ABCMdb

PMID: 15034580

Bienengraeber M, Olson TM, Selivanov VA, Kathmann EC, O'Cochlain F, Gao F, Karger AB, Ballew JD, Hodgson DM, Zingman LV, Pang YP, Alekseev AE, Terzic A
ABCC9 mutations identified in human dilated cardiomyopathy disrupt catalytic KATP channel gating.
Nat Genet. 2004 Apr;36(4):382-7. Epub 2004 Mar 21., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr Published online 21 March 2004; doi:10.1038/ng1329 L E T T E R S 382 VOLUME 36 | NUMBER 4 | APRIL 2004 NATURE GENETICS a b 1310 GEIKI...HRVSSIMDAGLVLVFSEGILVECDTVPNLLAHKNGLFSTLVMTNK* 1549 1310 GEIKI...HRVSSIMDAGLVLVFSEGIKCGV* 1527 4570-4572delTTAinsAAAT (Fs1524) 1310 GEIKI...HRVSSIMDTGLVLVFSEGILVECDTVPNLLAHKNGLFSTLVMTNK* 1549 4537G→A (A1513T) ...HRVSSIMDAGLVLVFSEGILVECDTGPNLLQHKNGLFSTLVMTNK* Rat ...HRVSSIVDAGLVLVFSEGILVECDTGPNLLQHKNGLFSTLVMTNK* Mouse ...HRVSSIVDADLVLVFSEGILVECDTGPNLLTHKNGLFSTLVMTNK* Rabbit ...HRVSSITDADLVLVFSEGILVECDTGPNLLTYRNGLFSTLVMTHK* Guinea pig T A G T G T G A G G T G A T T T T A T G G A G T G T G A G G T G T A A A T T A T G G Asp Cys Glu Val Leu Ile Gly Ter Val Gly Cys Lys Ile Gly G A T C G A T C T T G T T C C G G A C A T A G G T A T T A T T G T T C C G G A C G T A G G T A T T A Val Leu Gly Thr Asp Met Ile Val Leu Gly Ala Asp Met Ile G A T C G A T C COOH 3 4 5 0 1 2 3 Individual 2 3 4 5 Absorbance(mV) 0 1 2 3 Frameshift mutationNormal Missense mutationNormal NormalNormal Time (min)Time (min) Absorbance(mV) Human wild-type Individual 1 NBD1 NBD2 Exon 38 SUR2A Kir6.2   Stress tolerance of the heart requires high-fidelity metabolic sensing by ATP-sensitive potassium (KATP) channels that adjust membrane potential-dependent functions to match cellular energetic demand. Login to comment 11 ABCC9 p.Ala1513Thr Sequencing identified frameshift (Fs1524; individual 1) and missense (A1513T; individual 2) mutations. Login to comment 28 ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr The second mutated allele harbored a missense mutation (4537G→A) causing the amino acid substitution A1513T (Fig. 1a). Login to comment 30 ABCC9 p.Ala1513Thr The C terminus of SUR proteins contributes to KATP channel traf- ficking19,20, and Fs1524 and A1513T SUR2A mutants, reconstituted with Kir6.2, had reduced expression in the plasma membrane (Fig. 2a). Login to comment 34 ABCC9 p.Ala1513Thr (a) Fs1524 and A1513T reduced KATP channel trafficking by ~70% and ~30%, probed immunologically by SUR surface expression in Xenopus laevis oocytes. Login to comment 38 ABCC9 p.Ala1513Thr Missense A1513T (cyan) and frameshift L1524 (magenta) mutations frame the β-strand adjacent to Walker motifs that coordinate NBD2-mediated catalysis. Login to comment 44 ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr -6 -5 -4 -3 -2 0.0 0.2 0.4 0.6 0.8 1.0 WT IC50: 19 ± 2µM 0.0 0.1 0.2 0.3 0.4 0.5 WT A1513T Fs1524 a e NBD2 of SUR2A WB WA COOH c WT Fs1524 A1513T 0 30 60 90 b WB WA L1524 A1513 G1514 G1530 V1501 V1342 C1345 L1517 V1518 V1525 E1526 M1472 L1470 d T1502 L1540 E1541 Frameshiftmutation Missense mutation Log[ATP] (M) Singlechannelconductance(pS) Surfaceexpression(a.u.) A1513T IC50: 148 ± 9µM Fs1524 IC50: 51 ± 4µM Relativechannelactivity Table 1 Summary of clinical phenotypes Individual ABCC9 Family history Gender, LVEDD (mm)a EF (%)b Coronary Cardiac rhythmc Outcome mutation of DCM age at diagnosis (y) angiography Individual 1 Frameshift No Male, 55 65 (55) 23 Normal Ventricular Death from (Fs1524) tachycardia heart failure at age 60 y Individual 2 Missense Yes Female, 40 89 (52) 15 Normal Ventricular Under intensive (A1513T) tachycardia therapy Male, 54 81 (53) 13 Normal Ventricular Death from heart Father of individual 2 tachycardia failure at age 55 y aLVEDD, left ventricular end-diastolic dimension. Login to comment 48 ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr L E T T E R S 384 VOLUME 36 | NUMBER 4 | APRIL 2004 NATURE GENETICS -20 -10 0 10 20 40 0.1 0.2 0.3 0.4 0.5 0 5 10 15 20 0 2 4 6 NS 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8e d 209 84 40.6 31.9 18.5 WT MBP 84 84 b ATP Pi Blank MBP WT A1513T Fs1524 MBP-NBD2 c f Fluorescence/[TNP-ATP](a.u./µM) Total [TNP-ATP] (µM) WT Fs1524 A1513T Fluorescence (a.u.) ATPaseactivity -1 (min) [ADP] (mM) [ATP] -1 (mM) ATPaseactivity-1(min) WT Fs1524 A1513T WT Fs1524 A1513T Time (min) Anti-MBP Anti-SUR2A Fs1524 A1513T F(a.u.) a [Pi](µM) WT Fs1524 A1513T 0.00 0.0 1.0 2.0 3.0 4.0 0.12 0.10 0.08 0.06 0.04 0.02 0.00 1.00.50.0 0.20 .3.11.00.50.0-0.5-1.0 30 disrupt folding of the C-terminal β-strand and, thus, the tertiary organization of the adjacent second nucleotide binding domain (NBD2) pocket in SUR2A. Login to comment 49 ABCC9 p.Ala1513Thr ATP-induced KATP channel gating was aberrant in both channel mutants (Fig. 2e), suggesting that structural alterations induced by the mutations A1513T and Fs1524 of SUR2A distorted ATP-dependent pore regulation. Login to comment 51 ABCC9 p.Ala1513Thr The A1513T and Fs1524 mutations substantially diminished the maximal rate of the NBD2 ATPase reaction without altering the Michaelis-Menten constant of catalysis (Fig. 3d). Login to comment 52 ABCC9 p.Ala1513Thr A1513T reduced the product-dependent inhibition of the NBD2 ATPase more substantially than Fs1524 (Fig. 3e) but produced a less severe delay in the pre-steady state profile of product accumulation (Fig. 3f). Login to comment 53 ABCC9 p.Ala1513Thr Thus, the mutations A1513T and Fs1524 compromise ATP hydrolysis at SUR2A NBD2, generating distinct reaction kinetic defects. Login to comment 54 ABCC9 p.Ala1513Thr Aberrant catalytic properties in the A1513T and Fs1524 mutants translated into abnormal interconversion of discrete conformations in the NBD2 ATPase cycle (Fig. 4a). Login to comment 58 ABCC9 p.Ala1513Thr In contrast, the A1513T mutation delayed the ATPase cycle in the SUR-ADP conformation, by reducing the rate constant defining ADP dissociation (k4) by a factor of 2, and reduced the ADP association rate constant (k04) by a factor of 10 (Fig. 4a-c). Login to comment 59 ABCC9 p.Ala1513Thr The ATPase cycle in both A1513T and Fs1524 mutants was abnormally delayed in a posthydrolytic conformation, SUR-ADP-Pi or SUR-ADP. Login to comment 62 ABCC9 p.Ala1513Thr Consequently, alterations in hydrolysis-driven SUR2A conformational probability induced by A1513T and Fs1524 translated into abnormal ATP sensitivity of mutant channels (Fig. 2e). Login to comment 65 ABCC9 p.Ala1513Thr Compared to the wild type and at any given ATP level, A1513T and Fs1524 mutants were less responsive in ADP-induced redistribution of post- (Fig. 4e) and prehydrolytic (Fig. 4f) conformations. Login to comment 68 ABCC9 p.Ala1513Thr Thus, the mutations A1513T and Figure 3 SUR2A NBD2 mutants have normal ATP binding but altered ATPase properties. Login to comment 70 ABCC9 p.Ala1513Thr An antibody against the last 12 amino acids of SUR2A recognized the wild-type (WT) and A1513T mutant but not Fs1524, whereas an antibody raised against MBP reacted with all constructs. Login to comment 74 ABCC9 p.Ala1513Thr (c) The A1513T and Fs1524 NBD2 mutations reduced ATPase activity measured from γ-32P liberation after [γ-32P]ATP hydrolysis. Login to comment 76 ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr (d,e) ATP and ADP dependence of NBD2 ATPase activities measured by spectrophotometry showed vmax values of 9.98 ± 0.34 min-1 in wild-type (WT), 6.07 ± 0.18 min-1 in A1513T and 5.69 ± 0.29 min-1 in Fs1524 with Michaelis-Menten constants at 0.11 ± 0.02, 0.094 ± 0.013 and 0.084 ± 0.010 mM, respectively (equation 3). Login to comment 78 ABCC9 p.Ala1513Thr ABCC9 p.Ala1513Thr ADP-dependent inhibition of the NBD2 ATPase (at 2 mM ATP) was characterized by an ADP-dissociation constant (KADP) of 8.6 ± 0.3 µM in wild-type versus 24.9 ± 5.6 and 11.0 ± 1.5 in the A1513T and Fs1524 mutants, respectively (equation 5). Login to comment 79 ABCC9 p.Ala1513Thr (f) Both pre-steady state and steady-state reaction rates were altered by A1513T and Fs1524 mutations in stopped-flow experiments. Login to comment 104 ABCC9 p.Ala1513Thr (c) Rate-limiting steps in the SUR2A ATPase cycle are ADP dissociation (k4) for wild-type (WT), Pi dissociation (k3) for Fs1524 and abnormally increased k4 for A1513T. Login to comment 111 ABCC9 p.Ala1513Thr Both Fs1524 and A1513T diminished the ADP-responsiveness of PΣADP and PE-ATP. Login to comment 113 ABCC9 p.Ala1513Thr (g) ADP-scavenging creatine kinase (0.01 U ml-1, 5 mM creatine phosphate) accelerates the ATPase in the wild type (WT) but not in Fs1524 and A1513T mutants. Login to comment 115 ABCC9 p.Ala1513Thr (h,i) After coexpression of Kir6.2, channel activity, at 0.3 mM ATP, in the presence and absence of 0.3 mM ADP was measured in wild-type (WT; n = 5) SUR2A and in Fs1524 (n = 4) and A1513T (n = 6) SUR2A mutants in inside-out patches. Login to comment