INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

SEVERAL CLINICAL STUDIES suggest that diets containing long-chain n-3 fatty acids significantly reduce the incidence of sudden death from coronary heart disease (1, 13, 14, 34, 35). A diet high in fish oil, in contrast to saturated fat or monounsaturated olive oil, prevented ventricular fibrillation induced by coronary artery ligation in rats and increased the electrical ventricular fibrillation thresholds in marmosets (27, 28). Furthermore, an intravenous infusion of an emulsion, largely eicosapentaenoic acid (EPA) and docosahexaenoic acid [DHA, C22:6(n-3)], prevented ischemia-induced ventricular fibrillation in dogs (7, 8). Previous data (21) indicate that polyunsaturated long-chain fatty acids (PUFAs) reduced electrical excitability of rat cardiac myocytes by increasing the depolarizing current required to elicit an action potential and by markedly prolonging the relative refractory period. Activation of voltage-dependent Na⁺ channels leads to a rapid influx of Na⁺ and initiates an action potential in most cardiac myocytes. Extracellular application of free PUFAs significantly suppressed Na⁺ currents (I_Na,rat) and shifted the steady-state inactivation to more hyperpolarized potentials in cultured neonatal rat cardiomyocytes (41). PUFAs also inhibit Ca²⁺ (40) and K⁺ currents (9) in mammalian heart cells. These effects of PUFAs on ion channels may be critical for their antiarrhythmic action in vivo.

It is recognized that the human cardiac Na⁺ channel consists of one large -subunit that alone creates a functional membrane channel, which we have studied (43). In addition, there is also a small ₁-subunit (12). The physiological consequences of ₁-subunit modulation of the voltage-dependent Na⁺ channel are controversial in the literature (2, 19, 24, 25, 29, 31). Therefore, in this study we assessed the effects of coexpressing the ₁-subunit on the kinetics of the -subunit of the voltage-dependent human cardiac Na⁺ channel (hH1) transiently expressed in a mammalian cell line (HEK293t). We were surprised that the fatty acid specificity was lost in the -subunit expressed in HEK293t cells in which monounsaturated and even saturated fatty acid had some suppressing effects on I_Na, (43), whereas only PUFAs suppressed I_Na,rat in rat cardiac myocytes (41). Two further aims of this study were 1) to learn how the complete human myocardial Na⁺ channel (hH1) would be affected by the antiarrhythmic PUFAs and 2) to learn whether the addition of the ₁-subunit to the hH1 -subunit would reestablish the requirement that only PUFAs could modulate the fast voltage-dependent Na⁺ current in the rat.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials and solutions. Fatty acids obtained from Sigma (St. Louis, MO) were dissolved weekly in ethanol at 10 mM and stored under a nitrogen atmosphere at 20°C before use. The experimental concentration of fatty acids was obtained by dilution of the stocks and contained negligible ethanol, which at the dilution applied had no effect on Na⁺ currents. The pipette solution for recording the inward Na⁺ current contained (in mM) 100 CsCl, 40 CsOH, 1 MgCl₂, 1 CaCl₂, 11 EGTA, 10 HEPES, and 5 MgATP, pH 7.3. The bath solution contained (in mM) 60 NaCl, 40 N-methyl-D-glucamine, 10 CsCl, 1 MgCl₂, 1.8 CaCl₂, 10 HEPES, and 10 glucose, pH 7.4. The Tyrode solution contained (in mM) 137 NaCl, 5 KCl, 1 MgCl₂, 1.8 CaCl₂, 10 HEPES, and 10 glucose, pH 7.4.

Cell culture and transient transfection of Na⁺ channels. The method for the culture of HEK293t cells was as described previously (11). Briefly, cells were grown to 50% confluence in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 1% penicillin-streptomycin solution, 3 mM taurine, and 25 mM HEPES. Cells were split twice per week.

Electrophysiological recordings. During an experiment HEK293t cells plated in a culture dish were continuously superfused (1-2 ml/min) with the Tyrode solution. Recording glass electrodes had a resistance of 1-3 M when filled with the pipette solution and were connected via Ag-AgCl wire to an Axopatch 1D amplifier (Axon Instruments, Foster City, CA). A cell coated with CD8 beads in HEK293t cells was chosen for patch-clamp study. After a conventional gigaseal was formed, the capacitance of an electrode was compensated. Additional suction was used to form the whole cell configuration. Whole cell membrane capacitance was measured by using the method described previously (42). The average membrane capacitance was 37 ± 0.8 pF (n = 179) for HEK293t cells. Correction of cell capacitance and series resistance was then performed before application of experimental voltage-clamp protocols. After the whole cell configuration was formed, the cells were dialyzed for 5-10 min before data were acquired. With our internal and external recording solutions, we found that maintaining a tight seal for a relatively long period became difficult at holding potentials more negative than 90 mV. Therefore, we held the membrane potential at 90 mV in most experiments to ensure that cells would remain stable long enough for us to examine I_Na, or I_Na, from the same cell before, during, and after exposure to fatty acids. In addition, the amplitude and current-voltage (I-V) relationship curve were not altered when I_Na, or I_Na, was elicited by pulses from a holding potential of 150 mV or from 90 mV with a 400-ms hyperpolarizing prepulse to 160 mV. Our experiments show that the 400-ms hyperpolarizing prepulse to 160 mV was sufficient to remove fast and slow inactivation. Na⁺ currents were activated by 10- or 20-ms test pulses. Bath solutions with or without fatty acids were rapidly exchanged by using a modified puffer-pipette system (41). Experiments were conducted at 22-23°C.

Statistics. Data from two groups were analyzed by the unpaired Student's t-test. Variance analysis (ANOVA) was used to compare the difference derived from three or more group experiments. The level for statistical significance was set at P < 0.05. Data are presented as means ± SE. Depending on the experiment, some data were fit with a logistical equation, (A₁  A₂)/[1 + (x/x₀)^p + A₂], where x₀ is the center, p is power, A₁ is the initial y value, and A₂ is the final y value. Other data were fit by either a Boltzmann equation {1/[1 + exp(V_1/2  V)/k], where V_1/2 is the half-inactivation potential, V is the voltage potential, and k is the slope factor (in mV/e-fold change in current} or least-squares fitting (y = A₀ + A₁ exp^t/₁ + A₂ exp^t/₂, where t is time and ₁ and ₂ are the time constants of the fast and slow components of inactivation, respectively) (Origin 4.1, Microcal Software, Northampton, MA) with a single or double exponential function.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Voltage-dependent Na⁺ currents in HEK293t cells transfected with hH1 (I_Na,) or hH1 (I_Na,). Voltage-gated Na⁺ currents with fast activation and fast inactivation kinetics were evoked by depolarizing pulses in HEK293t cells transiently transfected with hH1 or cotransfected with hH1 and ₁-subunit (Fig. 1A). Functional association of ₁-subunit with hH1 significantly enhanced the peak current densities (Fig. 1B). The current densities (elicited by voltage pulses from 150 to 30 mV) were 74 ± 8 pA/pF for I_Na, (n = 27) and 106 ± 9 pA/pF for I_Na, (n = 39, P < 0.05; Fig. 1B), respectively. Compared with the current densities of 68 ± 7, 17 ± 3, and 0 ± 0 pA/pF for I_Na, (n = 27) elicited by the corresponding voltage commands from 120, 90, and 70 mV to 30 mV, the corresponding values of I_Na, (n = 39) were 105 ± 9, 85 ± 9, and 25 ± 5 pA/pF (Fig. 1B). Whereas >80% of peak I_Na, was elicited by pulses from 90 to 30 mV, the same voltage step evoked only 18% of peak I_Na,. In addition, a considerable amount of I_Na, (23%) was activated by voltage pulses from 70 to 30 mV, but no I_Na, was evoked with the same voltage protocol (Fig. 1A). These results indicate that coexpression of ₁-subunit modifies the voltage-dependent availability of Na⁺ channels.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Voltage-dependent activation of human cardiac Na+ currents (hH1). A: current traces were evoked by depolarizing voltage pulses from 150, 120, 90, and 70 mV to 30 mV (see protocols at top) for the complete hH1 ( + 1) and hH1 (). Note that no current was elicited by a pulse from 70 to 30 mV in a HEK293t cell expressing hH1 only. B: averaged peak current densities evoked by voltage pulses from different holding potentials. I, current. *P < 0.05; ** P < 0.01; *** P < 0.001.

Modification of channel activation and inactivation by the ₁-subunit. Figure 2A shows the original current traces, and Fig. 2B shows the I-V relationship of the voltage-dependent activation of hH1 and hH1. Na⁺ currents were activated at around 60 mV and reached the maximal amplitude at 30 mV for both I_Na, (n = 7) and I_Na, (n = 6). Although the general shape of the I-V relationship did not change, the midpoint of the normalized I-V curve (from 60 to 30 mV) for I_Na, had an ~10-mV shift in the positive direction. This result suggests that the ₁-subunit may modify the activation process of hH1 Na⁺ channels. Normalized whole cell activation conductance from peak Na⁺ currents confirmed the modulatory effect of the ₁-subunit on I_Na, activation, which caused an 8-mV positive shift at V_1/2 (P < 0.05, Fig. 2C). The average V_1/2 and k (slope) values for the fitted functions were 42.2 ± 0.81 and 5.3 ± 0.17 mV, respectively, for I_Na, and 50.1 ± 0.74 and 5.5 ± 0.56 mV, respectively, for I_Na,.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effects of coexpression of 1-subunits on the activation and inactivation of Na+ current in HEK293t cells. A: original current traces were recorded from HEK293t cells transfected with -subunit alone (INa,) or cotransfected with - and 1-subunits (INa,). Top: voltage protocol used for the activation of currents. After 400-ms hyperpolarizing pulses to 160 mV, Na+ currents were elicited by 10-ms test pulses from 90 to 50 mV with 5-mV increments. The membrane potential was held at 90 mV, and the pulse rate is 0.2 Hz. B: current-voltage relationships for INa, (, n = 6) and INa, ( + 1, n = 7) were normalized to their own maximal peak current. V, voltage. C: relative whole cell activation conductances for INa, and INa,. D: normalized fast steady-state inactivation was averaged for INa, (n = 9) and INa, (n = 6). Currents were elicited by 10-ms test pulses to 30 mV following 500-ms conditional prepulses varying from 160 to 10 mV in 10-mV increments every 10 s. The membrane potential of the cells was held at 90 mV. Data were fit with a Boltzmann equation.

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Coexpression of ₁-subunit with hH1 slows the recovery from inactivation. Voltage-activated cardiac Na⁺ channels may directly transit from the resting state to the inactivated state without opening of the channel. This process of inactivation is referred to as resting inactivation (6, 15, 18, 22, 33). To assess the effects of ₁-subunit coexpression on the development of resting inactivation of hH1 Na⁺ channels, a conditioning pulse to 65 mV with variable durations was followed by a 10-ms test pulse to 30 mV (Fig. 3A). We selected 65 mV as the conditioning voltage because the depolarization was large enough to ensure that the inactivation of both I_Na, and I_Na, neared completion but small enough to ensure that the channels did not open. Figure 3B shows that the amplitudes of I_Na, and I_Na, dramatically decreased as the duration (t) of conditioning pulses was prolonged, indicating that an increasing proportion of channels entered the inactivated state. The decay time constant of inactivation development was 26.2 ± 3.6 ms for I_Na, (n = 6) and 32.2 ± 4.2 ms for I_Na, (n = 7) (Fig. 3B). Our results indicate that coexpression of the ₁-subunit with hH1 does not significantly (P > 0.05) alter the development of inactivation.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effects of coexpression of 1-subunits on development of resting inactivation and recovery from inactivation. A: the pulse protocol was composed of a depolarizing pulse from 150 to 65 mV with various duration, followed by a 10-ms test pulse to 30 mV. The membrane potential was held at 150 mV with a pulse rate of 0.2 Hz. B: prolonging the time of prepulse reduced the normalized current amplitude for hH1 (, n = 6) and hH1 ( + 1, n = 7). Data were fit with a single exponential function. C: experimental protocol. The pulse protocol was composed of a 10-s depolarizing pulse from 120 to 65 mV, followed by a hyperpolarizing pulse to 140 mV with progressively longer durations and then a 10-ms test pulse to 30 mV. The membrane holding potential was 120 mV, and the rate of pulse was 0.1 Hz. D: time course of recovery of peak INa, (, n = 6) and INa, ( + 1, n = 8) from inactivation. Data were fit by a double exponential function. Because recovery of Na+ currents from inactivation occurred very fast, the inset expands the initial portion, the first 100 ms, of the recovery, which was slower for INa, ( + 1) than for INa, ().

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Voltage- and concentration-dependent suppression of I_Na, by EPA. It has been reported that coexpression with the ₁-subunit reduced the affinity of resting channels to lidocaine by a factor of two in oocytes (24). In a recent study (39), we showed that coexpression of hH1 with the ₁-subunit elicited a positive shift in state-dependent cocaine block of the Na⁺ channel. In another study (41, 43), we demonstrated that I_Na, was more sensitive to EPA than was I_Na,rat. To determine whether coexpression with the ₁-subunit had an effect on EPA block of hH1 channels similar to that observed in local anesthetic experiments and to determine whether the different sensitivity to PUFAs between I_Na, and I_Na,rat might result from a lack of the ₁-subunit, experiments were designed to look at the effects of EPA on I_Na,. The inhibition of I_Na, initiated within 20 s and reached the maximal effect within 3 min after application of 5 µM EPA. I_Na, returned toward the pretreatment level after washout of EPA with 0.2% fatty acid-free BSA solution. Figure 4 shows a voltage-dependent inhibition. The original current traces of I_Na, were evoked by single-step pulses from 150, 120, 90, and 70 mV to 30 mV in the absence (control) and presence (EPA) of 5 µM EPA (Fig. 4A). The reduction of I_Na, caused by 5 µM EPA at all of the tested voltage steps is statistically significant (n = 15, P < 0.001; Fig. 4B). In hH1, EPA (5 µM) inhibited I_Na,, evoked by a single-step voltage command from 150, 120, or 90 mV to 30 mV, by 67 ± 6, 82 ± 5, or 97 ± 1% (n = 15), respectively. Coexpression with the ₁-subunit decreased the degree of block by 5 µM EPA, because with the same voltage steps I_Na, was inhibited by 50 ± 5, 61 ± 5, or 90 ± 4% (n = 15), respectively. The inhibition is more profound at pulses from 90 to 30 mV. This result suggests that the EPA-induced suppression of I_Na, is voltage dependent.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Voltage-dependent suppression of INa, by extracellular application of eicosapentaenoic acid (EPA). A: original current traces of INa, in the absence (control) and presence (EPA) of 5 µM EPA were evoked by depolarizing pulses from 150, 120, 90, and 70 mV to 30 mV (see protocols at top). B: extracellular application of 5 µM EPA significantly reduced peak INa, densities (n = 15). ***P < 0.001. C: normalized voltage-dependent suppression of INa, by 5 µM EPA (n = 15).

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View larger version (18K): [in this window] [in a new window]   Fig. 5.   Concentration-dependent suppression of cardiac Na+ currents by EPA. EPA-induced suppression of INa, () and INa, ( + 1) is concentration dependent. Each data point represents the average value of at least 6 individual cells. After a hyperpolarizing pulse to 150 mV for 400 ms, a 10-ms test pulse to 30 mV was applied to activate the Na+ current. The membrane holding potential was 90 mV, and the rate of pulses was 0.1 Hz.

Effects of EPA on activation and inactivation of I_Na,. EPA (5 µM) block of I_Na, did not alter the I-V relationship, but the decrease in amplitude of I_Na, in the presence of 5 µM EPA was significantly different at the voltages from 40 to 30 mV (Fig. 6A, n = 7). The Na⁺ current was activated at 60 mV and achieved its maximal amplitude at 30 mV in either the absence or presence of 5 µM EPA. The inhibition was reversible after washout of EPA with the bath solution containing 0.2% BSA (data not shown). The activation curves calculated from normalized conductance were superimposed in the absence and presence of 5 µM EPA (n = 7, Fig. 6B). The 50% channel activation was at 42.2 ± 0.81 mV with a k value of 5.3 ± 0.17 mV for control and at 41.5 ± 0.21 mV with a k value of 6.0 ± 0.15 mV for 5 µM EPA.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effects of EPA on activation and inactivation of INa,. A: normalized current-voltage relationships in the absence (control) and presence (EPA, n = 8) of 5 µM EPA. Na+ currents were evoked by the same voltage protocol shown in Fig. 2A. B: relative whole cell activation conductance in the absence (control) and presence (EPA) of 5 µM EPA. The midpoint voltage (V1/2) of activation was 42.2 ± 0.81 mV with a slope factor of 5.3 ± 0.17 mV for control and 41.5 ± 0.21 mV with a slope of 6.0 ± 0.15 mV for EPA, respectively. C: effects of EPA on the normalized steady-state inactivation of INa, (n = 7) in the absence (control) and presence (EPA) of 5 µM EPA as well as during washout. Na+ currents were evoked by the same protocol shown in Fig. 2C. Data in B and C were fit by a Boltzmann equation.

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EPA-induced acceleration of inactivation development. To assess the effects of EPA on the development of resting inactivation of I_Na,, conditioning pulses to 65 mV with increasing durations were followed by a test pulse to 30 mV (Fig. 7A). Because the conditioning pulses to 65 mV did not evoke any current (Fig. 2), the development of inactivation proceeded directly from the resting or preactivated states. Figure 7B shows that increases in duration of the conditioning pulse gradually increased the population of hH1 channels into the inactivated state. EPA at 5 µM significantly accelerated the process of this transition. The data were well fit by a single exponential decay with a time constant of 32.2 ± 0.8 and 8.3 ± 0.8 ms for control and EPA (P < 0.01), respectively. It is interesting that compared with hH1 alone, coexpression of the ₁-subunit with hH1 reduced the rate of inactivation development in the presence of EPA (Fig. 7C). The time constant was significantly different between I_Na, (3.6 ± 0.2 ms, n = 6) and I_Na, (8.3 ± 0.8 ms, n = 7, P < 0.05; Fig. 7C). The data suggest that functional association of the ₁-subunit with hH1 reduces the effects of EPA on the development of resting inactivation of cardiac Na⁺ channels.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Acceleration of development of resting inactivation of INa, by EPA. A: the voltage protocol was composed of a prepulse from 150 to 65 mV with increasing durations and a 10-ms test pulse to 30 mV. B: time course of development of resting inactivation in the absence ( + 1, control) and presence ( + 1, EPA; n = 7) of 5 µM EPA. The time courses of resting inactivation of INa, were fit by a single exponential function with the time constant of 32.2 ± 0.8 ms for control and 8.3 ± 0.8 ms for EPA. C: comparison of development of resting inactivation of INa, () and INa, ( + 1) in the presence of 5 µM EPA. The time constant (fit by a single exponential function) of development of resting inactivation of INa, is 3.6 ± 0.2 ms (n = 6) in the presence of 5 µM EPA, which is significantly shorter than that of INa, (8.3 ± 0.8 ms; n = 7, P < 0.05).

EPA slows the recovery of I_Na, from inactivation. We examined the kinetics of recovery of I_Na, from inactivation in the absence and presence of EPA using a protocol similar to that shown in Fig. 3C. In these experiments we used 100 mV as the holding and recovery potential. Note from Fig. 7 that 10 s at 65 mV would more than suffice to convert all Na⁺ channels to the inactivated state. We determined the recovery from inactivation by increasing the duration (t) of recovery pulses and measuring the available current elicited by a test pulse to 30 mV. In control saline, the time course of recovery from inactivation of I_Na, was fit by a double exponential function, and most of the recovery was in the fast component (Fig. 8). The time constants for the recovery from inactivation of I_Na, in control saline were 9 ± 7 ms (₁; A₁ = 0.78) and 1,149 ± 256 ms (₂; A₂ = 0.22). In the presence of 5 µM EPA, the recovery from inactivation of I_Na, was also fit with a double exponential function (Fig. 8) with the time constants of 33 ± 7 ms (₁; A₁ = 0.65) and 5,363 ± 845 ms (₂; A₂ = 0.35). Both ₁ and ₂ are significantly slower in the presence of 5 µM EPA (P < 0.05). To obtain a better view of the fast component, the x-axis was plotted to 1 s (Fig. 8, inset). At the recovery potential of 100 mV, the time required for 50% channel recovery from the fast component of inactivation was significantly delayed, from 13 ± 1 ms for control to 63 ± 7 ms for 5 µM EPA (n = 9, P < 0.01), respectively. The time required for 50% recovery from the fast component of inactivation was also significantly prolonged when the recovery potential was 150 mV, from 5.7 ± 0.6 ms for control to 21.8 ± 1.6 ms for 5 µM EPA (n = 8, P < 0.01). These results indicate that EPA slows recovery of I_Na, from both the fast and slow components of inactivation.

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Kinetics of recovery from inactivation of INa,. The time course of recovery of peak INa, (n = 9) from inactivation is shown in the absence (control, ) and presence (EPA, ) of 5 µM EPA. The testing currents were normalized to their maximal values of INa, recorded before application of the protocol. Recovery of INa, from inactivation was markedly delayed for both fast and slow components in the presence of EPA. Data were fit by a double exponential function. Inset: fraction of recovery from inactivation during the first 1 s for control and EPA. The voltage protocol is similar to that of Fig. 3A, but with a holding and returning potential of 100 mV.

A higher efficacy of EPA on inactivated Na⁺ channels. Only closed resting channels are able to open in response to a depolarizing pulse. During a cycle of depolarization and repolarization, channels are in dynamic equilibrium between the resting and inactivated states. Therefore, current amplitude is proportional to the number of channels in the resting state before a depolarizing pulse. The EPA-induced suppression of I_Na, was voltage dependent (Fig. 4), and the midpoint of the steady-state inactivation of I_Na, was significantly shifted to the negative direction in the presence of EPA (Fig. 6). The voltage-dependent block and the negative shift in channel availability could result from preferential EPA action on inactivated channels compared with closed resting channels (3). Therefore, we tested the effects of EPA on the resting and inactivated states of Na⁺ channels. Figure 9, A and B, shows that 5 µM EPA suppressed superimposed Na⁺ currents evoked by depolarization pulses to 30 mV from the holding potentials of 150 and 70 mV; from the latter holding potential I_Na, was completely inhibited.

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Effects of EPA on resting and inactivated hH1 channels. Current traces were evoked by voltage steps from 150 to 30 mV (A) and from 70 to 30 mV (B) in the absence (control) and presence (EPA) of 5 µM EPA. C: suppression of resting () and inactivated () hH1 channels by EPA was concentration dependent. Data were fit by a logistical equation (see MATERIALS AND METHODS). Each value represents 6-15 cells (mean ± SE). Normalized current was calculated as INa,(EPA)/INa,(control) from the same corresponding cell.

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EPA block of resting hH1 channels. Several Na⁺ channel blockers show a tonic and frequency-dependent inhibition of voltage-gated Na⁺ channels (32). Figure 10A shows EPA-induced inhibition of I_Na, without and with predepolarizing pulses. The current was evoked by a voltage command from a holding potential of 140 mV to 30 mV every 5 s. After 10 control current traces were collected, 5 µM EPA solution was washed into the bath. Na⁺ currents were then recorded after 3 min of EPA perfusion. The amplitude of peak currents were elicited by a train of 30 pulses in the presence of 5 µM EPA. The amplitude of peak I_Na, elicited by the first pulse of the train was markedly reduced. This inhibition is referred to as tonic block, because the block developed at a holding potential of 140 mV and without opening channels. During subsequent pulses in the train, no additional block developed. The amplitude of peak I_Na, evoked by the 30th pulse was similar to the value evoked by the 1st pulse. Therefore, EPA-induced suppression of I_Na, was mainly due to fatty acid block of closed resting channels and did not require that the channels enter the open state to gain access to a "binding" site. The inhibition was removed after washout of EPA. In addition, the time course of the EPA-induced suppression of I_Na, in another group of experiments was not altered when the stimulating rate of pulses was altered from 0.1 to 1 Hz (data not shown). These results are consistent with our previous findings (40, 41, 43) that EPA-induced inhibition of cardiac Na⁺ and Ca²⁺ channels are time dependent, but not use dependent, in rat cardiac myocytes and in HEK293t cells transfected with hH1.

View larger version (20K): [in this window] [in a new window]   Fig. 10.   EPA-induced tonic block of INa,. A: time course of EPA-induced inhibition of INa, in the absence (control) and presence (EPA) of 5 µM EPA as well as during washout. Inset: original currents were evoked by pulses from 150 to 30 mV, and arrows indicate the time points when current traces were recorded. B: tonic block (1st pulse) and frequency block (30th pulse) of hH1 Na+ channels by 5 µM EPA (n = 9) are well overlapped. Data were fit by a logistical equation (see MATERIALS AND METHODS).

Fatty acid effects on Na⁺ currents in HEK293t cells transfected with hH1 or hH1 plus ₁-subunit. Table 1 summarizes the inhibitory effects of several fatty acids on I_Na, and I_Na,. EPA, DHA, -linolenic acid, conjugated linoleic acid (all 5 µM), and retinoic acid (10 µM) are significant inhibitors of these Na⁺ currents. EPA ethyl ester at 5 µM had no effect on either I_Na, or I_Na,. Monounsaturated and saturated fatty acids (5 µM) had no significant inhibitory effects on I_Na, but significantly inhibited I_Na,. This loss of the characteristic fatty acid specificity on I_Na, was recovered when the ₁-subunit was coexpressed in HEK293t cells.

View this table: [in this window] [in a new window]   Table 1.   Effects of fatty acids on Na+ currents in HEK293t cells transfected with hH1 or hH1 plus 1-subunit

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Modulation of hH1 channels by the ₁-subunit. In our present study, coexpression of the rat brain ₁-subunit with the hH1