KChannelDB: Extraction of mutation data from the literature

This data was extracted from Medline abstracts and full texts (when available) in an automated manner.

The table below describes the selected point mutation and provides links to other documents. The sentence(s) where the point mutation Y330L was found are listed after the table.

The mutated residues are also indicated in the family sequence alignments and hyperlinked to the corresponding mutation pages.

Point mutation Y330L in IRK11_HUMAN

Relevant sentences:

Y330

• 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 Y330C mutation reduced ATP inhibition both directly , by impairing ATP binding (and / or transduction) , and indirectly , by stabilizing the intrinsic open state of the 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 )

• Patients with the Y330C mutation presented with blood glucose levels of 2270 mM within 5 weeks of birth

• 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 1A shows that significant resting whole-cell K ATP currents were present in oocytes expressing homomeric (hom) Y330C channels

• 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

• Resting hetY330C currents were greater than wild-type but much less than for homY330C channels (Table IA and Figure 1A )

• Like homY330C channels , however , they were further activated by azide , to approximately the same amplitude as wild-type channels

• 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

• (a) Kir6.2 / SUR1 (open symbols , n = 10) , heterozygous (semifilled symbols , n = 6) and homomeric (filled symbols , n = 11) Kir6.2-Y330C / 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

• However , the Y330C mutation was associated with a marked increase in intrinsic P o(0) (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

• To explore this question further , we used a simple kinetic model of the hetY330C channel (Proks et al , 2004 )

• We assumed that the energy of the open state is simply the sum of the contributions of the four individual subunits in the channel (G 0 , j): (image) The IC50 of channels containing i mutant and 4-i wild-type subunits can be estimated as (for further details , see Proks et al , 2004 ) (image) where IC50 , wt is the IC50 measured for wild-type channels , IC50 , i is the IC50 for channels formed from i (0Y330C mutation

• 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)

• We next examined the single-channel kinetics of Kir6.2DeltaC-Y330C , expressed in the absence of SUR1

• Homomeric mutant channels displayed a very low sensitivity to MgATP , the IC50 for inhibition of homY330C channels being 2.8 mM

• 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

• (a) Kir6.2 / SUR1 (open symbols , n = 6) , heterozygous (semifilled symbols , n = 6) and homomeric (filled symbols , n = 7) Kir6.2-Y330C / SUR1 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 )

• 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 )

• 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

• In the case of hetY330C , however , the IC50 for MgATP inhibition is intermediate between that of homY330C and wild type (Table IA ): thus , it is likely that in this case fewer Kir6.2 subunits must be mutated in order for MgATP to stimulate channel activity

• 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 )

• Residues Y330 and F333 are shown in red

• The ability of the Y330C mutation to affect the ATP sensitivity of Kir6.2DeltaC , without altering gating , is consistent with its predicted proximity to I182

• Interestingly , mutation of Y330 to leucine was without effect on the ATP sensitivity of Kir6.2DeltaC (Antcliff et al , 2005 )

• How the Y330C mutation influences gating when SUR1 is present remains unclear

• However , the region of the C-domain in which Y330 resides lies very close to the N-domain of the adjacent Kir6.2 subunit

• The fact that the Y330C mutation only affects gating when SUR1 is present suggests that Y330 might lie close to SUR1 in the three-dimensional structure of the octameric K ATP channel complex , and is consistent with its predicted location on the outer surface of Kir6.2 (Figure 9A )

• Mutation of Y330 to cysteine impaired ATP binding / transduction , but the mechanism appears to differ from that of F333I

• Simulating an ensemble of Kir6.2 tetramers containing a binomial mixture of wild-type and mutant subunits exhibiting free energy additivity in their effects of gating produced an IC50 for hetY330C channels of 43 muM , which is more than twice that observed experimentally (20 muM)

• One possible explanation for this discrepancy is that the subunits do not assemble according to a perfect binomial distribution , and that there is a reduced number of Y330C subunits in the heterozygous population

• However , the reduced ATP sensitivity of Kir6.2DeltaC-Y330C channels argues that there may also be a direct effect on ATP binding / transduction at Kir6.2

• It is noteworthy that the decrease in ATP sensitivity observed for Y330C (compared to the wild type) is less for Kir6.2DeltaC (four-fold) than for full-length Kir6.2 coexpressed with SUR1 (20-fold)

• Consistent with this idea , Kir6.2DeltaC-Y330C channels had normal gating , whereas the P o(0) of Kir6.2-Y330C / SUR1 channels was increased

• The four-fold shift in the ATP sensitivity of Kir6.2DeltaC can be attributed solely to changes in ATP binding / transduction ; however , it is not correct to attribute all of the remaining shift in the ATP sensitivity of Kir6.2-Y330C / SUR1 to the observed changes in gating , as additional effects might also be involved

• 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

• This may explain why two out of the three patients with the Y330C mutation exhibited neurological problems

• 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

• This leads to an enhanced K ATP current , which inhibits insulin secretion , and in the case of the Y330C mutation may also reduce electrical activity in extrapancreatic tissues

F.Horn (kchanneldbcmbi.ru.nl), 17-Aug-2005