ABCMdb

PMID: 22020219

Babenko AP, Vaxillaire M
Mechanism of KATP hyperactivity and sulfonylurea tolerance due to a diabetogenic mutation in L0 helix of sulfonylurea receptor 1 (ABCC8).
FEBS Lett. 2011 Nov 16;585(22):3555-9. Epub 2011 Oct 19., [PubMed]

Sentences

No. Mutations Sentence Comment

12 ABCC8 p.Leu213Arg The first ND mutation found in the L0 linker, L213R, is in the middle of the hotspot region in the putative interface helix [14] and causes severe ND with neurological abnormalities [7]. Login to comment 14 ABCC8 p.Phe132Leu Consistent with our hypothesis, the diabetogenic F132L in TMD0 of SUR1 increased KATP activity in the absence of nucleotides [15]. Login to comment 22 ABCC8 p.Leu213Arg L213R mutation was introduced into hamster SUR1 cDNA. Login to comment 50 ABCC8 p.Leu213Arg Results and discussion Fig. 2 shows that L213R dramatically increases the Po in intact cells while not affecting i and slightly decreasing N. Login to comment 51 ABCC8 p.Leu213Arg ABCC8 p.Leu225Pro The latter effect is consistent with the small negative effect of L213R on the amount of mature receptor, which is in line with observations that a comparable amphipathic L0 helix of ABCC1 attaches to the membrane [23] and L225P in a less conserved portion of the cytoplasmic linker of SUR1 does not affect N [24] or surface expression of SUR1 in the same cell line [25]. Login to comment 52 ABCC8 p.Leu213Arg The results show that L213R can induce pathogenic currents in intact cells by hyperactivating KATP and support the notion that possible negative effects of some ND mutations on N (see also [25]) are overridden by their much stronger effect on PO. Login to comment 61 ABCC8 p.Leu213Arg L213R and other elements of the model discussed in the text are identified using color-coded labels. Login to comment 64 ABCC8 p.Leu213Arg L213R markedly elevates on-cell PO, does not affect the unitary conductance, and slightly decreases functional expression of (SUR1/KIR6.2)4 complexes without altering their labeling with 1 nM 125 I-azidoglibenclamide. Login to comment 69 ABCC8 p.Leu213Arg Analysis of these records (Fig. 3) demonstrated that L213R nearly saturates the channel intrinsic activity, POmax ? Login to comment 73 ABCC8 p.Leu213Arg To test this prediction we first compared the activities of the same macro-population of L213R channels in intact cells, in 1 mM MgATP, and in 1 mM ATP without Mg (Fig. 4A illustrates the protocol) vs similarly recorded activities of WT channels. Login to comment 75 ABCC8 p.Leu213Arg The results suggested that L213R does not alter the Mg-nucleotide stimulatory action but compromises the Mg-independent nucleotide inhibition of KATP. Login to comment 76 ABCC8 p.Leu213Arg Indeed, the steady-state inhibitory ATP dose-response (Fig. 4C) demonstrated that unlike A type mutations tested earlier under identical experimental conditions [7,11], L213R markedly ($20 times) increases IC50(ATP). Login to comment 78 ABCC8 p.Leu213Arg This biophysical mechanism of KATP hyperactivity, called B type mechanism for short, explains why pancreatic b-cells in the L213R patient did not release insulin (remained hyperpolarized) despite his very high blood glucose [7]. Login to comment 79 ABCC8 p.Leu213Arg A dose of glibenclamide above the FDA recommended dose allowed to transfer the L213R patient from insulin injections to SU therapy [7]. Login to comment 81 ABCC8 p.Leu213Arg Our first test (Fig. 5A) indicated reduced inhibition of L213R channels by glibenclamide, but the slow washout of the second generation SU made it difficult to estimate its steady-state effect corrected for rundown (see Section 2 and similar test for WT KATP in [22]). Login to comment 82 ABCC8 p.Leu213Arg Therefore we used the rapidly unbinding SU tolbutamide to quantify the effect of L213R on KATP inhibition by [SU] saturating its specific, but not non-specific, sites [22,31]. Login to comment 83 ABCC8 p.Leu213Arg We compared the SU inhibition of L213R vs WT KATP activity under non-stimulatory conditions, as well as in the presence of the lowest possible submembrane [MgATP] and the highest possible [MgADP] in resting b-cells [32,33], as we did earlier for A type mutant KATP [7,11]. Login to comment 84 ABCC8 p.Leu213Arg We found that unlike A type mutations, L213R compromises SU inhibition under non-stimulatory conditions (Fig. 5B, left bars). Login to comment 85 ABCC8 p.Leu213Arg While saturation of specific SU binding sites with 200 lM tolbutamide reduces spontaneous activity of WT KATP by about 60% [22,31], the same SU treatment of L213R channels produces significantly smaller inhibition. Login to comment 86 ABCC8 p.Leu213Arg Thus, L213R alters the nucleotide-independent component of SU action by uncoupling its high-affinity binding from the channel closure. Login to comment 93 ABCC8 p.Phe132Leu Consistent with the proposal, F132L in TMD0 and activating mutations in M0 alter intrinsic gating [15,39,40]. Login to comment 94 ABCC8 p.Leu213Arg Strong effects of L213R in L0 helix on TB/TIB and KATP inhibition provide a new evidence in support of our working mechanistic model (Fig. 1). Login to comment 95 ABCC8 p.Glu208Lys Only subtle and insignificant functional effects of E208K [25] are not surprising. Login to comment 96 ABCC8 p.Leu213Arg ABCC8 p.Glu208Lys Unlike L213R, E208K can be carried asymptomatically [41,42] and exchanges similarly hydrophilic side chains on the hydrophilic side of the submembrane amphipathic helix (Fig. 6). Login to comment 101 ABCC8 p.Leu213Arg L213R saturates intrinsic activity of KATP by altering its slow gating kinetics. Login to comment 102 ABCC8 p.Leu213Arg The top panel shows short segments of records of currents trough single WT and L213R KATP in inside-out patches in the nucleotide-free internal solution. Login to comment 104 ABCC8 p.Leu213Arg The next panels show the results of analysis of 10 L213R vs 10 WT KATP records like those shown in the top panel. Login to comment 109 ABCC8 p.Leu213Arg ABCC8 p.Leu213Arg L213R does not alter the direct proximity of L0 to Kir6.2 (Fig. 2 inset), but could disrupt optimized L0/M0 interactions by rotating the L0 helix along its axis (Fig. 6) as L213R changes the helical hydrophobic moment. Login to comment 113 ABCC8 p.Val324Met Previous studies showed that ND mutations in every major domain of the ABC core of SUR1 can increase KATP PO via A type mechanism (reviewed in [9,10]; see also [25] on V324M in TMD1). Login to comment 118 ABCC8 p.Leu213Arg L213R compromises inhibitory nucleotide action. Login to comment 119 ABCC8 p.Leu213Arg (A) L213R KATP currents under conditions indicated. Login to comment 122 ABCC8 p.Leu213Arg (C) The inhibitory ATP dose-response curve for L213R (IC50(ATP) = 101.7 ± 4.1 lM; Hill coefficient = 1.19 ± 0.04) vs those for WT and A type mutant KATP tested earlier under similar conditions (see [7,11] for details); n = 10 and R2 > 0.995 for each curve fit. Login to comment 124 ABCC8 p.Leu213Arg L213R attenuates inhibition of KATP by sulfonylureas. Login to comment 125 ABCC8 p.Leu213Arg (A) L213R KATP currents indicating their reduced response to the normally (tightly) binding, slowly dissociating, insulin secretagogue glibenclamide (Glb). Login to comment 128 ABCC8 p.Leu213Arg (B) The steady-state inhibition of L213R vs WT KATP by tolbutamide (Tlb) under conditions indicated; n = 10 for each bar. Fig. 6. Login to comment 131 ABCC8 p.Leu213Arg The expected L213R-induced rotation of L0 helix is indicated. Login to comment