Reference #1 (Tammaro P et al.): F333
- We tested the functional effects of two Kir6.2 mutations (Y330C , F333I) that cause permanent neonatal diabetes mellitus , by heterologous expression in Xenopus oocytes
- The F333I mutation altered ATP binding / transduction directly
- This effect was particularly dramatic for the Kir6.2-F333I mutation , and was abolished by SUR1 mutations that prevent MgATP binding / hydrolysis
- Further analysis of F333I heterozygous channels indicated that at least three SUR1 must bind / hydrolyse MgATP to open the mutant K ATP channel
- In this paper , we analyse the functional effects of the Y330C and F333I mutations , identified in Caucasian families from Norway , France and the USA (Sagen et al , 2004 ; Vaxillaire et al , 2004 )
- The patient with the F333I mutation presented with neonatal diabetes within 10 weeks of birth but had no additional symptoms (Sagen et al , 2004 )
- We show here that the Y330C and F333I mutations both impair the ability of ATP to block the K ATP channel (at Kir6.2) and enhance channel activation by MgATP (via SUR1)
- In the presence of 1 mM MgATP , the K ATP current is larger for heterozygous Y330C channels than for heterozygous F333I channels , and much greater than for wild-type channels , consistent with the difference in clinical phenotypes
- We analyse the molecular basis of the reduction in ATP inhibition and demonstrate that it is primarily due to impaired ATP binding / transduction (F333I) and / or secondary to a change in channel gating (Y330C)
- Results Effects on whole-cell currents We analysed the effects of the Y330C and F333I mutations on the metabolic regulation of the K ATP channel by the two-electrode voltage-clamp method
- Figure 1 Figure 1 (A) Whole-cell currents recorded from Xenopus oocytes coexpressing SUR1 and either Kir6.2 , Kir6.2-F333I or Kir6.2-Y330C , as indicated , in response to voltage steps of plusminus20 mV from a holding potential of -10 mV
- Table 1 Table 1 Mean data for wild-type and mutant Kir6.2 / SUR1 and Kir6.2DeltaC channels The results of similar experiments with hetF333I and homF333I channels are also shown in Figure 1
- Resting currents for homF333I channels were substantially larger , and those for hetF333I channels slightly larger , than wild-type channels (Table IA )
- Consistent with the less severe phenotype , hetF333I resting currents were smaller than those of hetY330C channels
- Both homF333I and homY330C channels were much less sensitive to ATP , being half maximally blocked (IC50) by 168 and 211 muM ATP , respectively , compared with 11 muM for wild-type channels (Table IA )
- Heterozygous channels were also significantly less sensitive to ATP , with IC50 equal to 20 muM (hetY330C) and 23 muM (hetF333I) (Figures 2A and B and Table IA )
- Figure 2 Figure 2 (A) Currents recorded in inside-out patches excised from Xenopus oocytes coexpressing SUR1 and either wild type Kir6.2 , Kir6.2-F333I or Kir6.2-Y330C , as indicated , in response to 3 s voltage ramps from -110 to +100 mV
- (b) Kir6.2 / SUR1 (open symbols , n = 10) , heterozygous (semifilled symbols , n = 5) , and homomeric (filled symbols , n = 6) Kir6.2-F333I / SUR1 channels
- As Figure 3 shows , the ATP sensitivity of Kir6.2DeltaC , expressed in the absence of SUR1 , was also impaired by the F333I and Y330C mutations , the IC50 being 124 , 6450 and 480 muM for wild-type , homF333I and homY330C channels , respectively (Table IB )
- This suggests that the altered ATP sensitivity of F333I and Y330C channels is , at least in part , intrinsic to Kir6.2 rather than due to impaired regulation by SUR1
- Figure 3 Figure 3 Mean relationship between [ATP] and K ATP conductance (G) , expressed relative to the conductance in the absence of nucleotide (G c) for Kir6.2DeltaC (circles , n = 7) , Kir6.2DeltaC-F333I (squares , n = 5) and Kir6.2DeltaC-Y330C (triangles , n = 6) channels
- We therefore examined the effect of the Y330C and F333I mutations on single-channel currents in the absence of ATP , where intrinsic gating can be assessed
- In the case of F333I channels , the open probability in the absence of ligand (P o(0)) was also unaffected (Figure 4 and Table IIA )
- This suggests that whereas the F333I mutation impairs ATP binding / transduction , the Y330C mutation exerts its effect on ATP sensitivity both directly , via changes in ATP binding / transduction , and indirectly , via an increase in P o(0)
- Table 2 Table 2 Mean single-channel data for wild-type and mutant Kir6.2 / SUR1 and Kir6.2DeltaC channels Figure 4 Figure 4 Single-channel currents recorded at -60 mV from inside-out membrane patches excised from oocytes expressing Kir6.2 / SUR1 , Kir6.2-F333I / SUR1 , Kir6.2-Y330C / SUR1 , Kir6.2DeltaC and Kir6.2DeltaC-Y330C
- Figure 5 Figure 5 Simulated ATP dose-inhibition curves (dashed lines) for heteromeric Kir6.2-Y330C / SUR1 (left) and Kir6.2-F333I / SUR1 (right) channels
- The predicted IC50 values are 43 muM (Kir6.2-Y330C / SUR1) and 25 muM (Kir6.2-F333I / SUR1)
- According to this model , the predicted IC50 , H for F333I is 25 muM , which is very close to the experimentally determined value of 23 muM (Figure 5 )
- In striking contrast , MgATP caused a dramatic stimulation of homF333I channels (Figure 6A )
- However , both heterozygous Y330C and F333I channels were blocked by MgATP: the IC50 for the hetY330C channel (116 muM) was about seven-fold greater than wild type (16 muM) , and that for the hetF333I channel (39 muM) was about 2.5-fold greater
- Figure 6 Figure 6 (A) Currents recorded in inside-out patches excised from Xenopus oocytes coexpressing SUR1 and either wild type , Kir6.2-F333I or Kir6.2-Y330C as indicated , in response to voltage ramps from -110 to +100 mV
- (b) Kir6.2 / SUR1 (open symbols , n = 6) and heterozygous Kir6.2-F333I / SUR1 (semifilled symbols , n = 6) channels
- At 1 mM ATP , this amounted to 4 , 10 and 20% of the maximal current for wild-type , hetF333I and hetY330C channels , respectively (Table IA )
- It is obvious that the ATP sensitivity of homF333I channels is dramatically less in the presence of Mg2+ than in the absence of the cation (compare Figures 2 and 6 ; see also Supplementary Figure 1 )
- The ATP sensitivity of homY330C channels is also reduced by Mg2+ (the IC50 increased approx17-fold ; Supplementary Figure 2B ) , although to a lesser extent than homF333I channels
- Interestingly , in the heterozygous condition , the shift in the IC50 for ATP inhibition produced by Mg2+ is greater for Y330C channels (approx6-fold) than for F333I channels (approx1.5-fold ; Supplementary Figures 2C and D )
- No difference in ATP sensitivity in the absence and presence of 2 mM Mg2+ was observed for Kir6.2DeltaC channels (Gribble et al , 1998 ) or Kir6.2DeltaC-F333I channels expressed in the absence of SUR1
- ATP (10 mM) blocked Kir6.2DeltaC-F333I currents by 70.2plusminus2% in the absence , and 71.1plusminus1% in the presence , of 2 mM Mg2+ (n = 4)
- Whichever explanation is correct , the F333I mutation must markedly enhance the stimulatory effect (of PIP2 and / or SUR1) on the channel and the Y330C mutation must do so to a lesser extent
- We have focused our analysis on the F333I mutation , as it has a much greater effect
- Activatory effect of F333I mutation in the presence of MgATP To determine if the stimulatory influence of SUR1 on Kir6.2 is modified by the F333I mutation , we coexpressed Kir6.2-F333I with a mutant form of SUR1 that does not support channel activation (Gribble et al , 1997b , 1998 )
- Figure 7A compares the effect of 1 mM MgATP on F333I channels containing either wild-type SUR1 (Figure 7Aa ) or SUR1-KA / KM (Figure 7Ab )
- The nucleotide produced a gradual increase in Kir6.2-F333I / SUR1 currents , of approximately five-fold
- In contrast , MgATP produced an immediate and substantial block of Kir6.2-F333I / SUR1-KA / KM currents , which was followed by a small time-dependent increase in current
- Figure 7 Figure 7 (A) Currents recorded in inside-out patches excised from Xenopus oocytes coexpressing Kir6.2-F333I and either SUR1 (a) or SUR1-KA / KM (b) in response to voltage ramps from -110 to +100 mV
- (B) Mean slope conductance (G) , expressed relative to the conductance in the absence of nucleotide (G c) , plotted against time , for Kir6.2-F333I / SUR1 (open circles , n = 5) and Kir6.2-F333I / SUR1-KA / KM (filled circles , n = 7)
- (C) Mean slope conductance (G) , expressed relative to the conductance in the absence of nucleotide (G c) , plotted against time for Kir6.2-F333I / SUR1-KA / KM
- (D) Currents recorded in inside-out patches excised from Xenopus oocytes coexpressing Kir6.2-F333I and SUR1 in response to voltage ramps from -110 to +100 mV
- The small time-dependent increase in Kir6.2-F333I / SUR1-KA / KM currents observed in the maintained presence of MgATP was prevented by 10 muM LY294002 (Figure 7C )
- The drug (10 muM) had no effect Kir6.2 / SUR1 or Kir6.2-F333I / SUR1-KA / KM currents when applied in the absence of ATP (n = 3) (Supplementary Figure 3 )
- This suggests that the very small activation of Kir6.2-F333I / SUR1-KA / KM channels produced by MgATP results from generation of PIP3
- The time course of activation of Kir6.2-F333I / SUR1 currents in the presence of MgATP is quite slow (Figure 7B )
- Another explanation for the greater stimulation of Kir6.2-F333I / SUR1 currents by MgATP is that the interaction between SUR1 and mutant Kir6.2-F333I subunits results in a conformational change in the MgATP-binding site on SUR1 that now allows MgATP to act directly as a positive channel modulator (rather than via hydrolysis to MgADP)
- Neither compound supported channel activation: Kir6.2-F333I / SUR1 currents were blocked by 45plusminus5% (n = 5) and 62plusminus2% (n = 6) with 1 mM AMP-PCP or AMP-PNP , respectively (Supplementary Figure 4 )
- It is evident that the F333I mutation produces a marked increase in the ability of MgATP to activate the homomeric mutant channel
- This effect is not due to a reduced ability of ATP to block Kir6.2 when Mg2+ is present , as MgATP and ATP blocked Kir6.2DeltaC-F333I channels with similar potency
- Instead , enhanced activation of homF333I channels involves MgATP binding / hydrolysis at the NBDs of SUR1 , as it was abolished when Kir6.2-F333I was coexpressed with SUR1-KA / KM
- MgADP activated homF333I channels , but the rate of activation was about 20-fold faster than for MgATP: it is possible that this reflects the fact that MgATP has to be hydrolysed to MgADP (by NBD2 of SUR1) in order to stimulate channel activity
- The fact that hetF333I channels were blocked , rather than strongly activated , by MgATP suggests that several (probably all four) Kir6.2 subunits must be mutated in order for MgATP to stimulate channel activity dramatically , and that heteromeric channels behave like wild type with regard to MgATP activation
- Because MgATP activation is conferred by SUR1 , which must interact with Kir6.2 to open the pore , these data argue that the F333I mutation influences the interaction of SUR1 with Kir6.2
- Activation of three or four SUR1 subunits is needed to open hetF333I K ATP channels The F333I mutation is the first Kir6.2 mutation reported to influence channel activation by Mg-nucleotides without affecting intrinsic gating
- Figure 8A shows the mixed population of channel types expected when wild-type and F333I Kir6.2 subunits are coexpressed with wild-type SUR1 , assuming they distribute randomly
- If activation of all four SUR1 subunits was required to open the channel , then only about 1 / 16 of channels in the heterozygous state would exhibit marked activation on exposure to MgATP (i.e. those containing four F333I subunits)
- On the other hand , if only one SUR must be activated to open the channel , then 15 / 16 channels will show the enhanced channel activation associated with the F333I subunit
- The fact that hetF333I channels are blocked rather than activated by MgATP (Figure 6 ) favours the former hypothesis
- (B) Schematic of the different channel types expected when wild-type and mutant SUR1 are coexpressed with Kir6.2-F333I (as in the heterozygous state)
- (C) Mean slope conductance (G) , expressed relative to the conductance in the absence of nucleotide (G c) , plotted against time , for the heterozygous mixture of channel types that occur when Kir6.2-F333I is coexpressed with both SUR1 and SUR1-KA / KM subunits (semifilled circles , n = 9)
- The blue line indicates the Kir6.2-F333I / SUR1 data and the pink line the Kir6.2-F333I / SUR1-KAKM data (same data as in Figure 7B)
- To test this idea further , we coexpressed Kir6.2-F333I with an equal amount of wild-type SUR1 and SUR1-KA / KM
- As Figure 8C shows , 1 mM MgATP substantially blocked Kir6.2-F333I / hetSUR1-KA / KM channels
- Our data indicate that (at least for channels containing Kir6.2-F333I mutant subunits) in order for MgATP to open the Kir6.2 pore , three or four SUR1 subunits probably must be activated (i.e. bind / hydrolyse MgATP) and transduce a conformational change to each Kir6.2 subunit in the tetramer
- It is interesting that F333I markedly enhances MgADP activation but does not affect gating , whereas Y330C produces a smaller effect on MgADP activation and also alters the ability of SUR1 to influence channel gating
- Discussion Both F333I and Y330C mutations result in a marked decrease in the sensitivity of the KATP channel to inhibition by ATP
- However , they appear to do so by somewhat different molecular mechanisms , with F333I causing impaired ATP binding / transduction , whereas Y330C both impairs ATP binding / transduction and also influences ATP inhibition indirectly , via an increase in intrinsic Po
- In addition , both mutations enhance MgATP activation by SUR1 , with F333I having a greater effect in the homomeric state and Y330C in the heterozygous state
- As Table I shows , in the absence of Mg2+ , both hetY330C and F333I channels are blocked to a similar extent by ATP , whereas in the presence of Mg2+ , ATP blocks hetF333I channels more strongly
- Structural considerations In a homology model of Kir6.2 (Antcliff et al , 2005 ) , residues Y330 and F333 both lie within the outer mouth of the ATP-binding pocket (Figure 9 )
- In particular , F333 sits within 3.0 A of the alpha phosphate of ATP (Trapp et al , 2003 ; Antcliff et al , 2005 )
- Previous studies have shown that mutation of F333 to leucine also strongly reduced K ATP channel inhibition (Antcliff et al , 2005 )
- Residues R201 , L181 , V202 and P254 also lie within 3.0 A of F333
- Homology modelling thus supports the idea that the F333I mutation interferes with ATP binding
- Residues Y330 and F333 are shown in red
- In our homology model , residue 330 lies within 3.0 A of several residues , including F333 and L181 in the C-terminal domain (C-domain) of the same subunit and H46 in the N-terminal domain (N-domain) of the adjacent subunit
- Molecular basis of reduced ATP sensitivity (in the absence of Mg2+) The Po(0) of homomeric channels containing the F333I mutation was not significantly different from wild type
- Since ATP binding to a single subunit is sufficient to close the channel and the number of mutant subunits in the tetramer follows the binomial distribution (Shyng and Nichols , 1997 ; Markworth et al , 2000 ) , F333I channels will exhibit a markedly reduced ATP sensitivity only when all four subunits are mutant (i.e. in about 1 / 16 of channels in the heterozygous state)
- Binomial analysis using the IC50 for wild-type and F333I subunits predicts an IC50 of 25 muM for hetF333I channels , close to that observed experimentally (23 muM)
- This is consistent with the F333I mutation (in the absence of Mg2+) principally affecting ATP binding
- The reduced ATP sensitivity observed for Kir6.2DeltaC-F333I channels is also harmonious with this idea
- Mutation of Y330 to cysteine impaired ATP binding / transduction , but the mechanism appears to differ from that of F333I
- Physiological implications Both F333I and Y330C mutations cause an increase in the K ATP current observed at 2 mM MgATP , a concentration close to that found in oocytes following metabolic poisoning (Gribble et al , 1997a , 2000 )
- It is noteworthy that in the presence of 1 mM MgATP , hetY330C currents are approximately twice as large as hetF333I currents , and the whole-cell currents are also correspondingly larger
- Values for Y330C and F333I were 10 and 3.5% , respectively
- Conclusion Analysis of two PNDM mutations , F333I and Y330C , demonstrates that the severity of the disease phenotype is correlated with a reduced sensitivity to MgATP
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Reference #1 (Tammaro P et al.): F333I
- We tested the functional effects of two Kir6.2 mutations (Y330C , F333I) that cause permanent neonatal diabetes mellitus , by heterologous expression in Xenopus oocytes
- The F333I mutation altered ATP binding / transduction directly
- This effect was particularly dramatic for the Kir6.2-F333I mutation , and was abolished by SUR1 mutations that prevent MgATP binding / hydrolysis
- Further analysis of F333I heterozygous channels indicated that at least three SUR1 must bind / hydrolyse MgATP to open the mutant K ATP channel
- In this paper , we analyse the functional effects of the Y330C and F333I mutations , identified in Caucasian families from Norway , France and the USA (Sagen et al , 2004 ; Vaxillaire et al , 2004 )
- The patient with the F333I mutation presented with neonatal diabetes within 10 weeks of birth but had no additional symptoms (Sagen et al , 2004 )
- We show here that the Y330C and F333I mutations both impair the ability of ATP to block the K ATP channel (at Kir6.2) and enhance channel activation by MgATP (via SUR1)
- In the presence of 1 mM MgATP , the K ATP current is larger for heterozygous Y330C channels than for heterozygous F333I channels , and much greater than for wild-type channels , consistent with the difference in clinical phenotypes
- We analyse the molecular basis of the reduction in ATP inhibition and demonstrate that it is primarily due to impaired ATP binding / transduction (F333I) and / or secondary to a change in channel gating (Y330C)
- Results Effects on whole-cell currents We analysed the effects of the Y330C and F333I mutations on the metabolic regulation of the K ATP channel by the two-electrode voltage-clamp method
- Figure 1 Figure 1 (A) Whole-cell currents recorded from Xenopus oocytes coexpressing SUR1 and either Kir6.2 , Kir6.2-F333I or Kir6.2-Y330C , as indicated , in response to voltage steps of plusminus20 mV from a holding potential of -10 mV
- Table 1 Table 1 Mean data for wild-type and mutant Kir6.2 / SUR1 and Kir6.2DeltaC channels The results of similar experiments with hetF333I and homF333I channels are also shown in Figure 1
- Resting currents for homF333I channels were substantially larger , and those for hetF333I channels slightly larger , than wild-type channels (Table IA )
- Consistent with the less severe phenotype , hetF333I resting currents were smaller than those of hetY330C channels
- Both homF333I and homY330C channels were much less sensitive to ATP , being half maximally blocked (IC50) by 168 and 211 muM ATP , respectively , compared with 11 muM for wild-type channels (Table IA )
- Heterozygous channels were also significantly less sensitive to ATP , with IC50 equal to 20 muM (hetY330C) and 23 muM (hetF333I) (Figures 2A and B and Table IA )
- Figure 2 Figure 2 (A) Currents recorded in inside-out patches excised from Xenopus oocytes coexpressing SUR1 and either wild type Kir6.2 , Kir6.2-F333I or Kir6.2-Y330C , as indicated , in response to 3 s voltage ramps from -110 to +100 mV
- (b) Kir6.2 / SUR1 (open symbols , n = 10) , heterozygous (semifilled symbols , n = 5) , and homomeric (filled symbols , n = 6) Kir6.2-F333I / SUR1 channels
- As Figure 3 shows , the ATP sensitivity of Kir6.2DeltaC , expressed in the absence of SUR1 , was also impaired by the F333I and Y330C mutations , the IC50 being 124 , 6450 and 480 muM for wild-type , homF333I and homY330C channels , respectively (Table IB )
- This suggests that the altered ATP sensitivity of F333I and Y330C channels is , at least in part , intrinsic to Kir6.2 rather than due to impaired regulation by SUR1
- Figure 3 Figure 3 Mean relationship between [ATP] and K ATP conductance (G) , expressed relative to the conductance in the absence of nucleotide (G c) for Kir6.2DeltaC (circles , n = 7) , Kir6.2DeltaC-F333I (squares , n = 5) and Kir6.2DeltaC-Y330C (triangles , n = 6) channels
- We therefore examined the effect of the Y330C and F333I mutations on single-channel currents in the absence of ATP , where intrinsic gating can be assessed
- In the case of F333I channels , the open probability in the absence of ligand (P o(0)) was also unaffected (Figure 4 and Table IIA )
- This suggests that whereas the F333I mutation impairs ATP binding / transduction , the Y330C mutation exerts its effect on ATP sensitivity both directly , via changes in ATP binding / transduction , and indirectly , via an increase in P o(0)
- Table 2 Table 2 Mean single-channel data for wild-type and mutant Kir6.2 / SUR1 and Kir6.2DeltaC channels Figure 4 Figure 4 Single-channel currents recorded at -60 mV from inside-out membrane patches excised from oocytes expressing Kir6.2 / SUR1 , Kir6.2-F333I / SUR1 , Kir6.2-Y330C / SUR1 , Kir6.2DeltaC and Kir6.2DeltaC-Y330C
- Figure 5 Figure 5 Simulated ATP dose-inhibition curves (dashed lines) for heteromeric Kir6.2-Y330C / SUR1 (left) and Kir6.2-F333I / SUR1 (right) channels
- The predicted IC50 values are 43 muM (Kir6.2-Y330C / SUR1) and 25 muM (Kir6.2-F333I / SUR1)
- According to this model , the predicted IC50 , H for F333I is 25 muM , which is very close to the experimentally determined value of 23 muM (Figure 5 )
- In striking contrast , MgATP caused a dramatic stimulation of homF333I channels (Figure 6A )
- However , both heterozygous Y330C and F333I channels were blocked by MgATP: the IC50 for the hetY330C channel (116 muM) was about seven-fold greater than wild type (16 muM) , and that for the hetF333I channel (39 muM) was about 2.5-fold greater
- Figure 6 Figure 6 (A) Currents recorded in inside-out patches excised from Xenopus oocytes coexpressing SUR1 and either wild type , Kir6.2-F333I or Kir6.2-Y330C as indicated , in response to voltage ramps from -110 to +100 mV
- (b) Kir6.2 / SUR1 (open symbols , n = 6) and heterozygous Kir6.2-F333I / SUR1 (semifilled symbols , n = 6) channels
- At 1 mM ATP , this amounted to 4 , 10 and 20% of the maximal current for wild-type , hetF333I and hetY330C channels , respectively (Table IA )
- It is obvious that the ATP sensitivity of homF333I channels is dramatically less in the presence of Mg2+ than in the absence of the cation (compare Figures 2 and 6 ; see also Supplementary Figure 1 )
- The ATP sensitivity of homY330C channels is also reduced by Mg2+ (the IC50 increased approx17-fold ; Supplementary Figure 2B ) , although to a lesser extent than homF333I channels
- Interestingly , in the heterozygous condition , the shift in the IC50 for ATP inhibition produced by Mg2+ is greater for Y330C channels (approx6-fold) than for F333I channels (approx1.5-fold ; Supplementary Figures 2C and D )
- No difference in ATP sensitivity in the absence and presence of 2 mM Mg2+ was observed for Kir6.2DeltaC channels (Gribble et al , 1998 ) or Kir6.2DeltaC-F333I channels expressed in the absence of SUR1
- ATP (10 mM) blocked Kir6.2DeltaC-F333I currents by 70.2plusminus2% in the absence , and 71.1plusminus1% in the presence , of 2 mM Mg2+ (n = 4)
- Whichever explanation is correct , the F333I mutation must markedly enhance the stimulatory effect (of PIP2 and / or SUR1) on the channel and the Y330C mutation must do so to a lesser extent
- We have focused our analysis on the F333I mutation , as it has a much greater effect
- Activatory effect of F333I mutation in the presence of MgATP To determine if the stimulatory influence of SUR1 on Kir6.2 is modified by the F333I mutation , we coexpressed Kir6.2-F333I with a mutant form of SUR1 that does not support channel activation (Gribble et al , 1997b , 1998 )
- Figure 7A compares the effect of 1 mM MgATP on F333I channels containing either wild-type SUR1 (Figure 7Aa ) or SUR1-KA / KM (Figure 7Ab )
- The nucleotide produced a gradual increase in Kir6.2-F333I / SUR1 currents , of approximately five-fold
- In contrast , MgATP produced an immediate and substantial block of Kir6.2-F333I / SUR1-KA / KM currents , which was followed by a small time-dependent increase in current
- Figure 7 Figure 7 (A) Currents recorded in inside-out patches excised from Xenopus oocytes coexpressing Kir6.2-F333I and either SUR1 (a) or SUR1-KA / KM (b) in response to voltage ramps from -110 to +100 mV
- (B) Mean slope conductance (G) , expressed relative to the conductance in the absence of nucleotide (G c) , plotted against time , for Kir6.2-F333I / SUR1 (open circles , n = 5) and Kir6.2-F333I / SUR1-KA / KM (filled circles , n = 7)
- (C) Mean slope conductance (G) , expressed relative to the conductance in the absence of nucleotide (G c) , plotted against time for Kir6.2-F333I / SUR1-KA / KM
- (D) Currents recorded in inside-out patches excised from Xenopus oocytes coexpressing Kir6.2-F333I and SUR1 in response to voltage ramps from -110 to +100 mV
- The small time-dependent increase in Kir6.2-F333I / SUR1-KA / KM currents observed in the maintained presence of MgATP was prevented by 10 muM LY294002 (Figure 7C )
- The drug (10 muM) had no effect Kir6.2 / SUR1 or Kir6.2-F333I / SUR1-KA / KM currents when applied in the absence of ATP (n = 3) (Supplementary Figure 3 )
- This suggests that the very small activation of Kir6.2-F333I / SUR1-KA / KM channels produced by MgATP results from generation of PIP3
- The time course of activation of Kir6.2-F333I / SUR1 currents in the presence of MgATP is quite slow (Figure 7B )
- Another explanation for the greater stimulation of Kir6.2-F333I / SUR1 currents by MgATP is that the interaction between SUR1 and mutant Kir6.2-F333I subunits results in a conformational change in the MgATP-binding site on SUR1 that now allows MgATP to act directly as a positive channel modulator (rather than via hydrolysis to MgADP)
- Neither compound supported channel activation: Kir6.2-F333I / SUR1 currents were blocked by 45plusminus5% (n = 5) and 62plusminus2% (n = 6) with 1 mM AMP-PCP or AMP-PNP , respectively (Supplementary Figure 4 )
- It is evident that the F333I mutation produces a marked increase in the ability of MgATP to activate the homomeric mutant channel
- This effect is not due to a reduced ability of ATP to block Kir6.2 when Mg2+ is present , as MgATP and ATP blocked Kir6.2DeltaC-F333I channels with similar potency
- Instead , enhanced activation of homF333I channels involves MgATP binding / hydrolysis at the NBDs of SUR1 , as it was abolished when Kir6.2-F333I was coexpressed with SUR1-KA / KM
- MgADP activated homF333I channels , but the rate of activation was about 20-fold faster than for MgATP: it is possible that this reflects the fact that MgATP has to be hydrolysed to MgADP (by NBD2 of SUR1) in order to stimulate channel activity
- The fact that hetF333I channels were blocked , rather than strongly activated , by MgATP suggests that several (probably all four) Kir6.2 subunits must be mutated in order for MgATP to stimulate channel activity dramatically , and that heteromeric channels behave like wild type with regard to MgATP activation
- Because MgATP activation is conferred by SUR1 , which must interact with Kir6.2 to open the pore , these data argue that the F333I mutation influences the interaction of SUR1 with Kir6.2
- Activation of three or four SUR1 subunits is needed to open hetF333I K ATP channels The F333I mutation is the first Kir6.2 mutation reported to influence channel activation by Mg-nucleotides without affecting intrinsic gating
- Figure 8A shows the mixed population of channel types expected when wild-type and F333I Kir6.2 subunits are coexpressed with wild-type SUR1 , assuming they distribute randomly
- If activation of all four SUR1 subunits was required to open the channel , then only about 1 / 16 of channels in the heterozygous state would exhibit marked activation on exposure to MgATP (i.e. those containing four F333I subunits)
- On the other hand , if only one SUR must be activated to open the channel , then 15 / 16 channels will show the enhanced channel activation associated with the F333I subunit
- The fact that hetF333I channels are blocked rather than activated by MgATP (Figure 6 ) favours the former hypothesis
- (B) Schematic of the different channel types expected when wild-type and mutant SUR1 are coexpressed with Kir6.2-F333I (as in the heterozygous state)
- (C) Mean slope conductance (G) , expressed relative to the conductance in the absence of nucleotide (G c) , plotted against time , for the heterozygous mixture of channel types that occur when Kir6.2-F333I is coexpressed with both SUR1 and SUR1-KA / KM subunits (semifilled circles , n = 9)
- The blue line indicates the Kir6.2-F333I / SUR1 data and the pink line the Kir6.2-F333I / SUR1-KAKM data (same data as in Figure 7B)
- To test this idea further , we coexpressed Kir6.2-F333I with an equal amount of wild-type SUR1 and SUR1-KA / KM
- As Figure 8C shows , 1 mM MgATP substantially blocked Kir6.2-F333I / hetSUR1-KA / KM channels
- Our data indicate that (at least for channels containing Kir6.2-F333I mutant subunits) in order for MgATP to open the Kir6.2 pore , three or four SUR1 subunits probably must be activated (i.e. bind / hydrolyse MgATP) and transduce a conformational change to each Kir6.2 subunit in the tetramer
- It is interesting that F333I markedly enhances MgADP activation but does not affect gating , whereas Y330C produces a smaller effect on MgADP activation and also alters the ability of SUR1 to influence channel gating
- Discussion Both F333I and Y330C mutations result in a marked decrease in the sensitivity of the KATP channel to inhibition by ATP
- However , they appear to do so by somewhat different molecular mechanisms , with F333I causing impaired ATP binding / transduction , whereas Y330C both impairs ATP binding / transduction and also influences ATP inhibition indirectly , via an increase in intrinsic Po
- In addition , both mutations enhance MgATP activation by SUR1 , with F333I having a greater effect in the homomeric state and Y330C in the heterozygous state
- As Table I shows , in the absence of Mg2+ , both hetY330C and F333I channels are blocked to a similar extent by ATP , whereas in the presence of Mg2+ , ATP blocks hetF333I channels more strongly
- Homology modelling thus supports the idea that the F333I mutation interferes with ATP binding
- Molecular basis of reduced ATP sensitivity (in the absence of Mg2+) The Po(0) of homomeric channels containing the F333I mutation was not significantly different from wild type
- Since ATP binding to a single subunit is sufficient to close the channel and the number of mutant subunits in the tetramer follows the binomial distribution (Shyng and Nichols , 1997 ; Markworth et al , 2000 ) , F333I channels will exhibit a markedly reduced ATP sensitivity only when all four subunits are mutant (i.e. in about 1 / 16 of channels in the heterozygous state)
- Binomial analysis using the IC50 for wild-type and F333I subunits predicts an IC50 of 25 muM for hetF333I channels , close to that observed experimentally (23 muM)
- This is consistent with the F333I mutation (in the absence of Mg2+) principally affecting ATP binding
- The reduced ATP sensitivity observed for Kir6.2DeltaC-F333I channels is also harmonious with this idea
- Mutation of Y330 to cysteine impaired ATP binding / transduction , but the mechanism appears to differ from that of F333I
- Physiological implications Both F333I and Y330C mutations cause an increase in the K ATP current observed at 2 mM MgATP , a concentration close to that found in oocytes following metabolic poisoning (Gribble et al , 1997a , 2000 )
- It is noteworthy that in the presence of 1 mM MgATP , hetY330C currents are approximately twice as large as hetF333I currents , and the whole-cell currents are also correspondingly larger
- Values for Y330C and F333I were 10 and 3.5% , respectively
- Conclusion Analysis of two PNDM mutations , F333I and Y330C , demonstrates that the severity of the disease phenotype is correlated with a reduced sensitivity to MgATP
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2005, KChannelDB.
cmbi.ru.nl), 17-Aug-2005