Intracellular ATP binding is required to activate the slowly activating K+ channel I(Ks)

Abstract

Gating of ion channels by ligands is fundamental to cellular function, and ATP serves as both an energy source and a signaling molecule that modulates ion channel and transporter functions. The slowly activating K(+) channel I(Ks) in cardiac myocytes is formed by KCNQ1 and KCNE1 subunits that conduct K(+) to repolarize the action potential. Here we show that intracellular ATP activates heterologously coexpressed KCNQ1 and KCNE1 as well as I(Ks) in cardiac myocytes by directly binding to the C terminus of KCNQ1 to allow the pore to open. The channel is most sensitive to ATP near its physiological concentration, and lowering ATP concentration in cardiac myocytes results in I(Ks) reduction and action potential prolongation. Multiple mutations that suppress I(Ks) by decreasing the ATP sensitivity of the channel are associated with the long QT (interval between the Q and T waves in electrocardiogram) syndrome that predisposes afflicted individuals to cardiac arrhythmia and sudden death. A cluster of basic and aromatic residues that may form a unique ATP binding site are identified; ATP activation of the wild-type channel and the effects of the mutations on ATP sensitivity are consistent with an allosteric mechanism. These results demonstrate the activation of an ion channel by intracellular ATP binding, and ATP-dependent gating allows I(Ks) to couple myocyte energy state to its electrophysiology in physiologic and pathologic conditions.

Keywords: heart failure; ischemia.

Fig. 1.

ATP-dependent activation of I_Ks channels. (A) KCNQ1 + KCNE1 (hI_Ks) currents in inside-out patches run down or up in 0 (red), 0.5 (green), and 5 mM (blue) [ATP] from that immediately after patch excision (black, I₀). Voltage pulses were +80 mV from a −80-mV holding potential. (Right) I_t, I₀ tail current amplitudes. (B) ATP dose–response of WT (black) and Q357R (red, also scaled to the WT currents at 20 mM ATP) I_Ks, and fits to the Hill equation (solid curves) with Hill coefficient 1 and 1.1, and EC₅₀ 1.66 and 9.60 mM, respectively. (C) Currents of WT (Left) and Q357R (Center) hI_Ks recorded from inside-out patches and G–V relations after patch excision in solutions containing various [ATP]. Solid curves are fits of Boltzmann equation (Right; Materials and Methods) with V_1/2 (mV) at 0.5, 5, and 20 mM [ATP], respectively: WT, 23.7 ± 3.5 (black), 25.6 ± 3.2 (purple), 25.4 ± 5.0 (blue); Q357R, 53.2 ± 2.5 (red), 30.8 ± 2.6 (green), and 26.2 ± 3.8 (cyan). (D) Normalized ATP dose–response of WT hI_Ks channel activation in control solution [PIP₂] = 100 µM (black), 5 µM PIP₂ (green), 100 µM Ca²⁺ (red), and S27D/S92D hI_Ks in control solution (blue; Materials and Methods). Five and 100 µM [PIP₂] are 50% and 100% of saturation for I_Ks channel activation, respectively (14). Dashed curves in B and C are the fittings of the model in Fig. 7A.

Fig. 2.

ATP activates I_Ks and shortens APs in cardiac myocytes. (A) Voltage protocol and current traces at different [ATP]s in the patch pipette in the absence (red) and presence (black) of chromanol 293B (10 µM). (B) I_Ks vs. [ATP]. n = 5, error bars are SD. Smooth curve: fitting to the data from 1 to 25 mM [ATP] with Hill equation; Hill coefficient = 1 and EC₅₀ = 1.4 mM. The 0 [ATP] data were not included in the curve fitting because 0 [ATP] inside the cell could not be achieved in live myocytes; the predicted value of [ATP] for that magnitude of I_Ks (gray open circle) is 0.64 mM. Potassium ATP at a concentration of 3 mM and 10 mM (red open circles) were also used in the pipette solution to substitute for MgATP. (C) AP traces in the absence and presence of chromanol 293B, with intracellular infusion of ATP (2–10 mM). (D) Summary of APD₉₀ at various [ATP]. (Upper) The APD for each cell is plotted in the absence and presence of chromanol 293B. (Lower) Average percentage increase in APD induced by chromanol 293B. Note a significantly larger APD prolongation induced by chromanol 293B at 5 or 10 mM ATP compared with that at 2 or 3 mM ATP (P < 0.01, individual t tests). Potassium ATP at two different concentrations (2 mM and 10 mM, in gray) were used to substitute for MgATP in the pipette solution.

Fig. 3.

ATP binding to KCNQ1. (A) hI_Ks tail current amplitude elicited by voltage pulses from −80 to +80 mV in intracellular solutions containing 1.5 mM of various nucleotides: ATP (blue), GTP (green), AMP–PNP (pink), ADP (red), AMP (purple), and no nucleotide control (black). (B) Western blot of channel proteins pulled down with avidin beads after UV light photo–cross-linking of the biotin-containing AB11 (Top) or after biotin treatment of intact cells to detect expression in the membrane (Middle). G_beta is a cytoplasmic protein. Antibodies against KCNQ1 (for lanes of uninjected, hI_Ks, and KCNQ1), KCNQ2 (KCNQ2), KCNQ3 (KCNQ3), and G_beta (low bands) were used. (C) Whole-cell currents of WT and chimeric KCNQ1 and KCNQ2/KCNQ3 channels (Upper) and time dependence of current amplitude after inside-out patch excision without application of intracellular ATP (Lower). WT hI_Ks (black), WT KCNQ2/KCNQ3 (blue), and Q2ctQ1/Q3ctQ1 (red). (D) Mutation scan of all cytosolic basic residues in KCNQ1. (Inset) Experimental design for ATP sensitivity screen. ATP dose–response curves of WT (red) and a hypothetical mutant hI_Ks with reduced ATP sensitivity (green). Three concentrations of ATP are used: 0.5, 1.5, and 20 mM.

Fig. 4.

ATP binds in the C terminus of KCNQ1. (A) ATP dose–response curve of WT and key mutant I_Ks channels. (B) The KCNQ1 motifs important for ATP interaction (Upper) and EC₅₀ of ATP dose–response of mutant hI_Ks channels (Lower). Due to insolubility of ATP above 20 mM, dose–response for some mutants did not reach saturation, leading to underestimated EC₅₀. Asterisk indicates no current expression. (C) Whole-cell currents of mutant hI_Ks. (D) Western blot to detect AB11 labeling of mutant I_Ks. (Upper) AB11 labeling of mutant I_Ks and G_β in the whole-cell lysate to indicate similar inputs. (Lower) Western blot probing for biotinylated mutant I_Ks and G_β in the membrane. G_β is a cytoplasmic protein.

Fig. 5.

ATP is not required for VSD movement. (A) VCF recordings of the WT and ATP-binding disruptive mutant KCNQ1 channels. Whole-cell current (Upper) and fluorescence signal (Lower) of WT (black), W379S (red), and R380S/R397W (green) in response to increasing voltages. (B) Superimposed fluorescence signal (Left) of WT (black), W379S (red), and R380S/R397W (green) in response to a series of voltage pulses with increasing voltages. The currents activation time constants in response to voltages (Right). (C) Steady-state fluorescence changes vs. voltage (F–V).

Fig. 6.

ATP is not required for VSD–PGD coupling. (A) VCF recordings of WT KCNQ1 (black), W379S (red), and R380S/R397W (green) with and without L353K. (B) Steady-state fluorescence change vs. voltage (F–V). Solid curves are fits of a Boltzmann equation with V_1/2. WT (black solid): −53.5 ± 1.1 mV; W379S (red solid): −56.2 ± 0.8 mV; R380S/R397W (green solid): −50.3 ± 0.9 mV; L353K (black open): −72.3 ± 0.8 mV; W379S/L353K (red open): −76.9 ± 0.9 mV; R380S/R397W/L353K (green open): −70.9 ± 1.1 mV.

Fig. 7.

ATP is required for pore opening. (A) The scheme of voltage and ATP-dependent activation of hI_Ks channels. Voltage sensor movements are simplified as one ATP-independent transition between the resting (V black) and activated (V red) state; the transition of the pore from closed (P black) to the open (P red) state can happen only after voltage sensor activation and ATP binding. K1: 300 M⁻¹, L(V): 4.2 × 10⁻⁴exp(0.94VF/RT) (V, voltage; F, Faraday constant; R, gas constant; and T, absolute temperature), and K2: 1,340 and 360 for WT and Q357R I_Ks, respectively, are obtained from fittings to ATP dose–responses (Fig. 1B) and G–V relations at various [ATP] (Fig. 1C). (B) VCF recordings and F–V relations of Q357R and Q357R/R380S/R397W. (C) Activation (Upper) and deactivation (Lower) kinetics of fluorescence for both WT and Q357R. (D) G–V relations of hI_Ks WT (black), R380S (green), K393M (blue), and R397W (red). Smooth curves in B–D are Boltzmann fits to the data.