K_ATP 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 K_ATP channels by assembling with the pore-forming Kir6.2 subunit (transmembrane domains M1 and M2). Analysis of exon 38 in ABCC9 genomic DNA, which encodes the C terminus of the SUR2A protein, identified abnormal chromatograms indicative of mutations in individuals with dilated cardiomyopathy (DCM). Sequencing identified frameshift (Fs1524; individual 1) and missense (A1513T; individual 2) mutations. The family of individual 1 was unavailable for segregation analysis. The mutation in individual 2 was not present in the proband’s mother, suggestive of inheritance from the affected father (DNA was unavailable). (b) SUR2A residues encoded by exon 38 in wild-type and mutant ABCC9 sequences in humans and other species.

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 the wild-type (WT) and A1513T mutant but not Fs1524, whereas an antibody raised against MBP reacted with all constructs. Molecular sizes are given in kDa. (b) The binding affinity of the fluorescent ATP analog TNP-ATP was similar for wild-type (WT) and mutant NBD2 in Scatchard analysis. Inset: Specific TNP-ATP binding-induced signal detected as difference between total and nonspecific (NS) fluorescence (F) of the ATP analog in the absence of NBD constructs. (c) The A1513T and Fs1524 NBD2 mutations reduced ATPase activity measured from γ-³²P liberation after [γ-³²P]ATP hydrolysis. WT, wild-type. (d,e) ATP and ADP dependence of NBD2 ATPase activities measured by spectrophotometry showed v_max 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). The ATP-dependence of the NBD2 ATPase was determined at 0 ADP in the presence of creatine kinase (0.01 U ml⁻¹) and creatine phosphate (5 mM). ADP-dependent inhibition of the NBD2 ATPase (at 2 mM ATP) was characterized by an ADP-dissociation constant (K_ADP) 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). (f) Both pre-steady state and steady-state reaction rates were altered by A1513T and Fs1524 mutations in stopped-flow experiments. Solid lines represent the fit of experimental data with the system of differential equations (equation 6), allowing evaluation of the rate kinetic constants of the NBD2 ATPase reaction. WT, wild-type.

SUR2A mutant proteins, coexpressed with Kir6.2, alter K_ATP channel function. (a) Fs1524 and A1513T reduced K_ATP channel trafficking by ∼70% and ∼30%, probed immunologically by SUR surface expression in Xenopus laevis oocytes. (b) Single channel conductance and inward rectification (not shown) of wild-type and mutant channels, expressed in HEK293 cells, were identical, indicating that biophysical pore properties were intact. (c,d) Atomic model of SUR2A NBD2. Red, α-helix; blue, β-strand; yellow, Walker motifs (W_A and W_B). Missense A1513T (cyan) and frameshift L1524 (magenta) mutations frame the β-strand adjacent to Walker motifs that coordinate NBD2-mediated catalysis. Representative hydrogen bonds that stabilize Walker A and the associated C terminus β-strand are indicated by dashed green lines. Red, oxygen atoms; blue, nitrogen; mustard, sulfur (Cys1345). (e) Abnormal ATP-induced K_ATP channel inhibition in mutants. Channel inhibition was expressed relative to activity in the absence of ATP and measured at −60 mV in inside-out patches. Solid curves represent Hill equation fits of experimental data, with the ATP concentration required for half-inhibition (IC₅₀) indicated for wild-type and mutant channels.

Altered kinetics of the ATPase cycle lead to disrupted metabolic decoding by K_ATP channels. (a) Rate constants defining the SUR2A ATPase reaction were derived from pre-steady state kinetics of P_i liberation in wild-type (WT) and mutant NBD2 constructs (n = 4 each). (b) Lifetime distribution of individual conformations (equation 10) in wild-type (WT) and mutant NBD2 ATPase cycle. (c) Rate-limiting steps in the SUR2A ATPase cycle are ADP dissociation (k₄) for wild-type (WT), P_i dissociation (k₃) for Fs1524 and abnormally increased k₄ for A1513T. Fs1524 has the lowest k₀₄ rate constant defining ADP association. (d) In the absence of ADP, both NBD2 mutants have lower probability to adopt ATP-bound (P_E-ATP) and higher probability to adopt ATP-bound (P_ΣADP) conformations in a wide range of ATP concentrations. P_ΣADP is the sum of E-ADP-P_i (P_E-ADP-Pi) and E-ADP (P_E-ADP) probabilities (equations 7-9). WT, wild-type. (e,f) ADP-induced modulation of probabilities in the NBD2 ATPase cycle intermediates determined as dP_ΣADP/d[ADP] and dP_E-ATP/d[ADP] derivatives at different ATP levels. In response to ADP, P_ΣADP increases whereas P_E-ATP decreases, defining the sign of respective derivatives. Both Fs1524 and A1513T diminished the ADP-responsiveness of P_ΣADP and P_E-ATP. WT, wild-type. (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. Rate of P_i liberation was measured at 2 mM ATP using spectrophotometry. (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. In addition to reduced ATP sensitivity, both mutants had blunted ADP channel response relative to K_ATP channel activity at zero nucleotide levels.