channels are heteromultimers composed of Kir6.2, an inwardly rectifying potassium channel pore, and the regulatory SUR2A subunit, an ATPase-harboring ATP-binding cassette protein1-4
. The SUR2A subunit recognizes and processes intracellular energetic signals, through its nucleotide binding domains, endowing the KATP
channel-enzyme complex with a metabolic decoding capacity5
. Potassium movement through Kir6.2 does not require energy expenditure6
, yet ATP hydrolysis at SUR2A is integral in the transduction of metabolic signals from cellular energetic pathways to the channel pore5,7
. In this way, KATP
channels set membrane excitability in response to stress challenge and preserve cellular energy-dependent functions8-12
. Thereby, the KATP
channel complex has a vital role in securing cellular homeostasis under stress12
channel dysregulation, recently recognized in a transgenic model of cardiomyopathy, has been associated with compromised stress tolerance13
. In fact, in the stressed heart, knockout of KATP
channel genes precipitates intracellular calcium overload predisposing to myocardial damage, arrhythmia and death preventable by calcium-channel blockade12
. Indeed, KATP
channel-deficient hearts are susceptible to calcium-dependent maladaptive remodeling under chronic hemodynamic load, progressing to congestive heart failure (G.C. Kane, F.O., D.M.H., T. Miki, S. Seino and A.T., unpublished data). Although improper myocellular calcium handling contributes to the pathogenesis of dilated cardiomyopathy14,15
, a malignant disorder characterized by heart failure and increased susceptibility to metabolic stressors16-18
, little is known about KATP
channels in human heart disease. Here, we report the first mutations in ABCC9
, encoding SUR2A. Identified in individuals with idiopathic dilated cardiomyopathy, these defects in the regulatory KATP
channel subunit disrupt catalysis-dependent gating and impair metabolic decoding, establishing a previously unrecognized mechanism of channel malfunction in human disease.
Scans for mutation in genomic DNA in a cohort of 323 individuals with idiopathic dilated cardiomyopathy identified two heterozygous mutations in exon 38 of ABCC9, which encodes the C-terminal domain of SUR2A specific to the cardiac splice variant of the regulatory KATP channel subunit (). Both individuals with mutations in ABCC9 had severely dilated hearts with compromised contractile function and rhythm disturbances (). DNA sequencing of one mutated allele identified a 3-bp deletion and 4-bp insertion mutation (4570-4572delTTAinsAAAT), causing a frameshift at Leu1524 and introducing four anomalous terminal residues followed by a premature stop codon (Fs1524; ). The second mutated allele harbored a missense mutation (4537G→A) causing the amino acid substitution A1513T (). The identified frameshift and missense mutations occurred in evolutionarily conserved domains of the C terminus of SUR2A (), and neither mutation was present in 500 unrelated control individuals.
Figure 1 KATP channel mutations in dilated cardiomyopathy. (a) The regulatory SUR2A subunit (nucleotide-binding domains NBD1 and NBD2 with Walker A and B motifs and a linker L region) forms cardiac KATP channels by assembling with the pore-forming Kir6.2 subunit (more ...)
Summary of clinical phenotypes
The C terminus of SUR proteins contributes to KATP
, and Fs1524 and A1513T SUR2A mutants, reconstituted with Kir6.2, had reduced expression in the plasma membrane (). Yet, mutant KATP
channel complexes formed functional channels with intact pore properties (). Structural molecular dynamics simulation showed that the residues Ala1513 and Leu1524 flank the C-terminal β-strand in close proximity to the signature Walker A motif (), required for coordination of nucleotides in the catalytic pocket of ATP-binding cassette proteins21,22
. Replacement of Ala1513 with a sterically larger and more hydrophilic threonine residue or truncation of the C terminus caused by the Fs1524 mutation would disrupt folding of the C-terminal β-strand and, thus, the tertiary organization of the adjacent second nucleotide binding domain (NBD2) pocket in SUR2A. ATP-induced KATP
channel gating was aberrant in both channel mutants (), suggesting that structural alterations induced by the mutations A1513T and Fs1524 of SUR2A distorted ATP-dependent pore regulation.
Figure 2 SUR2A mutant proteins, coexpressed with Kir6.2, alter KATP channel function. (a) Fs1524 and A1513T reduced KATP channel trafficking by ~70% and ~30%, probed immunologically by SUR surface expression in Xenopus laevis oocytes. (b) Single (more ...)
On further examination, purified wild-type and mutant NBD2 constructs had similar ATP binding but reduced ATP hydrolytic activities (). The A1513T and Fs1524 mutations substantially diminished the maximal rate of the NBD2 ATPase reaction without altering the Michaelis-Menten constant of catalysis (). A1513T reduced the product-dependent inhibition of the NBD2 ATPase more substantially than Fs1524 () but produced a less severe delay in the pre-steady state profile of product accumulation (). Thus, the mutations A1513T and Fs1524 compromise ATP hydrolysis at SUR2A NBD2, generating distinct reaction kinetic defects.
Figure 3 SUR2A NBD2 mutants have normal ATP binding but altered ATPase properties. (a) Affinity-purified mutant or wild-type SUR2A NBD2, cloned in-frame with maltose binding protein (MBP), on SDS gels. An antibody against the last 12 amino acids of SUR2A recognized (more ...)
Aberrant catalytic properties in the A1513T and Fs1524 mutants translated into abnormal interconversion of discrete conformations in the NBD2 ATPase cycle (). Each mutation doubled the rate constant of the SUR-ATP to SUR-ADP-Pi conversion (k2), reducing the lifetime of SUR2A in the prehydrolytic state (). Moreover, the rate constant of the SUR-ADP-Pi to SUR-ADP transition (k3) was one hundred times lower in the Fs1524 mutant, markedly extending the lifetime of the SUR-ADP-Pi conformation and ‘jamming’ the ATPase cycle (). Thus, in contrast to the catalytic reaction in the wild type, where the rate-limiting step is ADP dissociation (k4), the Fs1524 ATPase is characterized by rate-limiting Pi dissociation (k3; ). 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 ().
Figure 4 Altered kinetics of the ATPase cycle lead to disrupted metabolic decoding by KATP channels. (a) Rate constants defining the SUR2A ATPase reaction were derived from pre-steady state kinetics of Pi liberation in wild-type (WT) and mutant NBD2 constructs (more ...)
The ATPase cycle in both A1513T and Fs1524 mutants was abnormally delayed in a posthydrolytic conformation, SUR-ADP-Pi
or SUR-ADP. Although they had distinct patterns of lifetime distribution, each mutation diminished the likelihood that SUR2A could adopt a prehydrolytic conformation and increased the probability of posthydrolytic conformations (). Individual conformations of the SUR2A ATPase cycle have distinct impacts on KATP
channel regulation, with the prehydrolytic SUR-ATP state promoting channel closure and the posthydrolytic SUR-ADP-Pi
and SUR-ADP states favoring channel activation5
. Consequently, alterations in hydrolysis-driven SUR2A conformational probability induced by A1513T and Fs1524 translated into abnormal ATP sensitivity of mutant channels ().
Under metabolic stress, the increase in intracellular ADP favors posthydrolytic ADP-bound conformations associated with antagonism of ATP-induced KATP
channel pore inhibition5,10
. ADP-dependent KATP
channel regulation, and thus the channel’s stress responsiveness, are represented by the relative shift from prehydrolytic to posthydrolytic of the conformational probability (P) of the ATPase induced by changes in ADP (dP/d[ADP]). Compared to the wild type and at any given ATP level, A1513T and Fs1524 mutants were less responsive in ADP-induced redistribution of post- () and prehydrolytic () conformations. Accordingly, metabolic pathways, like the creatine kinase phosphotransfer system23
, effectively regulated ATPase activity of wild-type but not mutant SUR2A (). In fact, aberrant catalysis in mutant SUR2A generated defective KATP
channel phenotypes characterized by abnormal responses to both ATP () and ADP (), mediators of the cellular energetic state. Thus, the mutations A1513T and Fs1524 altered intrinsic catalytic properties of the SUR2A ATPase, compromising proper translation of cellular energetic signals into KATP
channel-mediated membrane electrical events.
channel mutations, identified here in two individuals with dilated cardiomyopathy, underscore the essential role of the intrinsic enzymatic reaction in cardiac channel pore regulation. Aberrant kinetics of hydrolysis at the regulatory channel subunit, despite unaltered nucleotide binding, produced defective metabolic signal decoding. Thus, nucleotide-dependent regulation of the stress-responsive KATP
channel is based not only on conventional competition between ATP and ADP at nucleotide binding domains24
but also on the profile of conformational interconversion driven by the catalytic cycle in the regulatory channel subunit. Traditionally linked to defects in ligand interaction, subunit trafficking or pore conductance10,20,25
, human cardiac KATP
channel dysfunction provoked by alterations in the catalytic module of the channel complex establishes a new mechanism for channelopathy. In this way, defective ion channel function would confer susceptibility to dilated cardiomyopathy.