reactions were analyzed on an abi 3730 capillary sequencer ( applied biosystems , warrington , u.k . ) .
sequences were compared with the reference sequences nm_000525 and nm_000352.2 , which incorporate the alternatively spliced residue in exon 17 ( l78208 , l78224 ) , using mutation surveyor 3.20 software ( softgenetics , state college , pa ) .
mutation testing was performed on parental dna extracted from peripheral leukocytes , and microsatellite analysis was used to confirm family relationships .
human kir6.2 ( genbank nm000525 ; e23 and i337 ) and rat sur1 ( genbank l40624 ) were used .
site - directed mutagenesis , synthesis of capped mrna , and preparation of xenopus laevis oocytes were performed as reported ( 33 ) .
oocytes were co - injected with 4 ng of wild - type or mutant sur1 mrna and 0.8 ng kir6.2 mrna , incubated in barth s solution and studied 14 days after injection . to simulate the heterozygous state ,
sur1 was coexpressed with a 1:1 mixture of wild - type and mutant kir6.2 . for each batch of oocytes ,
all homomeric or heterozygous mutations were injected to enable direct comparison of their effects and to control for batch - to - batch variation in expression levels ( see supplementary data ) .
whole - cell currents were recorded using a two - electrode voltage clamp in response to voltage steps of 20 mv from a holding potential of 10 mv , in a solution containing ( in mmol / l ) 90 kcl , 1 mgcl2 , 1.8 cacl2 , and 5 hepes ( ph 7.4 with koh ) ( 33 ) .
metabolic inhibition was induced by 3 mmol / l sodium azide , the katp channel opener diazoxide ( 340
mol / l ) was used to fully activate the channels , and tolbutamide ( 0.5
macroscopic currents were recorded from giant inside - out patches at 60 mv ; inward currents are shown as upward deflections of the current trace .
the pipette ( external ) solution contained ( in mmol / l ) 140 kcl , 1.2 mgcl2 , 2.6 cacl2 , and 10 hepes ( ph 7.4 with koh ) .
the intracellular solution contained ( in mmol / l ) 107 kcl , 11 egta , 2 mgcl2 , 1 cacl2 , and 10 hepes ( ph 7.2 with koh ) , as well as nucleotides as indicated .
the mg - free intracellular solution contained ( in mmol / l ) 107 kcl , 1 k2so4 , 10 egta , and 10 hepes ( ph 7.2 with koh ) , as well as nucleotides as indicated .
atp concentration - inhibition curves were constructed by alternating control and test solutions . to control for possible rundown , or activation by mgatp
, the conductance in the test solution was expressed as the mean of that in control solution before and after nucleotide application .
atp concentration - response curves were fit with : g / gc = 1/{1+[(atp)/ic50 ] } ( eq .
1 ) , where ( atp ) is the atp concentration , ic50 is the atp concentration at which inhibition is half maximal , and h is the slope factor . in some experiments ( figs
. 6 and 7 ) , we used a conditioning prepulse of variable [ mgatp ] , followed by a test pulse of mgatp .
current during the test pulse was expressed as a fraction of that in nucleotide - free solution .
the single - coding exon of kcnj11 and the 39 exons of abcc8 were amplified and sequenced as described ( 7 ) .
reactions were analyzed on an abi 3730 capillary sequencer ( applied biosystems , warrington , u.k . ) .
sequences were compared with the reference sequences nm_000525 and nm_000352.2 , which incorporate the alternatively spliced residue in exon 17 ( l78208 , l78224 ) , using mutation surveyor 3.20 software ( softgenetics , state college , pa ) .
mutation testing was performed on parental dna extracted from peripheral leukocytes , and microsatellite analysis was used to confirm family relationships .
human kir6.2 ( genbank nm000525 ; e23 and i337 ) and rat sur1 ( genbank l40624 ) were used .
site - directed mutagenesis , synthesis of capped mrna , and preparation of xenopus laevis oocytes were performed as reported ( 33 ) .
oocytes were co - injected with 4 ng of wild - type or mutant sur1 mrna and 0.8 ng kir6.2 mrna , incubated in barth s solution and studied 14 days after injection . to simulate the heterozygous state , sur1 was coexpressed with a 1:1 mixture of wild - type and mutant kir6.2 . for each batch of oocytes ,
all homomeric or heterozygous mutations were injected to enable direct comparison of their effects and to control for batch - to - batch variation in expression levels ( see supplementary data ) .
whole - cell currents were recorded using a two - electrode voltage clamp in response to voltage steps of 20 mv from a holding potential of 10 mv , in a solution containing ( in mmol / l ) 90 kcl , 1 mgcl2 , 1.8 cacl2 , and 5 hepes ( ph 7.4 with koh ) ( 33 ) .
metabolic inhibition was induced by 3 mmol / l sodium azide , the katp channel opener diazoxide ( 340
mol / l ) was used to fully activate the channels , and tolbutamide ( 0.5 mmol / l ) was used to block katp channels .
macroscopic currents were recorded from giant inside - out patches at 60 mv ; inward currents are shown as upward deflections of the current trace . the pipette ( external ) solution contained ( in mmol / l ) 140 kcl , 1.2 mgcl2 , 2.6 cacl2 , and 10 hepes ( ph 7.4 with koh ) .
the intracellular solution contained ( in mmol / l ) 107 kcl , 11 egta , 2 mgcl2 , 1 cacl2 , and 10 hepes ( ph 7.2 with koh ) , as well as nucleotides as indicated .
the mg - free intracellular solution contained ( in mmol / l ) 107 kcl , 1 k2so4 , 10 egta , and 10 hepes ( ph 7.2 with koh ) , as well as nucleotides as indicated .
to control for possible rundown , or activation by mgatp , the conductance in the test solution was expressed as the mean of that in control solution before and after nucleotide application .
atp concentration - response curves were fit with : g / gc = 1/{1+[(atp)/ic50 ] } ( eq .
1 ) , where ( atp ) is the atp concentration , ic50 is the atp concentration at which inhibition is half maximal , and h is the slope factor . in some experiments ( figs . 6 and 7 ) , we used a conditioning prepulse of variable [ mgatp ] , followed by a test pulse of mgatp .
current during the test pulse was expressed as a fraction of that in nucleotide - free solution .
we identified novel heterozygous abcc8 mutations , e1507d ( c.4521g > t ) and e1507 g ( c.4520a > g ) , in two male probands .
this numbering refers to the l78208 human abcc8 isoform that encodes 1,582 amino acids and contains an additional residue in nbd1 ; consequently , e1507 in our sequence is equivalent to e1506 in that of huopio et al .
proband 1 with the e1507d mutation weighed 2.9 kg at birth ( 42 weeks gestation ) and was diagnosed as insulin - dependent at 10 weeks . at 30 weeks , the diabetes remitted and insulin treatment was withdrawn .
proband 2 weighed 3.5 kg at birth ( 40 weeks gestation ) and was diagnosed with diabetes at the age of 8 weeks . after an e1507 g mutation was identified , the patient was successfully transferred from insulin to glibenclamide at the age of 6 months .
he was treated with glibenclamide ( 2.5 mg , once daily ) for 4 months , at which time the diabetes remitted .
a family history of diabetes had not been reported for either proband , and testing of parental samples demonstrated that the e1507d and e1507 g mutations had each arisen de novo in the proband .
we analyzed the effects of mutations at e1506 of rat sur1 ( equivalent to e1507 in the l78208 human abcc8 isoform ) on the metabolic regulation of the katp channel by measuring whole - cell currents .
wild - type ( kir6.2-sur1 ) channels expressed in xenopus oocytes are normally closed , due to the high intracellular atp concentration ( [ atp]i ) , but can be opened by lowering [ atp]i using a metabolic inhibitor , such as sodium azide ( fig .
the subsequent addition of the katp channel - opener diazoxide ( 0.34 mmol / l ) increased the current only slightly , suggesting wild - type katp channels are almost fully activated by 3 mmol / l sodium azide .
tolbutamide ( 0.5 mmol / l ) almost completely blocked the current , confirming it flows through katp channels .
c : representative whole - cell current amplitudes evoked by repeated voltage steps from 10 to 30 mv for wild - type ( a , wt ) , and homomeric ( b , home1506d ) or heterozygous ( c , hete1506d ) kir6.2/sur1-e1506d channels .
the bars indicate application of 3 mmol / l azide , 0.34 mmol / l diazoxide ( dz ) , and 0.5 mmol / l tolbutamide ( tb ) .
scale bars are 0.5 a ( y - axis ) and 400 s ( x - axis ) .
d and e : mean steady - state whole - cell katp current amplitudes for wild - type homomeric ( d ) and heterozygous ( e ) mutant channels .
currents were evoked by a voltage step from 10 to 30 mv before ( control ,  ) and after (  ) application of 3 mmol / l azide , in the presence of 3 mmol / l azide plus 0.34 mmol
/ l dz (  ) , and 3 azide + 0.5 mmol / l tb (  ) for wt and sur1 mutant channels , as indicated .
* p  0.05 , * * p  0.01 , * * * p  0.001 against wt ( student t test ) .
mutations that reduce the channel atp sensitivity normally increase the whole - cell current in the absence of metabolic inhibition , reflecting the fact that they are less blocked by resting [ atp]i .
a small but significant increase in resting current was observed for both homomeric kir6.2/sur1-e1506d ( home1506d ) and kir6.2/sur1-e1506 g ( home1506 g ) channels ( fig .
these currents were only slightly enhanced by metabolic inhibition but were further increased by diazoxide , suggesting azide - induced changes in nucleotide concentrations may be insufficient to fully activate the mutant channels . as previously reported ( 27 ) , kir6.2/sur1-e1506k ( home1506k ) currents were minimal in both the absence and presence of azide but were slightly increased by diazoxide .
the resting currents of heterozygous ( het ) e1506d and hete1506 g channels were significantly greater than wild - type channels , which may explain why these mutations cause neonatal diabetes .
azide strongly activated all heterozygous channels , and in the additional presence of diazoxide there was a further increase .
the maximal hete1506d current was not significantly different from the wild - type current , and that of het1506 g channels was only slightly smaller .
these results suggest that hete1506d and hete1506 g channels retain metabolic sensitivity and that their functional expression is similar to wild - type channels .
the maximal amplitude of hete1506k channels was reduced , as previously reported ( 27 ) .
tolbutamide ( 500 mol / l ) blocked hete1506d currents by 95  1% ( n
= 8) , hete1506 g currents by 95  1% ( n = 6 ) , and e1506k currents by 91  2% ( n = 6 ) compared with 96  1% ( n = 7 ) for wild - type channels . in the cell - attached configuration , the home1506d and home1506 g currents were both larger than wild - type or home1506k currents , consistent with the whole - cell resting currents ( supplementary fig
however , no significant difference was found between wild - type and mutant hete1506 channels .
there was no difference in the amplitude of wild - type and home1506k katp currents recorded after patch excision ( table 1 , supplementary fig .
2b ) , indicating the smaller amplitude of home1506k whole - cell currents is not attributable to reduced channel expression .
however , there was no difference in hete1506 g currents and a < 50% decrease in het1506d currents ( table 1 , supplementary fig .
this suggests that the neonatal diabetes mutations have little ( e1506d ) or no ( e1506 g ) effect on expression levels of the katp channel in the heterozygous state . to determine the molecular mechanism of action of e1506 mutations , we studied homomeric channels because the single population of channels simplifies the analysis .
atp sensitivity of wild - type and mutant channels data are mean  sem ; n = number of patches .
excised patch currents were measured immediately after excision into nucleotide - free solution p  0.05 , all against wild type ( student t test ) .
there was a slight reduction in mgatp block for home1506d and home1506 g channels but no effect for home1506k channels ( fig .
d ) ; the concentration of atp producing half - maximal inhibition ( ic50 ) was 21 mol / l for home1506d , 19 mol / l for home1506 g , 12 mol / l for home1506k , and 13 mol / l for wild - type ( e1506 ) channels ( table 1 ) .
the reduced atp sensitivity of neonatal diabetes channels may account for the increased resting whole - cell currents produced by these mutations .
a : representative inside - out patch currents recorded in response to mgatp ( 100 mol / l ) for channels composed of wt kir6.2 and wt or mutant sur1 , as indicated . the dashed lines
b : mean relationship between katp current ( i ) , expressed relative to that in the absence of nucleotide ( ic ) , and atp concentration in the presence ( b  d ) or absence ( e  g ) of 2 mmol / l mg for wt (  , - - - ) and mutant (  ,  ) channels .
1 , with the following parameters ( in mol / l for ic50 ) : b  d : wild - type ( n = 23 ) , ic50 = 12.2 , h = 1.05 ; e  g : wt ( n = 7 ) , ic50 = 8.3 , h = 1.09 .
b : kir6.2/sur1-e1506d ( n = 11 ) , ic50 = 20.1 , h = 1.05 .
c : kir6.2/sur1-e1506 g ( n = 10 ) , ic50 = 18.8 , h = 0.93 .
d : kir6.2/sur1-e1506k ( n = 11 ) , ic50 = 11.0 , h = 1.14 .
e : kir6.2/sur1-e1506d ( n = 7 ) , ic50 = 8.2 , h = 1.00 .
f : kir6.2/sur1-e1506 g ( n = 7 ) , ic50 = 6.9 , h = 0.86 .
g : sur1-e1506k ( n = 7 ) , ic50 = 6.4 , h = 1.00 .
this allows the effects of a mutation on atp inhibition at kir6.2 to be isolated from its action on mg - nucleotide stimulation at sur1 because atp does not interact with sur1 in the absence of mg ( 34 ) .
none of the home1506 mutant channels showed changes in atp sensitivity in the absence of mg ( fig .
g , table 1 ) , suggesting that the altered mgatp sensitivity of the home1506d and home1506 g channels is not a consequence of a reduced atp block at kir6.2 but may arise from enhanced mg - nucleotide activation at sur1 .
the lack of an effect of the home1506k mutation on channel inhibition by atp is in agreement with previous studies ( 27 ) .
as is well established , katp currents decline in amplitude after patch excision into nucleotide - free solution ( 35 ) .
activation was quantified by expressing the current in the presence of mgadp as a fraction of that in the control solution before nucleotide application .
wild - type katp currents were increased 3.5-fold by 100 mol / l mgadp ( fig . 3a and b ) . by contrast
, mgadp blocked all three mutant channels : by 77% for home1506k , 55% for home1506d , and 42% for home1506 g channels .
qualitatively similar results were observed in the presence of 100 mol / l mgatp ( fig .
the inability of mgadp to activate the home1506k channels in the presence or absence of atp has been reported previously ( 27 ) and probably underlies the failure of these channels to open upon metabolic inhibition .
a : representative inside - out patch currents recorded in response to application of mgadp ( 100 mol / l ) for channels composed of wt kir6.2 and wt or mutant sur1 , as indicated . the dashed lines
/ l mgadp activates wt - sur1 channels but blocks all the mutant e1506 channels .
b and c : mgadp activation of wt or mutant kir6.2/sur1-e1506 channels , as indicated .
b : katp current ( i ) in the presence of 100 mol / l mgadp is expressed relative to the current in the absence of nucleotides ( ic ) .
c : katp current ( i ) in the presence of 100 mol / l mgadp and 100 mol / l mgatp is expressed relative to the current in the presence of 100 mol / l mgatp alone ( iatp ) . *
p  0.05 , * * p  0.01 , * * * p  0.001 against wild type ( student t test ) .
previous studies have shown that in the absence of mg , 100 mol / l adp blocks wild - type katp channels by 60% ( 16 ) by binding to the inhibitory site on kir6.2 .
the fact that home1506d and home1506 g channels were blocked < 60% by 100 mol / l mgadp suggests that mgadp may retain some small stimulatory effect on these channels but that this is masked by inhibition at kir6.2 . to assess
if this is correct , we examined the effect of mgadp on a kir6.2-g334d background .
the g334d mutation abolishes block by atp ( 36 ) and thus allows the effects of mg - nucleotides on sur1 to be studied in isolation from their effects at kir6.2 .
kir6.2-g334d / sur1 ( g334d / e1506 ) channels were highly active in the on - cell configuration ( fig .
no increase in current was observed for the kir6.2-g334d / sur1-e1506d ( g334d / e1506d ) or kir6.2-g334d / sur1-e1506 g ( g334d / e1506 g ) channels ; in contrast , the kir6.2-g334d / sur1-e1506k ( g334d / e1506k ) currents increased 1.5-fold on excision .
this suggests that the g334d / e1506 and g334d / e1506k channels were partially blocked under resting conditions in the oocyte , whereas the g334d / e1506d and g334d / e1506 g channels were fully open .
larger amplitudes were found for the g334d / e1506 ( 8.5  1.9 na , n = 16 ) and g334d / e1506k ( 14  1.9 na , n = 9 ) currents than for the g334d / e1506d ( 1.0  0.2 na , n = 11 ) or g334d / e1506 g ( 0.8  0.2 na , n = 13 ) currents , again suggesting that the e1506d and e1506 g mutations may impair surface expression in the homomeric state . mgadp and mgatp activation of sur1-e1506 mutations on a kir6.2-g334d background .
mgadp and mgatp activation of katp channels composed of kir6.2-g334d and wt or mutant sur1 , as indicated .
a : the maximal katp current after patch excision is expressed relative to that in the cell - attached patch .
/ l mgadp ( b ) or 100 mol / l mgatp ( c ) is expressed relative to the current in the absence of nucleotides ( ic ) . *
p  0.05 , * * p  0.01 , * * * p  0.001 against wild type ( student t test ) .
mgadp ( 100 mol / l ) activated g334d / e1506 channels approximately fivefold ( fig . 4b , supplementary fig .
3 ) presumably reflects a lack of mgadp block at kir6.2 when g334 is mutated .
activation of e1506 mutant channels was greatly reduced , but not completely abolished , with all three channels showing an 1.5-fold increase in current in 100 mol / l mgadp .
interestingly , although mgatp ( 100 mol / l ) activated g334d / e1506 channels about as effectively as mgadp ( 100 mol / l ) , it was far more effective than mgadp at stimulating all three double - mutant channels ; mgatp increased the mutant currents 3-fold compared with 1.5-fold for mgadp ( fig . 4c , supplementary fig .
the difference in the extent of activation of wild - type and neonatal diabetes channels was not significant .
the time course of channel activation by mgatp and mgadp was also affected by mutations at e1506 of sur1 .
activation of g334d / e1506 channels could be fitted by one exponential with a time constant ( on ) of 1 s for mgadp and 2.5 s for mgatp ( fig .
in contrast , the time constant of activation by both mgadp and mgatp was increased to 20 s for all three sur1 mutant channels .
channels were composed of kir6.2-g334d and wild - type ( wt ) or mutant sur1 subunits , as indicated .
time constant of current activation ( on ) by 100 mol / l mgadp ( a ) or 100 mol / l mgatp ( b ) is shown .
time constant of current deactivation ( off ) after removal of 100 mol / l mgadp ( c ) or 100 mol / l mgatp ( d ) is shown . *
p  0.05 * * p  0.01 , * * * p  0.001 against wild - type , ( student t test ) .
the wt trace is interrupted to align the time point of atp removal with that of the e1506d trace .
the time constant of deactivation ( off ) for the g334d / e1506 channels was 3 s for mgadp and 5 s for mgatp ( fig .
5c and d ) . the off - rate of mgadp was not significantly different for the g334d / e1506k channels but was slower for the g334d / e1506d and g334d / e1506 g channels , with a off of 10 and 11 s , respectively .
the off - rate of mgatp was significantly less for all three mutant channels , with a off of 8.5 s for g334d / e1506k , 16 s for g334d / e1506 g , and 67 s for g334d / e1506d .
these results suggest that mgatp is far more efficient than mgadp at activating the e1506 mutant channels .
we explored this possibility further using channels composed of wild - type kir6.2 and wild - type or mutant sur1 .
we measured the atp concentration - inhibition curve after a 30-s exposure to 10 mmol / l mgatp ( fig .
such atp preconditioning reduced the atp sensitivity of the two neonatal diabetic mutant channels but had little or no effect on wild - type or e1506k channels , respectively ( table 1 ) . as a consequence , the ic50 for the atp block of the homomeric and heterozygous e1506d and e1506 g channels was significantly greater than wild - type , whereas that of hete1506k was no different , and that of home1506k was actually slightly smaller ( fig .
preconditioning pulses of > 300 mol / l mgatp markedly reduced the ability of 100 mol / l mgatp to block e1506d and e1506 g channels but had only a small effect on wild - type channels and no effect on the home1506k channels .
atp sensitivity of wild - type and mutant channels after preconditioning with 10 mmol / l mgatp .
a : representative home1506d currents recorded in response to different mgatp concentrations ( as indicated ) , preceded by a preconditioning pulse in 10 mmol / l mgatp solution ( indicated by the unlabeled solid line [  ] ) and followed by control ( nucleotide - free ) solution . the dashed lines ( - - - ) indicate the zero current .
b  d : mean relationship between mgatp concentration and katp current ( i ) , expressed relative to that in the absence of nucleotide ( ic ) , after 30-s preincubation in 10 mmol / l mgatp for wt (  , n = 9 ) , heterozygous (  ) , and homomeric mutant (  ) channels .
channels were composed of kir6.2 and wild - type or mutant sur1 subunits , as indicated .
c : home1506 g ( n = 7 ) , hete1506 g ( n = 7 ) .
d : home1506k ( n = 7 ) , hete1506k ( n = 5 ) .
1 , with the following parameters ( in mol / l for ic50 ) : wild - type , ic50 = 28 , h = 1.2 ; home1506d , ic50 = 78 , h = 1.2 ; hete1506d , ic50 = 45 , h = 1.2 ; home1506 g , ic50 = 105 , h = 1.3 ; hete1506 g , ic50 = 45 , h = 1.3 ; home1506k , ic50 = 18 , h = 1.6 ; hete1506k , ic50 = 26 , h = 1.4 .
a : representative home1506 g currents recorded in response to test pulses of 100 mol / l mgatp ( unlabeled solid line [  ] ) , preceded by a preconditioning pulse to a variable mgatp concentration ( 10 mol / l to 10 mmol / l , as indicated ) and followed by control ( nucleotide - free ) solution .
b : mgatp concentration during the conditioning prepulse plotted against the current during a 100 mol / l mgatp test pulse .
current is expressed as a fraction of that in control ( nucleotide - free ) solution after the test pulse for wild - type (  , n = 14 ) , home1506d (  , n = 15 ) , home1506 g (  , n = 10 ) , and home1506k (  , n = 8) channels . the lines are drawn through the points by eye .
pre - exposure to 10 mmol / l mgadp , which fully blocked the wild - type and mutant channels , did not affect the ability of 100 mol / l mgatp to block any of the four channel types ( data not shown ) .
we identified novel heterozygous abcc8 mutations , e1507d ( c.4521g > t ) and e1507 g ( c.4520a > g ) , in two male probands .
this numbering refers to the l78208 human abcc8 isoform that encodes 1,582 amino acids and contains an additional residue in nbd1 ; consequently , e1507 in our sequence is equivalent to e1506 in that of huopio et al .
proband 1 with the e1507d mutation weighed 2.9 kg at birth ( 42 weeks gestation ) and was diagnosed as insulin - dependent at 10 weeks . at 30 weeks , the diabetes remitted and insulin treatment was withdrawn .
proband 2 weighed 3.5 kg at birth ( 40 weeks gestation ) and was diagnosed with diabetes at the age of 8 weeks . after an e1507 g mutation was identified , the patient was successfully transferred from insulin to glibenclamide at the age of 6 months .
he was treated with glibenclamide ( 2.5 mg , once daily ) for 4 months , at which time the diabetes remitted .
a family history of diabetes had not been reported for either proband , and testing of parental samples demonstrated that the e1507d and e1507 g mutations had each arisen de novo in the proband .
we analyzed the effects of mutations at e1506 of rat sur1 ( equivalent to e1507 in the l78208 human abcc8 isoform ) on the metabolic regulation of the katp channel by measuring whole - cell currents .
wild - type ( kir6.2-sur1 ) channels expressed in xenopus oocytes are normally closed , due to the high intracellular atp concentration ( [ atp]i ) , but can be opened by lowering [ atp]i using a metabolic inhibitor , such as sodium azide ( fig .
the subsequent addition of the katp channel - opener diazoxide ( 0.34 mmol / l ) increased the current only slightly , suggesting wild - type katp channels are almost fully activated by 3 mmol / l sodium azide .
tolbutamide ( 0.5 mmol / l ) almost completely blocked the current , confirming it flows through katp channels .
c : representative whole - cell current amplitudes evoked by repeated voltage steps from 10 to 30 mv for wild - type ( a , wt ) , and homomeric ( b , home1506d ) or heterozygous ( c , hete1506d ) kir6.2/sur1-e1506d channels .
the bars indicate application of 3 mmol / l azide , 0.34 mmol / l diazoxide ( dz ) , and 0.5 mmol / l tolbutamide ( tb ) .
scale bars are 0.5 a ( y - axis ) and 400 s ( x - axis ) .
d and e : mean steady - state whole - cell katp current amplitudes for wild - type homomeric ( d ) and heterozygous ( e ) mutant channels .
currents were evoked by a voltage step from 10 to 30 mv before ( control ,  ) and after (  ) application of 3 mmol / l azide , in the presence of 3 mmol / l azide plus 0.34 mmol / l dz (  ) , and 3 azide + 0.5 mmol / l tb (  ) for wt and sur1 mutant channels , as indicated .
* p  0.05 , * * p  0.01 , * * * p  0.001 against wt ( student t test ) .
mutations that reduce the channel atp sensitivity normally increase the whole - cell current in the absence of metabolic inhibition , reflecting the fact that they are less blocked by resting [ atp]i .
a small but significant increase in resting current was observed for both homomeric kir6.2/sur1-e1506d ( home1506d ) and kir6.2/sur1-e1506 g ( home1506 g ) channels ( fig .
these currents were only slightly enhanced by metabolic inhibition but were further increased by diazoxide , suggesting azide - induced changes in nucleotide concentrations may be insufficient to fully activate the mutant channels . as previously reported ( 27 ) , kir6.2/sur1-e1506k ( home1506k ) currents were minimal in both the absence and presence of azide but were slightly increased by diazoxide .
the resting currents of heterozygous ( het ) e1506d and hete1506 g channels were significantly greater than wild - type channels , which may explain why these mutations cause neonatal diabetes .
azide strongly activated all heterozygous channels , and in the additional presence of diazoxide there was a further increase .
the maximal hete1506d current was not significantly different from the wild - type current , and that of het1506 g channels was only slightly smaller .
these results suggest that hete1506d and hete1506 g channels retain metabolic sensitivity and that their functional expression is similar to wild - type channels .
the maximal amplitude of hete1506k channels was reduced , as previously reported ( 27 ) .
tolbutamide ( 500 mol / l ) blocked hete1506d currents by 95  1% ( n = 8) , hete1506 g currents by 95  1% ( n = 6 ) , and e1506k currents by 91  2% ( n = 6 ) compared with 96  1% ( n = 7 ) for wild - type channels .
in the cell - attached configuration , the home1506d and home1506 g currents were both larger than wild - type or home1506k currents , consistent with the whole - cell resting currents ( supplementary fig .
however , no significant difference was found between wild - type and mutant hete1506 channels .
there was no difference in the amplitude of wild - type and home1506k katp currents recorded after patch excision ( table 1 , supplementary fig .
2b ) , indicating the smaller amplitude of home1506k whole - cell currents is not attributable to reduced channel expression .
in contrast , home1506d and home1506 g currents were both substantially smaller . however , there was no difference in hete1506 g currents and a < 50% decrease in het1506d currents ( table 1 , supplementary fig .
this suggests that the neonatal diabetes mutations have little ( e1506d ) or no ( e1506 g ) effect on expression levels of the katp channel in the heterozygous state . to determine the molecular mechanism of action of e1506 mutations , we studied homomeric channels because the single population of channels simplifies the analysis .
atp sensitivity of wild - type and mutant channels data are mean  sem ; n = number of patches .
excised patch currents were measured immediately after excision into nucleotide - free solution p  0.05 , all against wild type ( student t test ) .
there was a slight reduction in mgatp block for home1506d and home1506 g channels but no effect for home1506k channels ( fig .
d ) ; the concentration of atp producing half - maximal inhibition ( ic50 ) was 21 mol / l for home1506d , 19 mol / l for home1506 g , 12 mol / l for home1506k , and 13 mol / l for wild - type ( e1506 ) channels ( table 1 ) .
the reduced atp sensitivity of neonatal diabetes channels may account for the increased resting whole - cell currents produced by these mutations .
a : representative inside - out patch currents recorded in response to mgatp ( 100 mol / l ) for channels composed of wt kir6.2 and wt or mutant sur1 , as indicated . the dashed lines
b : mean relationship between katp current ( i ) , expressed relative to that in the absence of nucleotide ( ic ) , and atp concentration in the presence ( b  d ) or absence ( e  g ) of 2 mmol / l mg for wt (  , - - - ) and mutant (  ,  ) channels .
1 , with the following parameters ( in mol / l for ic50 ) : b  d : wild - type ( n = 23 ) , ic50 = 12.2 , h = 1.05 ; e  g : wt ( n = 7 ) , ic50 = 8.3 , h = 1.09 .
b : kir6.2/sur1-e1506d ( n = 11 ) , ic50 = 20.1 , h = 1.05 .
c : kir6.2/sur1-e1506 g ( n = 10 ) , ic50 = 18.8 , h = 0.93 .
d : kir6.2/sur1-e1506k ( n = 11 ) , ic50 = 11.0 , h = 1.14 .
e : kir6.2/sur1-e1506d ( n = 7 ) , ic50 = 8.2 , h = 1.00 .
f : kir6.2/sur1-e1506 g ( n = 7 ) , ic50 = 6.9 , h = 0.86 .
g : sur1-e1506k ( n = 7 ) , ic50 = 6.4 , h = 1.00 .
this allows the effects of a mutation on atp inhibition at kir6.2 to be isolated from its action on mg - nucleotide stimulation at sur1 because atp does not interact with sur1 in the absence of mg ( 34 ) .
none of the home1506 mutant channels showed changes in atp sensitivity in the absence of mg ( fig .
g , table 1 ) , suggesting that the altered mgatp sensitivity of the home1506d and home1506 g channels is not a consequence of a reduced atp block at kir6.2 but may arise from enhanced mg - nucleotide activation at sur1 .
the lack of an effect of the home1506k mutation on channel inhibition by atp is in agreement with previous studies ( 27 ) .
as is well established , katp currents decline in amplitude after patch excision into nucleotide - free solution ( 35 ) .
activation was quantified by expressing the current in the presence of mgadp as a fraction of that in the control solution before nucleotide application .
wild - type katp currents were increased 3.5-fold by 100 mol / l mgadp ( fig . 3a and b ) . by contrast
, mgadp blocked all three mutant channels : by 77% for home1506k , 55% for home1506d , and 42% for home1506 g channels .
qualitatively similar results were observed in the presence of 100 mol / l mgatp ( fig .
the inability of mgadp to activate the home1506k channels in the presence or absence of atp has been reported previously ( 27 ) and probably underlies the failure of these channels to open upon metabolic inhibition .
a : representative inside - out patch currents recorded in response to application of mgadp ( 100 mol / l ) for channels composed of wt kir6.2 and wt or mutant sur1 , as indicated . the dashed lines
note that 100 mol / l mgadp activates wt - sur1 channels but blocks all the mutant e1506 channels .
b and c : mgadp activation of wt or mutant kir6.2/sur1-e1506 channels , as indicated .
b : katp current ( i ) in the presence of 100 mol / l mgadp is expressed relative to the current in the absence of nucleotides ( ic ) .
c : katp current ( i ) in the presence of 100 mol / l mgadp and 100 mol / l mgatp is expressed relative to the current in the presence of 100 mol / l mgatp alone ( iatp ) . *
p  0.05 , * * p  0.01 , * * * p  0.001 against wild type ( student t test ) .
previous studies have shown that in the absence of mg , 100 mol / l adp blocks wild - type katp channels by 60% ( 16 ) by binding to the inhibitory site on kir6.2 .
the fact that home1506d and home1506 g channels were blocked < 60% by 100 mol / l mgadp suggests that mgadp may retain some small stimulatory effect on these channels but that this is masked by inhibition at kir6.2 . to assess
if this is correct , we examined the effect of mgadp on a kir6.2-g334d background .
the g334d mutation abolishes block by atp ( 36 ) and thus allows the effects of mg - nucleotides on sur1 to be studied in isolation from their effects at kir6.2 .
kir6.2-g334d / sur1 ( g334d / e1506 ) channels were highly active in the on - cell configuration ( fig .
no increase in current was observed for the kir6.2-g334d / sur1-e1506d ( g334d / e1506d ) or kir6.2-g334d / sur1-e1506 g ( g334d / e1506 g ) channels ; in contrast , the kir6.2-g334d / sur1-e1506k ( g334d / e1506k ) currents increased 1.5-fold on excision .
this suggests that the g334d / e1506 and g334d / e1506k channels were partially blocked under resting conditions in the oocyte , whereas the g334d / e1506d and g334d / e1506 g channels were fully open .
larger amplitudes were found for the g334d / e1506 ( 8.5  1.9 na , n = 16 ) and g334d / e1506k ( 14  1.9 na , n = 9 ) currents than for the g334d / e1506d ( 1.0  0.2 na , n = 11 ) or g334d / e1506 g
( 0.8  0.2 na , n = 13 ) currents , again suggesting that the e1506d and e1506 g mutations may impair surface expression in the homomeric state . mgadp and mgatp activation of sur1-e1506 mutations on a kir6.2-g334d background .
mgadp and mgatp activation of katp channels composed of kir6.2-g334d and wt or mutant sur1 , as indicated .
a : the maximal katp current after patch excision is expressed relative to that in the cell - attached patch .
katp current ( i ) in the presence of 100 mol / l mgadp ( b ) or 100 mol / l mgatp ( c ) is expressed relative to the current in the absence of nucleotides ( ic ) .
* p  0.05 , * * p  0.01 , * * * p  0.001 against wild type ( student t test ) .
mgadp ( 100 mol / l ) activated g334d / e1506 channels approximately fivefold ( fig .
3 ) presumably reflects a lack of mgadp block at kir6.2 when g334 is mutated .
activation of e1506 mutant channels was greatly reduced , but not completely abolished , with all three channels showing an 1.5-fold increase in current in 100 mol / l mgadp .
interestingly , although mgatp ( 100 mol / l ) activated g334d / e1506 channels about as effectively as mgadp ( 100 mol / l ) , it was far more effective than mgadp at stimulating all three double - mutant channels ; mgatp increased the mutant currents 3-fold compared with 1.5-fold for mgadp ( fig . 4c , supplementary fig .
the difference in the extent of activation of wild - type and neonatal diabetes channels was not significant .
the time course of channel activation by mgatp and mgadp was also affected by mutations at e1506 of sur1 .
activation of g334d / e1506 channels could be fitted by one exponential with a time constant ( on ) of 1 s for mgadp and 2.5 s for mgatp ( fig .
in contrast , the time constant of activation by both mgadp and mgatp was increased to 20 s for all three sur1 mutant channels .
channels were composed of kir6.2-g334d and wild - type ( wt ) or mutant sur1 subunits , as indicated .
time constant of current activation ( on ) by 100 mol / l mgadp ( a ) or 100 mol / l mgatp ( b ) is shown .
p  0.05 * * p  0.01 , * * * p  0.001 against wild - type , ( student t test ) .
e : representative current traces for wt and kir6.2/sur1-e1506d ( e1506d ) channels . the horizontal bar indicates the duration of application of 100 mol / l mgatp .
the wt trace is interrupted to align the time point of atp removal with that of the e1506d trace .
the time constant of deactivation ( off ) for the g334d / e1506 channels was 3 s for mgadp and 5 s for mgatp ( fig .
5c and d ) . the off - rate of mgadp was not significantly different for the g334d / e1506k channels but was slower for the g334d / e1506d and g334d / e1506 g channels , with a off of 10 and 11 s , respectively .
the off - rate of mgatp was significantly less for all three mutant channels , with a off of 8.5 s for g334d / e1506k , 16 s for g334d / e1506 g , and 67 s for g334d / e1506d .
these results suggest that mgatp is far more efficient than mgadp at activating the e1506 mutant channels .
we explored this possibility further using channels composed of wild - type kir6.2 and wild - type or mutant sur1 .
we measured the atp concentration - inhibition curve after a 30-s exposure to 10 mmol / l mgatp ( fig .
such atp preconditioning reduced the atp sensitivity of the two neonatal diabetic mutant channels but had little or no effect on wild - type or e1506k channels , respectively ( table 1 ) .
as a consequence , the ic50 for the atp block of the homomeric and heterozygous e1506d and e1506 g channels was significantly greater than wild - type , whereas that of hete1506k was no different , and that of home1506k was actually slightly smaller ( fig .
preconditioning pulses of > 300 mol / l mgatp markedly reduced the ability of 100 mol / l mgatp to block e1506d and e1506 g channels but had only a small effect on wild - type channels and no effect on the home1506k channels .
atp sensitivity of wild - type and mutant channels after preconditioning with 10 mmol / l mgatp .
a : representative home1506d currents recorded in response to different mgatp concentrations ( as indicated ) , preceded by a preconditioning pulse in 10 mmol / l mgatp solution ( indicated by the unlabeled solid line [  ] ) and followed by control ( nucleotide - free ) solution .
the dashed lines ( - - - ) indicate the zero current . b  d : mean relationship between mgatp concentration and katp current ( i ) , expressed relative to that in the absence of nucleotide ( ic ) , after 30-s preincubation in 10 mmol / l mgatp for wt (  , n = 9 ) , heterozygous (  ) , and homomeric mutant (  ) channels .
channels were composed of kir6.2 and wild - type or mutant sur1 subunits , as indicated .
c : home1506 g ( n = 7 ) , hete1506 g ( n = 7 ) .
d : home1506k ( n = 7 ) , hete1506k ( n = 5 ) .
1 , with the following parameters ( in mol / l for ic50 ) : wild - type , ic50 = 28 , h = 1.2 ; home1506d , ic50 = 78 , h = 1.2 ; hete1506d , ic50 = 45 , h = 1.2 ; home1506 g , ic50 = 105 , h = 1.3 ; hete1506 g , ic50 = 45 , h = 1.3 ; home1506k , ic50 = 18 , h = 1.6 ; hete1506k , ic50 = 26 , h = 1.4 .
a : representative home1506 g currents recorded in response to test pulses of 100 mol / l mgatp ( unlabeled solid line [  ] ) , preceded by a preconditioning pulse to a variable mgatp concentration ( 10 mol / l to 10 mmol / l , as indicated ) and followed by control ( nucleotide - free ) solution .
b : mgatp concentration during the conditioning prepulse plotted against the current during a 100 mol / l mgatp test pulse .
current is expressed as a fraction of that in control ( nucleotide - free ) solution after the test pulse for wild - type (  , n = 14 ) , home1506d (  , n = 15 ) , home1506 g (  , n = 10 ) , and home1506k (  , n = 8) channels .
pre - exposure to 10 mmol / l mgadp , which fully blocked the wild - type and mutant channels , did not affect the ability of 100 mol / l mgatp to block any of the four channel types ( data not shown ) .
our results provide a functional explanation for why mutation of e1506 in sur1 to lysine ( k ) results in hyperinsulinism , whereas mutation of the same residue to aspartate ( d ) or glycine ( g ) causes neonatal diabetes .
this occurs because the mutations have opposite effects on the whole - cell katp currents ; hete1506d and hete1506 g increase resting whole - cell currents , whereas the hete1506k channels show no resting whole - cell currents and are activated less in response to metabolic inhibition .
an increase in the resting -cell katp current would be expected to produce -cell hyperpolarization and reduce ca influx and insulin secretion evoked by glucose , thereby predisposing to diabetes .
in contrast , a reduction in katp current will cause depolarization , maintained ca influx , and persistent insulin secretion , giving rise to hyperinsulinism .
as reported for most other neonatal diabetes mutations in sur1 ( 18,21 ) , the increase in the resting whole - cell currents was relatively small .
such small changes are consistent with the relatively mild , transient form of diabetes and the absence of neurological symptoms ( 6,18 ) .
resting currents in the pancreatic -cell are likely larger than those measured in the oocyte , perhaps because of lower atp levels .
it is not evident why the diabetes remits , but as is the case for other sur1 mutations causing transient neonatal diabetes ( 29 ) , it seems likely the diabetes may later relapse and patients should be monitored for this possibility .
sulfonylureas are an effective therapy for most patients with neonatal diabetes caused by katp channel mutations and are now the treatment of choice for this disease ( 24 ) .
we observed that tolbutamide blocked hete1506d and hete1506 g currents as much as wild - type katp channels , which explains why our patients could be successfully treated with glibenclamide .
so why does the mutation of e1506 to k abolish resting whole - cell katp currents , whereas the mutation to e and g enhances them ?
we observed that the neonatal diabetic mutant channels are substantially less sensitive to atp inhibition if they have been previously exposed to physiological atp concentrations of 110 mmol / l ( 37 ) . in contrast , this effect was very small for wild - type channels and absent for e1506k channels .
these results are consistent with the differences in the resting whole - cell current amplitude we measured in intact oocytes and suggest that neonatal diabetic channels have larger resting currents because they are less sensitive to atp
. metabolic inhibition will result in a fall in atp and an increase in mgadp , both of which will affect the response of the katp channel .
both reduced atp inhibition at kir6.2 and increased mgadp activation at sur1 will contribute to the increase in wild - type current and , to a lesser extent , the heterozygous channels .
the impaired response of the home1506k channels may be explained by their markedly reduced mgadp activation , as previously suggested ( 27 ) .
we suggest the small increase in the home1506d and home1506 g currents is because their reduced mgadp activation is outweighed by their greatly reduced atp sensitivity , allowing the channels to open in response to falling atp levels .
because atp sensitivity did not change in the absence of mg , it appears that the mutations do not reduce nucleotide block at kir6.2 .
e1506 lies within nbs2 of sur1 , close to the -phosphate of atp and its associated mg atom ( 38 ) .
the equivalent residue in other abc transporters is known to be involved in nucleotide binding and atpase hydrolysis ( 32 ) and its mutation to aspartate dramatically reduces atpase activity and the initial p ( pi ) release in abc proteins ( 39,40 ) .
thus mutation of e1506 in sur1 is likely to alter the conformation that mg nucleotides adopt in the nbs and thereby impair 1 ) nucleotide binding , and/or 2 ) atpase activity , and/or 3 ) the mechanism by which mgadp occupation at nbs2 is translated into channel opening .
it seems possible that the e1506 mutations trap nbs2 in different conformational states of the atpase cycle ( fig .
7c ) that either promote channel activity ( e , g ) or do not ( k ) .
all three mutations markedly reduce mgadp activation of the channel , arguing that occupancy of this state is reduced and/or the translation of the mgadp - bound state into channel activity is impaired .
mutation of the equivalent residue in mrp1 to aspartate does not alter mgadp binding ( 32 ) , which suggests that sur1-e1506d might bind mgadp but that binding no longer leads to channel activation .
all mutations support mgatp activation of the channel , as indicated by the use of a kir6.2 atp - insensitive mutation .
it is not possible to distinguish whether the channel is activated by occupancy of the mgatp - bound state or the mgadp + pi state , or both ( fig .
nevertheless , the ability of mgatp , but not mgadp , to stimulate mutant channels almost as effectively as wild - type channels argues that one or both of these states can do so . the slower off - rate of mgatp seen with the neonatal diabetes mutations ( especially e1506d ) may indicate that the channel becomes trapped in a particular state of the reaction cycle that is associated with increased channel activity ; for example , the mgatp - bound or mgadp + pi state .
the off - rate of mgadp was much faster than that of mgatp for the e1506d channels , and mgadp had little stimulatory effect , which supports arguments that it can not be the mgadp - bound state .
the most striking difference between the e1506k and e1506g / e1506d channels is that shown in figs .
6 and 7 : pre - exposure to millimolar concentrations of mgatp desensitizes the channel to subsequent inhibition by a lower atp concentration
. this can not be due to an effect of mgatp on kinases , for example , because this should be similar for all channels .
first , exposure to a saturating atp concentration for an extended period may force the channels into a state in which nbs1 and nbs2 on all four subunits are both occupied by nucleotide , and this may lead to greater channel activation .
second , it may relate to the slower off - rate of mgatp of neonatal diabetes channels , which could maintain the channel in an activated state for an extended period , so impairing atp block . as shown previously , mg - nucleotide activation not only enhances channel activity per se but also reduces atp inhibition at kir6.2 ( 41 ) . in the -cell , where atp levels are normally within the millimolar range ,
so why does the mutation of e1506 to k abolish resting whole - cell katp currents , whereas the mutation to e and g enhances them ?
we observed that the neonatal diabetic mutant channels are substantially less sensitive to atp inhibition if they have been previously exposed to physiological atp concentrations of 110 mmol / l ( 37 ) . in contrast , this effect was very small for wild - type channels and absent for e1506k channels .
these results are consistent with the differences in the resting whole - cell current amplitude we measured in intact oocytes and suggest that neonatal diabetic channels have larger resting currents because they are less sensitive to atp .
metabolic inhibition will result in a fall in atp and an increase in mgadp , both of which will affect the response of the katp channel .
both reduced atp inhibition at kir6.2 and increased mgadp activation at sur1 will contribute to the increase in wild - type current and , to a lesser extent , the heterozygous channels .
the impaired response of the home1506k channels may be explained by their markedly reduced mgadp activation , as previously suggested ( 27 ) .
we suggest the small increase in the home1506d and home1506 g currents is because their reduced mgadp activation is outweighed by their greatly reduced atp sensitivity , allowing the channels to open in response to falling atp levels .
because atp sensitivity did not change in the absence of mg , it appears that the mutations do not reduce nucleotide block at kir6.2 .
e1506 lies within nbs2 of sur1 , close to the -phosphate of atp and its associated mg atom ( 38 ) .
the equivalent residue in other abc transporters is known to be involved in nucleotide binding and atpase hydrolysis ( 32 ) and its mutation to aspartate dramatically reduces atpase activity and the initial p ( pi ) release in abc proteins ( 39,40 )
. thus mutation of e1506 in sur1 is likely to alter the conformation that mg nucleotides adopt in the nbs and thereby impair 1 ) nucleotide binding , and/or 2 ) atpase activity , and/or 3 ) the mechanism by which mgadp occupation at nbs2 is translated into channel opening .
it seems possible that the e1506 mutations trap nbs2 in different conformational states of the atpase cycle ( fig .
7c ) that either promote channel activity ( e , g ) or do not ( k ) .
all three mutations markedly reduce mgadp activation of the channel , arguing that occupancy of this state is reduced and/or the translation of the mgadp - bound state into channel activity is impaired .
mutation of the equivalent residue in mrp1 to aspartate does not alter mgadp binding ( 32 ) , which suggests that sur1-e1506d might bind mgadp but that binding no longer leads to channel activation .
all mutations support mgatp activation of the channel , as indicated by the use of a kir6.2 atp - insensitive mutation .
it is not possible to distinguish whether the channel is activated by occupancy of the mgatp - bound state or the mgadp + pi state , or both ( fig .
nevertheless , the ability of mgatp , but not mgadp , to stimulate mutant channels almost as effectively as wild - type channels argues that one or both of these states can do so . the slower off - rate of mgatp seen with the neonatal diabetes mutations ( especially e1506d ) may indicate that the channel becomes trapped in a particular state of the reaction cycle that is associated with increased channel activity ; for example , the mgatp - bound or mgadp + pi state .
the off - rate of mgadp was much faster than that of mgatp for the e1506d channels , and mgadp had little stimulatory effect , which supports arguments that it can not be the mgadp - bound state .
the most striking difference between the e1506k and e1506g / e1506d channels is that shown in figs .
6 and 7 : pre - exposure to millimolar concentrations of mgatp desensitizes the channel to subsequent inhibition by a lower atp concentration
. this can not be due to an effect of mgatp on kinases , for example , because this should be similar for all channels .
first , exposure to a saturating atp concentration for an extended period may force the channels into a state in which nbs1 and nbs2 on all four subunits are both occupied by nucleotide , and this may lead to greater channel activation .
second , it may relate to the slower off - rate of mgatp of neonatal diabetes channels , which could maintain the channel in an activated state for an extended period , so impairing atp block . as shown previously , mg - nucleotide activation
not only enhances channel activity per se but also reduces atp inhibition at kir6.2 ( 41 ) . in the -cell , where atp levels are normally within the millimolar range
, this mechanism would result in reduced atp inhibition and thus larger resting katp currents .
 objectivetwo novel mutations ( e1506d , e1506 g ) in the nucleotide - binding domain 2 ( nbd2 ) of the atp - sensitive k+ channel ( katp channel ) sulfonylurea receptor 1 ( sur1 ) subunit were detected heterozygously in patients with neonatal diabetes . 
 a mutation at the same residue ( e1506k ) was previously shown to cause congenital hyperinsulinemia . 
 we sought to understand why mutations at the same residue can cause either neonatal diabetes or hyperinsulinemia.research design and methodsneonatal diabetic patients were sequenced for mutations in abcc8 ( sur1 ) and kcnj11 ( kir6.2 ) . 
 wild - type and mutant katp channels were expressed in xenopus laevis oocytes and studied with electrophysiological methods.resultsoocytes expressing neonatal diabetes mutant channels had larger resting whole - cell katp currents than wild - type , consistent with the patients diabetes . 
 conversely , no e1506k currents were recorded at rest or after metabolic inhibition , as expected for a mutation causing hyperinsulinemia . 
 katp channels are activated by mg - nucleotides ( via sur1 ) and blocked by atp ( via kir6.2 ) . 
 all mutations decreased channel activation by mgadp but had little effect on mgatp activation , as assessed using an atp - insensitive kir6.2 subunit . importantly , using wild - type kir6.2 , a 30-s preconditioning exposure to physiological mgatp concentrations ( > 300 mol / l ) caused a marked reduction in the atp sensitivity of neonatal diabetic channels , a small decrease in that of wild - type channels , and no change for e1506k channels . 
 this difference in mgatp inhibition may explain the difference in resting whole - cell currents found for the neonatal diabetes and hyperinsulinemia mutations.conclusionsmutations in the same residue can cause either hyperinsulinemia or neonatal diabetes . 
 differentially altered nucleotide regulation by nbd2 of sur1 can explain the respective clinical phenotypes . 
RESEARCH DESIGN AND METHODS
Mutation detection.
Molecular biology and oocyte preparation.
Electrophysiology.
RESULTS
Patient characteristics and genetics.
Effects on whole-cell K
Effects on K
Effects on MgADP activation of K
Effects of MgATP and MgADP activation on E1506 mutants expressed on the Kir6.2-G334D background.
Effects on MgATP activation of the K
DISCUSSION
Effects of mutations on whole-cell currents.
Molecular mechanism.