NM P Death at 14 mo 6 2 3 Second cousins Downloaded from www.nejm.org at UC SHARED JOURNAL COLLECTION on May 19 , 2004
Clinical Characteristics of Patients with Mutant Kir6.2.* Characteristic ISPAD 19 ISPAD 22 ISPAD 41 ISPAD 44 ISPAD 54 BR 1 ISPAD 55 ISPAD 25 ISPAD 27 ISPAD 43 Proband Mother Proband Proband Proband Proband Proband Brother Father Proband Proband Proband Proband Sex M F F M F M M M M M M M F Mutation R201H R201H R201H R201C R201H V59M R201H R201H R201H V59M V59G Q52R I296L Birth Weight (g) Percentile for weight Gestation (wk) 2550 9 38 2280 0.4 40 2280 1.3 39 2200 1.5 38 2125 2.5 38 1440 <0.1 37 3080 16 40 3120 18 40 2500 NA NA 2700 1.4 41 1850 29 33 2400 11 37 2550 2.5 40 Findings on presentation Clinical abnormality Hyper- glycemia Keto- acidosis Keto- acidosis Hyper- glycemia Keto- acidosis Hyper- glycemia Hyper- glycemia Hyper- glycemia Hyper- glycemia Hyper- glycemia Hyper- glycemia Hyper- glycemia Hyper- glycemia Glucose (mmol / liter) 15 54 54 36 33 16 23 30 NA 35.1 18 34.5 18.8 Pancreatic autoantibodies Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Age diabetes diagnosed (wk) Birth 6 6 4 15 Birth <4 <3 12 5 1 5 26 Current status Age (yr) 8 36 3 12 2 2 5 3 46 2 4 1.2 (died) ; 0.8 (last assessed) 17 Percentile for height 75 10 10 98 1025 75 10 10 <5 85 1.5 3 <3 Percentile for weight 91 75 25 98 1025 >98 10 10 6 50 1 3 <3 Insulin dose (U / kg / day) 0.6 0.61.0 0.61.0 1.6 0.71.0 1.3 0.7 0.71.3 NA§ 0.3 0.30.4 0.20.5 0.20.4 Glycosylated hemoglobin (%) 8.7 7.410.0 7.410.0 7.0 7.7 9.2 NA NA NA 7.0 9.011.0 9.0 9.010.0 C-peptide (pmol / liter) <170 <200 <200 <100 NA <94 <200 <200 400¶ 613 603 <165 60 Paired glucose (mmol / liter)¿ 18.1 12.0 12.0 16.3 16.3 13.0 13.8 6.1 5.7 16.2 17.2 18.8 Peak C-peptide with glucagon (pmol / liter) <170 <200 <200 NA NA NA NA NA NA NA <165 <165 NA Downloaded from www.nejm.org at UC SHARED JOURNAL COLLECTION on May 19 , 2004
The mutations were a glutamine-to-arginine substitution at position 52 (Q52R) , a valine-to-glycine substitution at position 59 (V59G) , a valine-to-methionine substitution at position 59 (V59M) , an arginine-tohistidine substitution at position 201 (R201H) , an arginine-to-cysteine substitution at position 201 (R201C) , and an isoleucine-to-leucine substitution at position 296 (I296L)
Reference #3 (Gloyn AL et al.): R201C
We describe a family in which two affected paternal half-siblings were found to be heterozygous for the previously reported R201C mutation
Reference #4 (Vaxillaire M et al.): R201C
We identified in nine patients seven heterozygous nonsynonymous mutations: three of them (V59M , R201C , and R201H) were already described , and the four novel mutations resulted in an amino acid change of Kir6.2 at positions F35L , G53N , E322K , and Y330C
Three of them (V59M , R201C , and R201H) have been previously described (5 ) , and the four novel mutations resulted in amino acid substitutions at the following positions: F35L , G53N , E322K , and Y330C (Table 1 )
Reference #5 (Edghill EL et al.): R201C
One patient was found to be heterozygous for the missense mutation R201C
The potential mutations were the substitution of arginine by cystine at codon 201 (CGT>TGT , R201C) and the substitution of arginine by cystine at codon 176 (CGC>TGC , R176C)
R201C is likely to be an etiological mutation , as it has previously been reported in a subject with PNDM (1 )
The R201C mutation has not been studied , but other substitutions at this site have also shown a reduction of ATP sensitivity (13 )
The clinical characteristics of the subjects with R201C and R176C are shown in Table 1
The subject with the R201C KCNJ11 mutation has similar characteristics to previously described patients with activating KCNJ11 mutations (1 ): she was born with a low birth weight (2.67 kg) and diagnosed with diabetes at 5 weeks of age , and GAD and IA-2 antibody testing performed at age 26 years were negative
View this table: [in this window] [in a new window] TABLE 1 Clinical details and investigations for patients BDA1 and BDA2 with R201C and R176C KCNJ11 mutations HLA typing of the patient with the R201C KCNJ11 mutation identified her genotype as DRB1*02-DQB1*0502 / DRB1*03-DQB1*0201
Reference #6 (Proks P et al.): R201C
To determine the molecular basis of these different phenotypes , we expressed wild-type or mutant (R201C , Q52R , or V59G) Kir6.2 / sulfonylurea receptor 1 channels in Xenopus oocytes
All mutations increased resting whole-cell K ATP currents by reducing channel inhibition by ATP , but , in the simulated heterozygous state , mutations causing PNDM alone (R201C) produced smaller K ATP currents and less change in ATP sensitivity than mutations associated with severe disease (Q52R and V59G)
Two mutations in the same residue (R201H and R201C) , which lies in the putative ATP-binding site (7 , 8 ) , cause mild disease
In this paper , we compare the functional effects of mutations associated with severe disease (Q52R and V59G) with those that cause mild disease (R201C and R201H)
In contrast , significant resting whole-cell K + currents are present in oocytes expressing homomeric Q52R , V59G , or R201C (homQ52R , homV59G , and homR201C , respectively) (Fig. 2 and Table 1 )
However , homR201C channels were enhanced by azide , suggesting that they are more ATP-sensitive than homV59G and homQ52R channels
For heterozygous channels (hetV59G , hetQ52R , and hetR201C) , hetV59G and hetQ52R resting whole-cell currents were of similar amplitude , constituting ~65% of their homomeric values , but were far larger than wild-type currents (Fig. 2B )
Resting hetR201C currents were smaller than hetQ52R and hetV59G currents but still significantly larger than wild-type (Fig. 2 and Table 1 )
All homomeric mutant channels were substantially less sensitive to intracellular ATP than wild type , the order of potency being WT > Q52R > R201C > V59G (Table 1 )
In particular , hetQ52R and hetV59G channels were half-maximally blocked by ATP concentrations of 20-30 µM , whereas the ATP sensitivity of hetR201C channels was higher (11 µM) (Table 1 )
(B) (Upper Left) Mean relationship between [ATP] and KATP conductance , G , expressed relative to the conductance in the absence of nucleotide , Gc , for Kir6.2 / SUR1 (open blue circles , n = 6) , and heterozygous (filled red circles , n = 6) and homomeric (filled black squares , n = 6) Kir6.2-R201C / SUR1 channels
For heterozygous R201C , IC50 = 10.4 µM and h = 1.0
For homomeric R201C , IC50 = 102 µM and h = 1.3
(Lower Right) Mean relationship between [ATP] and KATP conductance expressed relative to the conductance in the absence of nucleotide for wild type (open blue circles) and hetR201C (filled red circles) , hetQ52R (filled black squares) , and hetV59G (filled green hexagons) channels
The latter value is close to that found for hetR201C , which also causes mild disease , and significantly less than that found for mutations associated with severe disease
Although the ATP concentration causing half-maximal block of hetR201C channels is only slightly greater than that found for wild-type channels (11 µM vs
Because [ATP] i is unlikely to fall below 0.1 mM in beta cells , even when extracellular glucose is low (12 ) , the difference in wild-type and mutant K ATP current at this (and higher) ATP concentration probably explains why the R201C and R201H mutations result in neonatal diabetes
This may explain why mutations that produce a small decrease in the ATP sensitivity of heterozygous channels (R201C and R201H) result in neonatal diabetes alone , whereas those causing a greater reduction (Q52R and V59G) are associated with severe disease
Why does the Q52R mutation cause a greater shift in the ATP sensitivity of the heterozygous channel than the R201C mutation , despite the similar ATP sensitivities of the homomeric channels? To answer this question , we recorded single-channel currents in inside-out membrane patches in ATP-free solution , where the intrinsic gating of the channel can be assessed
Thus , the P O(0) of homomeric channels containing the R201C mutation was not significantly different from wild-type , whereas that of homQ52R and homV59G channels was substantially greater (Table 1 )
This finding explains why mutations in the ATP-binding site , like R201C , cause only a small shift in the ATP concentration-inhibition curve of heterozygous channels
In the case of the V59G channels , simulations suggest that the mutation must affect both gating and ATP-binding / transduction and indicate that the latter effect is largely suppressed in heterozygous channels containing wild-type subunits (as is the case for hetR201C)
The results we have described thus far predict that the second mutation at V59 (V59M) , which is also associated with severe disease , will have an increased intrinsic open probability , whereas the R201H mutation , which , like R201C , causes PNDM alone , will not
We observed that 500 µM tolbutamide blocked azide-activated whole-cell currents by 89 ± 1% (n = 12) , 65 ± 5% (n = 9) , and 41 ± 2% (n = 9) for hetR201C , hetQ52R , and hetV59G channels , respectively
Reference #2 (Gloyn AL et al.): R201H
These partial pedigrees show families with the Q52R , V59G , V59M , R201C , R201H , and I296L mutations
NM ISPAD 55 (V59M) NN NN NN NM P ISPAD 43 (I296L) NN NN NM P ISPAD 41 (R201C) NN NN NM P ISPAD 27 (Q52R) NN NN NM NA P ISPAD 19 (R201H) NN NN NN NN NN NM P ISPAD 22 (R201H) NN NN NM P BR 1 (R201H) NN NM NM NM P ISPAD 25 (V59G) NN NN NM P ISPAD 44 (R201H) NN NN NN NM P ISPAD 54 (V59M) NN NN NM P Death at 14 mo 6 2 3 Second cousins Downloaded from www.nejm.org at UC SHARED JOURNAL COLLECTION on May 19 , 2004
f u n c t i o n a l s t u d i e s Wild-type mouse Kir6.2 or Kir6.2 in which histidine replaced arginine at position 201 (R201H) was coexpressed with rat SUR1 (containing exon 17) in Xenopus laevis oocytes , and K ATP currents were recorded as previously described
19 , 20 To si mulate the effect of heterozygosity , we injected oocytes with SUR1 and a 1:1 mixture of Kir6.2 and Kir6.2R201H messenger RNA (mRNA)
Clinical Characteristics of Patients with Mutant Kir6.2.* Characteristic ISPAD 19 ISPAD 22 ISPAD 41 ISPAD 44 ISPAD 54 BR 1 ISPAD 55 ISPAD 25 ISPAD 27 ISPAD 43 Proband Mother Proband Proband Proband Proband Proband Brother Father Proband Proband Proband Proband Sex M F F M F M M M M M M M F Mutation R201HR201HR201H R201C R201H V59M R201HR201HR201H V59M V59G Q52R I296L Birth Weight (g) Percentile for weight Gestation (wk) 2550 9 38 2280 0.4 40 2280 1.3 39 2200 1.5 38 2125 2.5 38 1440 <0.1 37 3080 16 40 3120 18 40 2500 NA NA 2700 1.4 41 1850 29 33 2400 11 37 2550 2.5 40 Findings on presentation Clinical abnormality Hyper- glycemia Keto- acidosis Keto- acidosis Hyper- glycemia Keto- acidosis Hyper- glycemia Hyper- glycemia Hyper- glycemia Hyper- glycemia Hyper- glycemia Hyper- glycemia Hyper- glycemia Hyper- glycemia Glucose (mmol / liter) 15 54 54 36 33 16 23 30 NA 35.1 18 34.5 18.8 Pancreatic autoantibodies Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Age diabetes diagnosed (wk) Birth 6 6 4 15 Birth <4 <3 12 5 1 5 26 Current status Age (yr) 8 36 3 12 2 2 5 3 46 2 4 1.2 (died) ; 0.8 (last assessed) 17 Percentile for height 75 10 10 98 1025 75 10 10 <5 85 1.5 3 <3 Percentile for weight 91 75 25 98 1025 >98 10 10 6 50 1 3 <3 Insulin dose (U / kg / day) 0.6 0.61.0 0.61.0 1.6 0.71.0 1.3 0.7 0.71.3 NA§ 0.3 0.30.4 0.20.5 0.20.4 Glycosylated hemoglobin (%) 8.7 7.410.0 7.410.0 7.0 7.7 9.2 NA NA NA 7.0 9.011.0 9.0 9.010.0 C-peptide (pmol / liter) <170 <200 <200 <100 NA <94 <200 <200 400¶ 613 603 <165 60 Paired glucose (mmol / liter)¿ 18.1 12.0 12.0 16.3 16.3 13.0 13.8 6.1 5.7 16.2 17.2 18.8 Peak C-peptide with glucagon (pmol / liter) <170 <200 <200 NA NA NA NA NA NA NA <165 <165 NA Downloaded from www.nejm.org at UC SHARED JOURNAL COLLECTION on May 19 , 2004
The mutations were a glutamine-to-arginine substitution at position 52 (Q52R) , a valine-to-glycine substitution at position 59 (V59G) , a valine-to-methionine substitution at position 59 (V59M) , an arginine-tohistidine substitution at position 201 (R201H) , an arginine-to-cysteine substitution at position 201 (R201C) , and an isoleucine-to-leucine substitution at position 296 (I296L)
The R201H missense mutation was identified in 4 of these 10 probands , and the V59M missense mutation was detected in 2
In contrast , significant resting currents were recorded from oocytes expressing Kir6.2R201HSUR1 (P<0.01 for the comparison with wild-type Kir6.2)
These data suggest that metabolism causes less blockade of Kir6.2-R201HSUR1 channels than it does of wild-type K ATP channels
Kir6.2-R201HSUR1 channels were considerably less sensitive than wild-type channels to intracellular ATP ; mutant channels were half maximally blocked at an ATP concentration of Figure 4
To si mulate the effect of heterozygosity , we injected oocytes with SUR1 and a 1:1 mixture of Kir6.2 and Kir6.2-R201H mRNA
The K ATP currents of mutant channels were further activated by azide and blocked by tolbutamide , to an extent Current (µA) 8 6 4 2 0 Kir6.2SUR1 Kir6.2-R201HSUR1 Kir6.2 plus Kir6.2-R201HSUR1 K AT P Current Relative to Current in Absence of Nucleotide 0.8 1.0 0.6 0.4 0.2 0.0 10¡7 10¡6 10¡5 10¡4 10¡3 10¡2 ATP (mol / liter) Kir6.2SUR1 Kir6.2-R201HSUR1 100 µM ATP 100 µM ATP 20 sec 100 sec 20 nA 1 nA -->mM azide -->mM azide 500 µM tolbutamide 500 µM tolbutamide 2 µA Kir6.2SUR1 1:1 mixture Kir6.2-R201HSUR1 A B C Control Azide plus tolbutamide Azide Kir6.2SUR1 Kir6.2-R201HSUR1 D E Figure 5
Whole-cell currents were recorded in a two-electrode voltage clamp from intact oocytes expressing Kir6.2SUR1 (Panel A) or Kir6.2-R201HSUR1 (Panel B) in response to voltage steps of ±20 mV from a holding potential of ¡10 mV
Oocytes were injected with messenger RNA encoding SUR1 plus either Kir6.2 (six oocytes) , Kir6.2-R201H (seven) , or a 1:1 mixture of Kir6.2 and Kir6.2-R201H (seven)
Panel E shows the relation between the ATP concentration and the K ATP current , expressed relative to the current in the absence of nucleotide , for Kir6.2SUR1 in six oocytes (open circles) , Kir6.2-R201H SUR1 in six oocytes (solid circles) , and a 1:1 mixture of Kir6.2-R201H and Kir6.2 coexpressed with SUR1 in six oocytes (diamonds)
The lines are the best fit of the Hill equation to the mean data: for Kir6.2SUR1 , the concentration at which inhibition is half maximal (IC 50 ) is 6.6 µM , and the Hill coefficient (h) is 1.1 ; for Kir6.2-R201H SUR1 , IC 50 is 245 µM and h is 2
For the 1:1 mixture , the line is the best fit to the following equation: a(1÷[1+ ([ATP]÷245 µM) 2 ]) +(1¡a)(1÷[1+([ATP]÷IC 50 ) h ]) , where [ATP] is the ATP concentration , IC 50 is 7.6 µM , h is 1.6 , and a (the fraction of the homomeric R201H channels) is 0.04
The most common mutation (resulting in the R201H substitution) occurs within a CpG dinucleotide , a 'hot spot' for mutations in mammalian genes
This probably explains the recurrent finding of the R201H mutation in unrelated families from different countries
Functional analysis of the most common mutation , R201H , showed that the homozygous mutation led to markedly reduced sensitivity to ATP
Of note , one patient with an R201H mutation (the proband's father in family BR 1) , who had always been treated with tolbutamide , had C-peptide levels in the normal range and good glycemic control
Reference #4 (Vaxillaire M et al.): R201H
We identified in nine patients seven heterozygous nonsynonymous mutations: three of them (V59M , R201C , and R201H) were already described , and the four novel mutations resulted in an amino acid change of Kir6.2 at positions F35L , G53N , E322K , and Y330C
Three of them (V59M , R201C , and R201H) have been previously described (5 ) , and the four novel mutations resulted in amino acid substitutions at the following positions: F35L , G53N , E322K , and Y330C (Table 1 )
The R201H mutation was found in three unrelated probands and occurred at a highly conserved codon , supporting a critical role for residue 201 in the channel function and ATP binding (5 , 8 )
Reference #5 (Edghill EL et al.): R201H
The amino acid position R201 has been reported as the most common mutated residue (R201H) in KCNJ11 PNDM subjects
Reference #6 (Proks P et al.): R201H
Two mutations in the same residue (R201H and R201C) , which lies in the putative ATP-binding site (7 , 8 ) , cause mild disease
However , all patients identified to date are heterozygotes , and no obvious loss of ATP sensitivity was observed when the heterozygous state was simulated by coinjection of R201H and wild-type Kir6.2 (4 )
In this paper , we compare the functional effects of mutations associated with severe disease (Q52R and V59G) with those that cause mild disease (R201C and R201H)
In previous studies using the mouse Kir6.2 clone , we were unable to detect a significant change in the ATP sensitivity of the heterozygous channel population with the R201H mutation (4 )
We therefore tested the effect of the R201H mutation in the human Kir6.2 clone
We found that ATP produced a half-maximal block of homomeric R201H channels at 298 ± 25 µM (n = 5) , and of the heterozygous channel population at 12.5 ± 1.1 µM (n = 5)
Because [ATP] i is unlikely to fall below 0.1 mM in beta cells , even when extracellular glucose is low (12 ) , the difference in wild-type and mutant K ATP current at this (and higher) ATP concentration probably explains why the R201C and R201H mutations result in neonatal diabetes
This may explain why mutations that produce a small decrease in the ATP sensitivity of heterozygous channels (R201C and R201H) result in neonatal diabetes alone , whereas those causing a greater reduction (Q52R and V59G) are associated with severe disease
Because subjects with mild mutations , such as R201H , do not show any insulin increment with i.v
However , consistent with this idea , R201H subjects do produce insulin in response to sulfonylureas (4 , 6 ) , but no insulin increment was seen in either of the two patients with neurological features who have been tested (E
The results we have described thus far predict that the second mutation at V59 (V59M) , which is also associated with severe disease , will have an increased intrinsic open probability , whereas the R201H mutation , which , like R201C , causes PNDM alone , will not
Consistent with this idea , the P O(0) of homV59M channels was 0.83 ± 0.1 (n = 5) , significantly different from wild-type (P > 0.001) , whereas there was no significant difference in the P O(0) between wild-type and R201H channels
Reference #7 (Tammaro P et al.): R201H
Consistent with this idea , the mutation R201H (which causes PNDM alone) produces a reduction in the ATP sensitivity of the KATP channel , and a larger resting KATP current when expressed in heterologous systems (Gloyn et al , 2004 )
Thus , the I296L mutation associated with DEND syndrome (Proks et al , 2005 ) causes a larger resting current than mutations that produce neonatal diabetes alone (R201H ; Gloyn et al , 2005 ) or cause transient neonatal diabetes (G35S / R , I182V ; Gloyn et al , 2005 )
At 3 mM ATP , which lies with the range found in pancreatic beta-cells in the presence of glucose (Detimary et al , 1995 ) , the K ATP current amplitude was 32 , 8 , 4 , 6 and 5% of that in the absence of nucleotide for I296L , R201H , G35S , G35R and I182V , respectively (Gloyn et al , 2005 ; Proks et al , 2005 )
2005, KChannelDB.
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