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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Charles A. Dana Research Institute and Harvard-Thorndike Laboratory, and Cardiovascular Division, Department of Medicine, Beth Israel Deaconess Medical Center, Boston; 2Department of Medicine, Massachusetts General Hospital, Boston; 3Department of Anesthesia, Brigham and Women’s Hospital, Boston; 4Harvard Medical School, Boston, Massachusetts; and 5Department of Biology, State University of New York at Albany, Albany, New York
Submitted 17 June 2005 ; accepted in final form 20 September 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-subunit (hH1) of human cardiac Na+ channels (hNav1.5) plus 
1-subunits. Extracellular application of 5 µM eicosapentaenoic acid (EPA; C20:5n-3) significantly inhibited INa. The late portion of INa (INa late, measured near the end of each pulse) was almost completely suppressed. INa returned to the pretreated level after washout of EPA. The inhibitory effect of EPA on INa was concentration dependent, with IC50 values of 4.0 ± 0.4 µM for INa peak (INa peak) and 0.9 ± 0.1 µM for INa late. EPA shifted the steady-state inactivation of INa peak by –19 mV in the hyperpolarizing direction. EPA accelerated the process of resting inactivation of the mutant channel and delayed the recovery of the mutated Na+ channel from resting inactivation. Other polyunsaturated fatty acids, docosahexaenoic acid, linolenic acid, arachidonic acid, and linoleic acid, all at 5 µM concentration, also significantly inhibited INa. In contrast, the monounsaturated fatty acid oleic acid or the saturated fatty acids stearic acid and palmitic acid at 5 µM concentration had no effect on INa. Our data demonstrate that the double mutations at the 409 and 410 sites in the D1–S6 region of hH1 induce inactivation-deficient INa and that n-3 PUFAs inhibit mutant INa. human cardiac sodium channel
How the mutant L409C/A410W produces an inactivation-deficient channel, which it does (14), must be different from that produced by the inactivation-dependent mutant IFMQ3. It has been postulated that the mutant L409C/A410W composes part of the receptor site for the docking of the inactivation particle (15). Thus it prevents the Na+ channel inactivation, not by the same action as does the mutant IFM3Q but by disabling the receptor of the inactivation particle so that the inactivation particle is unable to close the Na+ channel.
Mammalian cardiomyocytes show a small, persistent INa, which hypoxia enhances to induce cardiac arrhythmias (5). Blockage of persistent INa in ventricular cardiomyocytes of failing human hearts normalized prolonged action potential and ceased soon after depolarization (10). In the present study, therefore, we investigated the effects of the n-3 PUFAs on persistent INa in HEK-293t cells transfected with the inactivation-deficient mutant (L409C/A410W) of the -subunit of human cardiac Na+ channel. The extracellular application of PUFAs significantly and reversibly inhibited the INa of the mutant.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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50% confluence, transfection of the wild-type cardiac Na+ channel (hNav1.5; 4 µg) or a mutant (3 µg) of the -subunit of the human cardiac Na+ channel (hH1) plus the rat Na+ channel 1-subunit (20 µg) and CD8 cDNA (1 µg) was performed using a calcium phosphate precipitation method (20, 21). Expression of Na+ channels was adequate for current recording. The transfected cells were replated 15 h after transfection in 35-mm dishes (which also served as recording chambers) and were incubated at 37°C in a 5% CO2 incubator. Transfection-positive cells were identified using immunobeads (CD8-Dynabeads M-450; Dynal, Oslo, Norway).
Recording of cardiac INa.
HEK-293t cells coated with CD8 beads were chosen for patch-clamp studies. The pipette solution contained (in mM) 100 CsCl, 40 CsOH, 1 MgCl2, 1 CaCl2, 11 EGTA, 5 MgATP, and 10 HEPES, pH 7.3 with CsOH. The bath solution contained (in mM) 30 NaCl, 100 N-methyl-D-glucamine, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose (pH adjusted to 7.4 with HCl). Glass electrodes (World Precision Instruments, Sarasota, FL) had a resistance of 1 M
when filled with the pipette solution. Whole cell current was recorded according to experimental protocols similar to those used in our previous study (20). Fatty acids (Sigma) were dissolved weekly in 100% ethanol at 10 mM concentration and stored in a nitrogen atmosphere at –20°C before use. The experimental concentration of fatty acids was obtained by diluting the stocks and contained a negligible amount of ethanol, which alone had no effect on the mutated INa. Extracellular solution with various concentrations of fatty acids was exchanged with a rapid perfusion system (18). Experiments were conducted at 22–23°C.
Statistical analysis.
INa values were measured at the points of maximal activated current (INa peak) and residual current near the end of each test pulse (INa late). Activation and steady-state inactivation curves were fitted using a Boltzmann equation, {1/[1 + exp(V – V)/k]}, in which V is the midpoint voltage of the function and k is the slope factor (in mV/e-fold change in current). Concentration-dependent data were fitted using a logistical equation, {(A1 – A2)/[1 + (x/x0)p + A2]}, in which x0 is the center, p is power, A1 is initial y-axis value, and A2 is final y-axis value. The time constant (
) of inactivation was analyzed using least-squares fitting (y = A0 + A1exp–t/
1) (Origin version 6.0 software; Microcal Software, Northampton, MA) with a single exponential function. Data are presented as means ± SE. Results derived from two groups were analyzed using the unpaired Student’s t-test. Statistical differences among the results obtained from three or more experimental groups were determined using ANOVA. P < 0.05 was set as the level for statistical significance.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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plus 1-subunit (L409C/A410W + 1) (Fig. 1A). More than 65% of persistent INa were observed at the end of 400-ms test pulses in HEK-293t cells transfected with inactivation-deficient mutants plus 1-subunits, whereas wild-type INa were almost completely inactivated at the end of 40-ms test pulses (wild-type + 1) (Fig. 1A). INa were activated at approximately –60 mV and reached maximal amplitude at –30 mV for both the mutant and wild-type hH1. To compare the current-voltage relationships between the mutant and the wild-type cardiac Na+ channels, the peak INa amplitudes were normalized to their corresponding maximal currents and plotted against different voltages. Figure 1B shows the similarities in the current-voltage relationship curves of the inactivation-deficient mutant (n = 15) and the wild type (n = 7) of hH1 plus 1-subunits. Normalized whole cell activation conductance curves calculated from peak INa remained comparable between the L409C/A410W mutant and wild-type hH1 (Fig. 1C). The average V and k (slope) values for the fitted functions were –42.2 ± 0.17 mV and 8.6 ± 0.30 mV, respectively, for the mutant (n = 15) and –43.0 ± 0.11 mV and 6.1 ± 0.09 mV, respectively, for the wild type (n = 7) (P > 0.05). These results demonstrate that the double mutations at the 409 and 410 sites in the D1–S6 region of hH1 Na+ channels induce inactivation-deficient channels with persistent INa, but the activation process is not altered.
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Na+ channels were completely inactivated when the prepulse voltages were depolarized to more than –50 mV (Fig. 2C). In contrast, the double mutations at the 409 and 410 sites of hH1 significantly shifted the V of the steady-state inactivation in the hyperpolarization direction with a V value of –90.6 ± 1.7 mV (n = 17, 
= –14.5 mV) (P < 0.05) (Fig. 2C) and a k value of 9.7 ± 1.0 mV. A significant portion of noninactivated currents of L409C/A410W mutant Na+ channels was observed when prepulse voltages were depolarized to more than –50 mV and even up to 80 mV (Fig. 2C). These results suggest that the hH1 mutant induces a significant hyperpolarizing shift of steady-state inactivation and generates a significant portion of noninactivated currents even at positive prepulse voltages.
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Na+ channels. To determine whether the n-3 PUFAs inhibited the long-lasting, persistent INa, we investigated the effects of EPA on INa in HEK-293t cells transfected with the mutant L409C/A410W of hH1 plus 1-subunits. Extracellular application of 5 µM EPA significantly inhibited both INa peak and INa late within 10 s and reached the maximal effect within 7 min (Fig. 3). INa returned to the pretreatment level after washout of EPA with 0.2% fatty acid-free BSA solution. Figure 3 shows the time course of inhibitory effects of 5 µM EPA on INa peak and INa late in a HEK-293t cell expressing L409C/A410W plus 1-subunits. INa late was more sensitive to the inhibitory effect of EPA and was almost completely inhibited, whereas INa peak was inhibited by 60%.
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plus 1-subunits in HEK-293t cells was similar: 4.0 ± 0.4 µM for L409C/A410W and 3.9 ± 0.3 µM for the wild type, respectively. However, INa late of the mutant was more sensitive to EPA, with IC50 of 0.9 ± 0.1 µM (Fig. 4).
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of inactivation for INa in HEK-293t cells transfected with the wild-type or inactivation-deficient mutant of hH1 plus 1-subunits. INa were elicited using the same protocol shown in Fig. 1. Compared with wild-type hH1 Na+ channels (n = 6) (Fig. 6), inactivation values of INa were significantly prolonged in HEK-293t cells transfected with inactivation-deficient Na+ channels (Fig. 6) (n = 19). The inactivation of the mutated currents elicited by pulses in a range from –25 mV to 40 mV were significantly reduced in the presence of 5 µM EPA (Fig. 6) (n = 12). The decreased inactivation of INa of the mutant in the presence of EPA, however, were still much greater than those of wild-type INa. The effects of 5 µM EPA on the inactivation were not obvious for wild-type INa (Fig. 6) (n = 6). These results suggest that EPA enhances phenotypic inactivation of inactivation-deficient Na+ channels but not that of the wild type.
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25%) of INa peak with prepulses depolarized above –50 mV, which inactivated all wild-type Na+ channels (Fig. 2C). Extracellular perfusion of 5 µM EPA significantly reduced INa peak, including complete inhibition of the noninactivated persistent portion (Fig. 7, B and C). The normalized steady-state inactivation curve of INa peak was significantly shifted to the negative direction in the presence of 5 µM EPA. The V of the steady-state inactivation curve was shifted from –90.3 ± 1.7 mV for the control (k = 9.6 ± 1.1 mV, n = 16) to –109.3 ± 0.5 mV for EPA (k = 10.1 ± 0.4 mV, n = 9) (P < 0.001). After washout of EPA with 0.2% fatty acid-free BSA solution, the steady-state inactivation curve was shifted back toward the control. These results demonstrate that EPA significantly shifted the steady-state inactivation of INa peak by –19 mV, which is similar to our previous finding of a –22-mV shift for the wild-type hH1 Na+ channel (20). In addition, EPA eliminates the noninactivated persistent portion of the steady-state inactivation curve of the mutant channel.
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t) of conditioning pulses was prolonged, indicating that an increasing proportion of channels was entering the inactivated state. However, an 50% portion of INa was not inactivated even with the longest conditioning pulse tested, 120 ms (Fig. 8C) (n = 5), at which the INa of wild-type hH1 Na+ channels were completely inactivated (Fig. 8C). Our results indicate that the L409C/A410W mutant of the hH1 Na+ channel significantly alters the development of resting inactivation and induces a significant portion of noninactivated currents.
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15% of mutant channels in the presence of EPA were not inactivated when the duration of the conditioning pulse was set at 120 ms (Fig. 8C). The slope of the resting inactivation of the mutant was superimposed with that of the wild-type Na+ channel in the presence of 5 µM EPA (Fig. 8C), except for the noninactivated portion of the mutant. The data suggest that the double mutations of hH1 result in incomplete resting inactivation of the channel and that EPA decreases the noninactivated portion of the current.
Delayed recovery from inactivation of INa by EPA.
To determine whether the mutations at the 409 and 410 sites of hH1 affect recovery from resting inactivation, the available currents elicited by 50-ms test pulses to –30 mV were measured (Fig. 9, A and B). The t of recovery from inactivation of INa peak was fitted using a single exponential function (Fig. 9C). The for recovery from inactivation of the mutant current was 600.7 ± 48.0 ms for control (A1 = –1.04) (Fig. 9C) and 927.1 ± 54 ms for 5 µM EPA (A1 = –1.05) (Fig. 9C) (n = 5; P < 0.01). Compared with the mutant, the values for wild-type hH1 Na+ channels were significantly (P < 0.01) smaller: 10.8 ± 1.8 ms for control (A1 = –0.96) (Fig. 9C) and 120.0 ± 13.6 ms for 5 µM EPA (A1 = –0.81) (Fig. 9C) (n = 8). These results indicate that the mutant of L409C/A410W delays recovery from resting inactivation and that EPA further slows recovery.
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plus 1-subunits. Figure 10 shows that extracellular application of one of the PUFAs at 5 µM concentration significantly blocked the mutant channel. In contrast, the monounsaturated fatty acid oleic acid (OA; C18:1n-9, n = 6) or either of the saturated fatty acids stearic acid (SA; C18:0, n = 6) or palmitic acid (PA; C16:0, n = 9) at 5 µM concentration had no significant inhibitory effect on the mutant channel in HEK-293t cells. These results are consistent with our previous findings that only PUFAs, not monounsaturated or saturated fatty acids, have inhibitory effects on cardiac INa (19, 20).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-subunit of human cardiac Na+ channels causes a long-lasting, persistent INa and that n-3 PUFAs significantly inhibited INa in HEK-293t cells transfected with the inactivation-deficient mutant. The effect of PUFAs on INa late was even greater than that on INa peak (Figs. 3, 5, and 7). The persistent INa current has been observed in adult mammalian ventricular cardiomyocytes (14), and hypoxia has been shown to enhance its amplitude (5). Increased Na+ influx during hypoxia increases intracellular Na+ concentration ([Na+]i), which in turn activates the reversal mode of the Na+/Ca2+ exchanger so that intracellular Ca2+ concentration ([Ca2+]i) level increases as well. An increase in the persistent INa and [Ca 2+]i level can cause arrhythmias and irreversible cell damage (5). Blockade of voltage-gated Na+ channels has long been accepted an effective therapy for patients with many types of cardiac arrhythmia. A recent study showed that blocking persistent INa late in ventricular cardiomyocytes of patients with heart failure ceased soon after depolarization (10). The inhibition of INa late by n-3 PUFAs thus might have potential therapeutic value in certain patients with ischemia-induced arrhythmia.
Traditional local anesthetics act on common structural determinants at the D4–S6 segment of the Na+ channel -subunit (13). Certain mutations (F1760K and Y1767K) in this region of hH1 Na+ channels were found eliminate the inhibitory effects of lidocaine and cocaine on cardiac INa in HEK-293t cells transfected with these mutants, but they did not alter the inhibition of INa by n-3 PUFAs. In contrast, the mutant N406K in the D1–S6 region greatly attenuated the effects of the n-3 PUFAs on cardiac INa (21). These results indicate that EPA may bind to a region (D1–S6) different from the one to which local anesthetics bind (D4–S6).
Because the sites of the mutant L409C/A410W are close to N406, EPA might possibly bind to a region near the mutation sites of the inactivation-deficient Na+ channels and thus modify the behavior of the inactivation gate so that it more closely resembles a normal inactivation process.
Our present results show that the fish oil n-3 PUFAs significantly shifted the curve of the steady-state inactivation in a hyperpolarizing direction and eliminated the portion of noninactivated currents (Fig. 7). EPA at 5 µM concentration inhibited INa peak values by 50%, but INa late was essentially abolished. This finding shows that EPA essentially abolishes the persistent INa of the mutant and results in phenotypic restoration of the inactivation in the inactivation-deficient mutant. Therefore, in the presence of EPA, the inactivation-deficient Na+ channel behaves similarly to inactivation in wild-type Na+ channels. The EPA-induced inhibition of the mutant INa had no effect on the activation of mutant Na+ channels (Fig. 5). The antiarrhythmic drug flecainide also inhibited the mutant current without altering the activation of inactivation-deficient mutants of skeletal muscle Na+ channels (15). EPA, however, significantly shifted the steady-state inactivation of the mutant INa by –19 mV, which is similar to our previous finding of a –22-mV shift for wild-type hH1 plus 1-subunits of Na+ channels (20). Typically, this action is limited to PUFAs and is not produced by monounsaturated or saturated fatty acids as shown in Fig. 10 (19, 20).
It seems that any cardiac dysfunction that results in prolonged INa enhances the opportunity for cardiac arrhythmias to occur. Long QT-3 syndromes, e.g., LQT-3/KPQ, have persistent INa late (12). Patients with these presentations, too, might potentially benefit from treatment with n-3 fatty acids, which block persistent INa late. After binding, the fatty acids may block Na+ channels or induce the channels to enter an inactive state and stabilize. The ability to inhibit persistent INa and stabilize Na+ channels in their inactivated state has clinical implications for potential therapeutic use of fish oil n-3 PUFAs.
Arrhythmias that arise from enhanced persistent INa in patients with ischemia can cause sudden cardiac death (5). We have shown that the n-3 PUFAs, by blocking persistent INa, may be able to prevent these fatal arrhythmias as has been shown in clinical trials (1, 11). The beneficial effects of n-3 PUFAs on certain cardiac arrhythmias may result from the inhibition of persistent INa by enhancement of channel inactivation and stabilization of the inactivation gate.
The results of the present study indicate that the blocking action of fish oil n-3 PUFAs on Na+ channels in a mutant produced an inactivation-deficient channel, presumably by disabling the receptor of the inactivation particle, so that the inactivation particle was unable to close the Na+ channel. The intracellular linker between domains 3 and 4 is known to be essential for the fast inactivation of the Na+ channel, and deletion of this region also causes persistence of INa during depolarization (2). We do not know whether the n-3 PUFAs have any or no blocking effect on a disabled, inactivated Na+ channel such as that produced in the mutant IFM/3Q and did not address that issue in this study.
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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clone, Drs. L. L. Isom and W. A. Catterall for the rat brain 1-subunit clone, and Dr. S. C. Cannon for the CD8 clone and the HEK-293t cell line. Present address of Y.-F. Xiao: Cardiac Rhythm Management, Medtronic, 7000 Central Ave. NE, Minneapolis, MN 55432.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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2. Catterall WA. From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26: 13–25, 2000.[CrossRef][ISI][Medline]
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4. Grant AO, Chandra R, Keller C, Carboni M, and Starmer CF. Block of wild-type and inactivation-deficient cardiac sodium channels IFM/QQQ stably expressed in mammalian cells. Biophys J 79: 3019–3035, 2000.
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7. Horn R, Patlak J, and Stevens CF. Sodium channels need not open before they inactivate. Nature 291: 426–427, 1981.[CrossRef][Medline]
8. Kang JX and Leaf A. Effects of long-chain polyunsaturated fatty acids on the contraction of neonatal rat cardiac myocytes. Proc Natl Acad Sci USA 91: 9886–9890, 1994.
9. Lawrence JH, Yue DT, Rose WC, and Marbán E. Sodium channel inactivation from resting states in guinea-pig ventricular myocytes. J Physiol 443: 629–650, 1991.
10. Maltsev VA, Sabbah HN, Higgins RS, Silverman N, Lesch M, and Undrovinas AI. Novel, ultraslow inactivating sodium current in human ventricular cardiomyocytes. Circulation 98: 2545–2552, 1998.
11. Marchioli R, Barzi F, Bomba E, Chieffo C, Di Gregorio D, Di Mascio R, Franzosi MG, Geraci E, Levantesi G, Maggioni AP, Mantini L, Marfisi RM, Mastrogiuseppe G, Mininni N, Nicolosi GL, Santini M, Schweiger C, Tavazzi L, Tognoni G, Tucci C, and Valagussa F; GISSI-Prevenzione Investigators. Early protection against sudden death by n-3 polyunsaturated fatty acids after myocardial infarction: time-course analysis of the results of the Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto Miocardico (GISSI)-Prevenzione. Circulation 105: 1897–1903, 2002.
12. Nagatomo T, January CT, and Makielski JC. Preferential block of late sodium current in the LQT3 KPQ mutant by the class IC antiarrhythmic flecainide. Mol Pharmacol 57: 101–107, 2000.
13. Ragsdale DS, McPhee JC, Scheuer T, and Catterall WA. Common molecular determinants of local anesthetic, antiarrhythmic, and anticonvulsant block of voltage-gated Na+ channels. Proc Natl Acad Sci USA 93: 9270–9275, 1996.
14. Saint DA, Ju YK, and Gage PW. A persistent sodium current in rat ventricular myocytes. J Physiol 453: 219–231, 1992.
15. Wang GK, Russell C, and Wang SY. State-dependent block of wild-type and inactivation-deficient Na+ channels by flecainide. J Gen Physiol 122: 365–374, 2003.
16. Wang K, Bonner SY, Russell C, and Wang GK. Tryptophan scanning of D1S6 and D4S6 C-termini in voltage-gated sodium channels. Biophys J 85: 911–920, 2003.
17. West JW, Patton DE, Scheuer T, Wang Y, Goldin AL, and Catterall WA. A cluster of hydrophobic amino acids required for fast Na+-channel inactivation. Proc Natl Acad Sci USA 89: 10910–10914, 1992.
18. Xiao YF, Huang L, and Morgan JP. Cytochrome P450: a novel system modulating Ca2+ channels and contraction in mammalian heart cells. J Physiol 508: 777–792, 1998.
19. Xiao YF, Kang JX, Morgan JP, and Leaf A. Blocking effects of polyunsaturated fatty acids on Na+ channels of neonatal rat ventricular myocytes. Proc Natl Acad Sci USA 92: 11000–11004, 1995.
20. Xiao YF, Wright SN, Wang GK, Morgan JP, and Leaf A. Coexpression with 1-subunit modifies the kinetics and fatty acid block of hH1 Na+ channels. Am J Physiol Heart Circ Physiol 279: H35–H46, 2000.
21. Xiao YF, Ke Q, Wang SY, Auktor K, Yang Y, Wang GK, Morgan JP, and Leaf A. Single point mutations affect fatty acid block of human myocardial sodium channel subunit Na+ channels. Proc Natl Acad Sci USA 98: 3606–3611, 2001.
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) and wild type Na+ channels (
) fitted using a Boltzmann equation.
) and late portion of INa (INa late, measured near the end of each pulse; 
) measured at dotted lines in A and B after wash-in and washout of 5 µM EPA. INa late was almost completely inhibited by EPA. Break in x-axis in C represents time required to collect data regarding current-voltage relationship and steady-state inactivation of INa in the presence of 5 µM EPA.
