ABCMdb

PMID: 20810569

Zhou Q, Garin I, Castano L, Argente J, Munoz-Calvo MT, Perez de Nanclares G, Shyng SL
Neonatal diabetes caused by mutations in sulfonylurea receptor 1: interplay between expression and Mg-nucleotide gating defects of ATP-sensitive potassium channels.
J Clin Endocrinol Metab. 2010 Dec;95(12):E473-8. Epub 2010 Sep 1., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC8 p.Val324Met ABCC8 p.Glu208Lys Objective: The objective of the study was to determine the mechanisms by which two SUR1 mutations, E208K and V324M, associated with transient neonatal diabetes affect KATP channel function. Login to comment 2 ABCC8 p.Val324Met ABCC8 p.Glu208Lys Design: E208K or V324M mutant SUR1 was coexpressed with Kir6.2 in COS cells, and expression and gatingpropertiesoftheresultingchannelswereassessedbiochemicallyandelectrophysiologically. Login to comment 3 ABCC8 p.Val324Met ABCC8 p.Glu208Lys Results: Both E208K and V324M augment channel response to MgADP stimulation without altering sensitivity to ATP4- or sulfonylureas. Login to comment 4 ABCC8 p.Val324Met ABCC8 p.Glu208Lys Surprisingly, whereas E208K causes only a small increase in MgADP response consistent with the mild transient diabetes phenotype, V324M causes a severe activating gating defect. Login to comment 5 ABCC8 p.Val324Met ABCC8 p.Glu208Lys Unlike E208K, V324M also impairs channel expression at the cell surface, which is expected to dampen its functional impact on beta-cells. Login to comment 6 ABCC8 p.Val324Met ABCC8 p.Glu208Lys When either mutation was combined with a mutation in the second nucleotide binding domain of SUR1 previously shown to abolish Mg-nucleotide response, the activating effect of E208K and V324M was also abolished. Login to comment 7 ABCC8 p.Val324Met ABCC8 p.Glu208Lys Moreover, combination of E208K and V324M results in channels with Mg-nucleotide sensitivity greater than that seen in individual mutations alone. Login to comment 8 ABCC8 p.Val324Met ABCC8 p.Glu208Lys Conclusion: The results demonstrate that E208K and V324M, located in distinct domains of SUR1, enhance transduction of Mg-nucleotide stimulation from the SUR1 nucleotide binding folds to Kir6.2. Login to comment 22 ABCC8 p.Val324Met ABCC8 p.Glu208Lys We conducted functional analyses of two SUR1 mutations, E208K and V324M, identified in transient ND (7, 8). Login to comment 23 ABCC8 p.Val324Met ABCC8 p.Glu208Lys E208K and V324M located in L0 and TMD1, respectively, cause channel overactivity by enhancing MgADP responsivity, establishing their causal role in ND. Login to comment 24 ABCC8 p.Val324Met ABCC8 p.Val324Met ABCC8 p.Val324Met ABCC8 p.Glu208Lys The enhancement effect on MgADP responsivity is greater in V324M than E208K; however, surface expression of the V324M mutant is significantly reduced, suggesting that the greater gain-of-function gating defect caused by V324M is offset by lower surface expression. Login to comment 25 ABCC8 p.Val324Met ABCC8 p.Glu208Lys When combined with a SUR1-NBF2 mutation known to abolish MgADP responsivity, effects of E208K and V324M were also abolished, indicating that these residues are involved in transducing the effect of Mg-nucleotides to channel gating. Login to comment 35 ABCC8 p.Val324Met ABCC8 p.Glu208Lys Results Mutations and clinical data E208K and V324M are SUR1 mutations identified in patients diagnosed with ND (7, 8, 12) (Supplemental Table 1). Login to comment 36 ABCC8 p.Val324Met The most relevant data of the patient with a V324M mutation, not previously reported, is that at 1 month and 17 d of age she was admitted to the hospital due to general hypotonia with severe hyperglycemia (88.4 mmol/liter) and positive ketonemia. Login to comment 40 ABCC8 p.Val324Met ABCC8 p.Glu208Lys No functional studies have been conducted on either E208K or V324M. Login to comment 41 ABCC8 p.Val324Met ABCC8 p.Glu208Lys Functional analysis of mutant channels Because not all carriers are symptomatic (7, 8, 12), it is important to determine whether E208K and V324M affect KATP channel function. Login to comment 42 ABCC8 p.Val324Met ABCC8 p.Val324Met ABCC8 p.Glu208Lys In 86 Rbϩ efflux assays, whereas WT channels exhibited the expected low activity due to inhibition by high intracellular ATP, both E208K and V324M channels yielded greater efflux, with V324M being the most active (Fig. 1A). Login to comment 46 ABCC8 p.Val324Met ABCC8 p.Glu208Lys Functional characterization of E208K- and V324M-SUR1 mutations. Login to comment 49 ABCC8 p.Val324Met ABCC8 p.Val324Met ABCC8 p.Val324Met ABCC8 p.Glu208Lys ABCC8 p.Glu208Lys Both E208K and V324M exhibited higher efflux than WT, with the V324M mutant showing the highest activity, confirming that E208K- and V324M-SUR1 are activating KATP channel mutations. Login to comment 52 ABCC8 p.Val324Met ABCC8 p.Glu208Lys ABCC8 p.Leu225Pro The mature form of SUR1 is clearly reduced in V324M compared with WT, E208K, or L225P. Login to comment 55 ABCC8 p.Val324Met The expression level of V324M is significantly reduced compared with WT (n ϭ 3; *, P Ͻ 0.05 by Student`s t test). Login to comment 57 ABCC8 p.Val324Met ABCC8 p.Leu225Pro Representative recordings of WT, V324M, and L225P channels are shown. Login to comment 60 ABCC8 p.Glu208Lys D, Similar to panel C, except that the concentrations of ATP and ADP were different, and comparisons between WT and E208K were made. Login to comment 63 ABCC8 p.Val324Met ABCC8 p.Glu208Lys Shown are representative recordings of WT, E208K, and V324M channels in response to 10 or 100 nM glibenclamide. Login to comment 69 ABCC8 p.Val324Met ABCC8 p.Val324Met ABCC8 p.Glu208Lys Because exit of SUR1 from the ER requires coassembly with Kir6.2, abundance of the upper band correlates with the amount of SUR1 at the cell surface that haspassedERqualitycontrol(9,14).Figure1Bshowsthat whereas in E208K-SUR1 both bands were similar in intensity to WT-SUR1, V324M-SUR1 had a clearly reduced upper band, suggesting that V324M may impair channel trafficking from ER to the plasma membrane. Login to comment 70 ABCC8 p.Val324Met ABCC8 p.Val324Met ABCC8 p.Glu208Lys Immunofluorescence staining of surface channels showed that V324M is indeed expressed at a reduced level compared with WT or E208K, although staining of permeabilized cells indicated that total V324M SUR1 protein levels were not significantly reduced (Supplemental Fig. 2). Login to comment 71 ABCC8 p.Val324Met ABCC8 p.Glu208Lys Surface expression was further quantified by chemiluminescence assays that confirmed markedly reduced surface expression of V324M, in contrast to E208K (56.3 Ϯ 6.3 vs. 93.9 Ϯ 10.1% of WT channels, respectively; Fig. 1B). Login to comment 72 ABCC8 p.Val324Met ABCC8 p.Glu208Lys Unaltered or reduced surface expression of E208K or V324M suggests that overactivity results from abnormal channel gating. Login to comment 74 ABCC8 p.Val324Met ABCC8 p.Glu208Lys ATP dose-response studies showed that E208K and V324M do not affect channel ATP4- sensi- tivity (Supplemental Fig. 3). Login to comment 77 ABCC8 p.Val324Met ABCC8 p.Leu225Pro These experiments showed that V324M markedly increases the Mg-nucleotide response (most evident at the 0.5/0.1 mM ATP/ADP ratio; Fig. 1C), even more so than L225P, another SUR1 mutation reported to cause permanent ND by a similar gating abnormality (15). Login to comment 78 ABCC8 p.Glu208Lys In contrast, E208K subtly increased Mg-nucleotide sensitivity that was statistically significant only in the 0.1/0.1 mM ATP/ADP ratio (compare Figs. 1D and 2B). Login to comment 79 ABCC8 p.Val324Met ABCC8 p.Glu208Lys These results indicate that E208K and V324M cause ND by hypersensitizing channels to Mg-nucleotide stimulation. Login to comment 81 ABCC8 p.Val324Met ABCC8 p.Glu208Lys To determine whether E208K or V324M affects channel sensitivity to sulfonylureas, we tested channel response to 10 or 100 nM glibenclamide. Login to comment 82 ABCC8 p.Val324Met ABCC8 p.Glu208Lys E208K and V324M were inhibited by glibenclamide similar to WT channels at both concentrations (Fig. 1E). Login to comment 83 ABCC8 p.Val324Met The effectiveness of glibenclamide in inhibiting V324M channels is consistent with the clinical observation reported previously that recurrent diabetes in a patient bearing this mutation was successfully treated with glibenclamide (8). Login to comment 84 ABCC8 p.Val324Met ABCC8 p.Glu208Lys E208K and V324M enhance transduction of MgADP stimulation Stimulation of KATP channels by Mg-nucleotides originates from Mg-nucleotide interactions with NBFs of SUR1. Login to comment 85 ABCC8 p.Val324Met ABCC8 p.Glu208Lys E208K and V324M are outside the NBFs, in the TMD0-L0 and TMD1 domains, respectively (Supplemental Fig. 1). Login to comment 88 ABCC8 p.Val324Met ABCC8 p.Val324Met ABCC8 p.Glu208Lys ABCC8 p.Glu208Lys Combining E208K or V324M with E1507K completely abrogated the enhancement effects of E208K or V324M on Mg-nucleotide responses (Fig. 2A). Login to comment 90 ABCC8 p.Val324Met ABCC8 p.Glu208Lys E208K and V324M enhance channel response to MgADP by affecting transduction of the MgADP effect to Kir6.2. Login to comment 93 ABCC8 p.Val324Met ABCC8 p.Glu208Lys A, The E1507K inactivating mutation in NBF2 completely abolished the activating effects of E208K or V324M. Login to comment 96 ABCC8 p.Val324Met ABCC8 p.Val324Met ABCC8 p.Val324Met ABCC8 p.Glu208Lys ABCC8 p.Glu208Lys ABCC8 p.Glu208Lys ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro ABCC8 p.Leu225Pro Each bar is the mean Ϯ SEM of 30 (WT), eight (E208K), six (L225P), five (V324M), nine (E208K/L225P), six (E208K/V324M), and six (L225P/V324M) patches. Login to comment 99 ABCC8 p.Val324Met ABCC8 p.Val324Met ABCC8 p.Val324Met ABCC8 p.Glu208Lys ABCC8 p.Glu208Lys ABCC8 p.Glu208Lys (#), P Ͻ 0.05 between E208K/V324M and E208K but the difference between E208K/V324M and V324M is not significant. Login to comment 100 ABCC8 p.Val324Met ABCC8 p.Glu208Lys ABCC8 p.Leu225Pro To determine the relationship between the activating effect caused by V324M in TMD1 and that caused by E208K or L225P in TMD0-L0, we compared Mg-nucleotide responsivity in channels harboring a combination of these mutations. Login to comment 101 ABCC8 p.Val324Met ABCC8 p.Glu208Lys ABCC8 p.Leu225Pro Currents measured in 0.1 mM ATP (free [Mg2ϩ ] ϳ1 mM) showed that channels harboring double mutations were more active than those with only one mutation such that the effects of E208K, L225P, or V324M are additive. Login to comment 104 ABCC8 p.Val324Met ABCC8 p.Glu208Lys Discussion We show that two heterozygous SUR1 mutations, E208K and V324M, identified in patients with transient ND cause KATP channel overactivity by enhancing channel responsivity to Mg-nucleotides without affecting ATP4- or glibenclamide sensitivity. Login to comment 106 ABCC8 p.Val324Met ABCC8 p.Val324Met ABCC8 p.Glu208Lys ABCC8 p.Glu208Lys ABCC8 p.Leu225Pro Although V324M renders a marked increase in Mg-nucleotide response, even more so than L225P previously reported to cause permanent ND (15), the effect of E208K is subtle, only statistically significant at 0.1 mM ATP/0.1 mM ADP; yet both E208K and V324M are associated with transient ND. Login to comment 108 ABCC8 p.Val324Met ABCC8 p.Leu225Pro We previously proposed that in ND surface expression efficiency of a mutant modifies the extent of manifestation of the gating defect to determine diseaseoutcome(18,19).Supportingthis,whereasE208K (and also L225P; Fig. 1B) exhibited surface expression similar to WT, V324M was expressed at only approximately 50% that of WT (Fig. 1B). Login to comment 109 ABCC8 p.Val324Met The severe gating defect caused by V324M is likely negated by low expression of the mutant on the beta-cell surface, resulting in a less severe diabetes phenotype than one would predict based solely on gating defects. Login to comment 110 ABCC8 p.Glu208Lys Of note, asymptomatic carriers have been described for both E208K and V324M(7, 12). Login to comment 112 ABCC8 p.Val324Met What environmental and genetic factors underlie the wide variations in disease presentation for V324M is an important question to address in the future. Login to comment 113 ABCC8 p.Val324Met ABCC8 p.Glu208Lys The activating effects of E208K and V324M were abrogated by a NBF2 mutation that abolishes the MgADP response. Login to comment 114 ABCC8 p.Leu225Pro This suggests that similar to L225P (15), the two mutations affect transduction of MgATP binding/hydrolysis signal from the SUR1-NBFs to Kir6.2. Login to comment 115 ABCC8 p.Glu208Lys E208K is in the TMD0-L0 domain that has been proposed to serve as a coupling module to transduce effects of Mg-nucleotide stimulation to Kir6.2 (4-6). Login to comment 117 ABCC8 p.Val324Met ABCC8 p.Glu208Lys ABCC8 p.Leu225Pro That the effect of E208K or L225P and of V324M are additive suggests that there may be multiple transduction pathways to regulate Kir6.2 gating. Login to comment 118 ABCC8 p.Val324Met Finally, V324M has normal glibenclamide sensitivity despite a markedly increased Mg-nucleotide response. Login to comment 120 ABCC8 p.Val324Met Sulfonylureas may block the activating effect of V324M by preventing the initial steps in the NBFs. Login to comment 121 ABCC8 p.Val324Met ABCC8 p.Leu225Pro Alternatively, transduction of the glibenclamide blocking effect may employ a pathway separate from that affected by V324M, as suggested for the L225P mutation (15). Login to comment