Activation of ATP-sensitive K(+) channels by epoxyeicosatrienoic acids in rat cardiac ventricular myocytes

Abstract

1. We examined the effects of epoxyeicosatrienoic acids (EETs), which are cytochrome P450 metabolites of arachidonic acid (AA), on the activities of the ATP-sensitive K(+) (K(ATP)) channels of rat cardiac myocytes, using the inside-out patch-clamp technique. 2. In the presence of 100 microM cytoplasmic ATP, the K(ATP) channel open probability (P(o)) was increased by 240 +/- 60 % with 0.1 microM 11,12-EET and by 400 +/- 54 % with 5 microM 11,12-EET (n = 5-10, P < 0.05 vs. control), whereas neither 5 microM AA nor 5 microM 11,12-dihydroxyeicosatrienoic acid (DHET), which is the epoxide hydrolysis product of 11,12-EET, had any effect on P(o). 3. The half-maximal activating concentration (EC(50)) was 18.9 +/- 2.6 nM for 11,12-EET (n = 5) and 19.1 +/- 4.8 nM for 8,9-EET (n = 5, P = n.s. vs. 11,12-EET). Furthermore, 11,12-EET failed to alter the inhibition of K(ATP) channels by glyburide. 4. Application of 11,12-EET markedly decreased the channel sensitivity to cytoplasmic ATP. The half-maximal inhibitory concentration of ATP (IC(50)) was increased from 21.2 +/- 2.0 microM at baseline to 240 +/- 60 microM with 0.1 microM 11,12-EET (n = 5, P < 0.05 vs. control) and to 780 +/- 30 microM with 5 microM 11,12-EET (n = 11, P < 0.05 vs. control). 5. Increasing the ATP concentration increased the number of kinetically distinguishable closed states, promoting prolonged closure durations. 11,12-EET antagonized the effects of ATP on the kinetics of the K(ATP) channels in a dose- and voltage-dependent manner. 11,12-EET (1 microM) reduced the apparent association rate constant of ATP to the channel by 135-fold. 6. Application of 5 microM 11,12-EET resulted in hyperpolarization of the resting membrane potential in isolated cardiac myocytes, which could be blocked by glyburide. 7. These results suggest that EETs are potent activators of the cardiac K(ATP) channels, modulating channel behaviour by reducing the channel sensitivity to ATP. Thus, EETs could be important endogenous regulators of cardiac electrical excitability.

Figure 1. Activation of cardiac K_ATP channels by 11,12-EET

A, macroscopic K_ATP currents of rat ventricular myocytes were recorded continuously from a membrane potential of -60 mV in an inside-out patch. B, dose-dependent inhibition of K_ATP activity by ATP (1-5 mm), in the presence of 0 (left) or 5 μm (right) 11,12-EET. Dotted lines in A and B represent the closed (C) channel level. C, relationship of the normalized open probability (P_o) plotted against the ATP concentration in the presence of 0 (•, n = 5), 0.1 μm (▪, n = 5), or 5 μm (▴, n = 10) 11,12-EET. Each point represents the mean ±s.e.m., and the continuous lines represent the best fits by a Hill equation.

Figure 2. Comparing the effects of AA, 8,9-EET, 11,12-EET and 11,12-DHET on cardiac K_ATP channels

A, raw current tracings showing the dose-dependent effect of 11,12-EET (0.001-1 μm) on the K_ATP channel activity. Single channel K_ATP currents were recorded at -60 mV in the presence of 100 μm cytoplasmic ATP. B, dose-response relationships of 8,9-EET (○) and 11,12-EET (•) on K_ATP channel activation. K_ATP currents were recorded at -60 mV in the presence of various concentrations of EETs. Data are presented as means ±s.e.m. (n = 5), and the continuous lines represent the best fits by a Hill equation. C, bar graph comparing the effects (percentage change of P_o) of ethanol control, 5 μm AA, 5 μm 11,12-EET and 5 μm 11,12-DHET. P_o was measured at -60 mV, and the data are presented as means ±s.e.m., *P < 0.05 vs. Control.

Figure 3. Effect of ATP on cardiac K_ATP channel kinetics

Single K_ATP channel activity was recorded at -60 mV in the presence of 1 μm (A), 10 μm (B), 100 μm (C) and 1000 μm (D) cytoplasmic ATP. Representative K_ATP channel tracings are shown on the left with selected segments expanded to show more details. The corresponding channel open dwell-time histograms and the closed dwell-time histograms are displayed to the right of the tracings. The dashed lines represent the distribution of the exponential components determined by the likelihood ratio test. The continuous line represents the best fit of data using TAC software. The values of the open (τ_o) and closed (τ_c) time constants are displayed above each histogram. The relative weight of each closed time constant is represented in parentheses as a percentage of the total, so that the sum of all weights is 100%.

Figure 4. Effects of ATP on the mean open time, the intraburst closed time and the mean interburst duration of the K_ATP channels

Reciprocals of the K_ATP channel mean open time, τ_o (A), the intraburst closed time, τ_c1(B), and the mean interburst duration (C). D, the mean burst duration plotted against cytoplasmic ATP concentration. In D the effects of 11,12-EET (1 μm, ♦) on the reciprocal of the mean burst duration were compared with controls (⋄) that contained no added EET. Data were fitted using the equation 1/σ= (k_c1o/(k_c1o+k_oc1))k_A[ATP]. Since the flickering transitions are fast and reach equilibrium, then k_oc1/k_c1o should be a constant, independent of [ATP]. Moreover, to allow long burst durations, k_c1o should be faster or greater than k_oc1. Hence, our data suggest the following relationship: 0 < k_oc1 < k_c1o≤ 1.5 ms⁻¹, which suggests 0 < k_c1o/(k_c1o+k_oc1) ≤ 0.5 ms⁻¹; thus, 1/σ= 0.5k_A[ATP]. From the slopes of these curves, the ATP apparent association rate constants with the K_ATP channels could be estimated. Data are represented as means ±s.e.m. (n = 3).

Figure 5. Voltage-dependent effects of 11,12-EET on the cardiac K_ATP channels

A, single K_ATP channel currents were recorded at membrane potentials from -80 to +80 mV in the presence of 100 μm cytoplasmic ATP and 0.1 μm 11,12-EET. B, the K_ATP channel current-voltage relationships are plotted, showing the characteristic weak inward rectification at strong depolarizations. Continuous lines represent the best fits using equations described in Methods. Single channel conductance (γ) was 80.9 ± 0.5 pS (n = 4) in the presence of 100 μm ATP (○, control) and was not altered by the addition of 0.1 μm 11,12-EET (79.6 ± 0.6 pS, •, p = n.s. vs. control). C, K_ATP channel P_o in the presence of 100 μm ATP (^) or 100 μm ATP plus 0.1 μm 11,12-EET (•) is plotted against membrane potential (-80 to +80 mV; n = 3).

Figure 6. Dose-dependent effects of 11,12-EET on cardiac K_ATP channel kinetics

Single K_ATP channel activities were recorded at -60 mV in the presence of 100 μm ATP and 0 μm (A), 0.01 μm (B), 0.1 μm (C) and 1 μm (D) 11,12-EET. Representative K_ATP channel tracings are shown on the left with selected segments enlarged to show more details. The corresponding channel open time histograms and the closed time histograms are shown to the right of the tracings. The values of the open and closed time constants are displayed above each histogram. The relative weight of each closed time constant is given in parentheses and the sum of all weights is equal to 100%.

Figure 7. Effects of 11,12-EET on the mean open time, the intraburst closed time and the mean interburst duration of the K_ATP channels

Reciprocals of the K_ATP channel mean open time, τ_o (A), the intraburst closed time, τ_c1 (B) and the mean interburst duration (C), which are plotted against 11,12-EET concentration. Experiments were performed at -60 mV in the presence of 100 μm ATP. Data are presented as means ±s.e.m. (n = 4).

Figure 8. Comparison of the transition rate constants between 10 μm ATP alone and 100 μm ATP plus 1 μm 11,12-EET

The transition rates between the channel open and closed states were calculated and compared for 10 μm ATP alone (Control; open symbols and dotted lines) and 100 μm ATP plus 1 μm 11,12-EET (filled symbols and continuous lines). The transition rate constants from the intraburst closed state to the open state, k_c1o (A), the open state to the intraburst closed state, k_oc1 (B), the open state to the interburst closed state, k_A (C) and the interburst closed state to the open state, k_B (D) (n = 3) are plotted against membrane potential (-20 to -80 mV). The data were fitted using a single exponential equation. The charge movement associated with each rate constant was estimated by fitting the voltage dependence of each transition rate to the following exponential function: k = k_oexp(zδFV/RT), where k_o represents the value of the voltage-dependent rate constants at 0 mV, F is the Faraday constant, R is the universal gas constant, T is the absolute temperature, and zδ represents the associated gating charges.

Figure 9. Effect of 11,12-EET on the resting membrane potential in cardiac myocytes

A, recordings of action potential from an isolated cardiac myocyte at 37 °C using current-clamp techniques. The patch-clamp pipette contained 5 mm ATP and action potentials were elicited by 1 nA stimuli at 0.5 Hz. Tracings at baseline (Control), with 11,12-EET (5 μm), and with 11,12-EET (5 μm) plus glyburide (2 μm) are superimposed for comparison. B, bar graph comparing the resting membrane potentials of isolated cardiac myocytes under control conditions, and in the presence of 11,12-EET (5 μm) and 11,12-EET (5 μm) plus glyburide (2 μm). The data are presented as means ±s.e.m., n = 7, *P < 0.05vs. Control.