Pharmacological modulation of human cardiac Na+ channels

European Journal of Pharmacology - Molecular Pharmacology Section, 266 (1994) 245-254 © 1994 Elsevier Science B.V. All rights reserved 0922-4106/94/$07.00

245

EJPMOL 90574

Pharmacological modulation of human cardiac Na + channels D o u g l a s S. K r a f t e a,,, K a t h l e e n D a v i s o n a, N a n c y D u g r e n i e r a, K i m b e r l y E s t e p b, K u r t J o s e f b, R o b e r t L. B a r c h i c,O,e, R o l a n d G. K a l l e n c, Paul J. Silver a a n d A l a n M. E z r i n ~ Departments of a Vascular and Biochemical Pharmacology and b Medicinal Chemistry, Sterling Winthrop, Inc., 1250 S. Collegerille Rd., P.O. Box 5000, CoUegeviUe, PA 19426-0900, USA, and the c Department of Biochemistry and Biophysics, a Neurology and e The David Mahoney Institute of Neurological Sciences, Uniuersity of Pennsyh,ania, Philadelphia, PA 19104, USA Received 16 September 1993; revised MS received 12 October 1993; accepted 15 October 1993

Pharmacological modulation of human sodium current was examined in Xenopus oocytes expressing human heart Na ÷ channels. Na ÷ currents activated near - 5 0 mV with maximum current amplitudes observed at - 2 0 mV. Steady-state inactivation was characterized by a 1111/2value of - 5 7 + 0.5 mV and a slope factor (k) of 7.3 + 0.3 mV. Sodium currents were blocked by tetrodotoxin with an IC50 value of 1.8/zM. These properties are consistent with those of Na ÷ channels expressed in mammalian myocardial cells. We have investigated the effects of several pharmacological agents which, with the exception of lidocaine, have not been characterized against cRNA-derived Na ÷ channels expressed in Xenopus oocytes. Lidocaine, quinidine and flecainide blocked resting Na + channels with IC50 values of 521 /zM, 198 /xM, and 41 /zM, respectively. Use-dependent block was also observed for all three agents, but concentrations necessary to induce block were higher than expected for quinidine and flecainide. This may reflect differences arising due to expression in the Xenopus oocyte system or could be a true difference in the interaction between human cardiac Na ÷ channels and these drugs compared to other mammalian Na + channels. Importantly, however, this result would not have been predicted based upon previous studies of mammalian cardiac Na + channels. The effects of DPI 201-106, RWJ 24517, and BDF 9148 were also tested and all three agents slowed a n d / o r removed Na ÷ current inactivation, reduced peak current amplitudes, and induced use-dependent block. These data suggest that the a-subunit is the site of interaction between cardiac Na ÷ channels and Class I antiarrhythmic drugs as well as inactivation modifiers such as DPI 201-106. Na + channel (human); Oocyte, Class I antiarrhythmic; DPI 201-106

1. Introduction Cardiac sodium (Na +) channels are transmembrane proteins essential for normal impulse propagation in the heart and it has also been suggested that Na ÷ channels are active during the plateau of the cardiac action potential (Coraboeuf et al., 1979). Consistent with this latter activity, recent reports have demonstrated the existence of a Na ÷ channel with the appropriate electrophysiological properties to be active during the cardiac action potential plateau (Saint et al., 1992; Bkaily et al., 1988; Bkaily et al., 1991). In addition, pharmacological activation of Na ÷ channels during the action potential plateau has been reported to prolong action potential duration (Lee, 1992). Given the central roles Na ÷ channels play in the heart, aberrant function can lead to direct electrical disturbances

* Corresponding author. Tel.: 215-983-7126; Fax: 215-983-6900.

SSDI 0 9 2 2 - 4 1 0 6 ( 9 3 ) E 0 1 7 0 - O

and, consequently, influence cardiac performance. A better understanding of pharmacological modulation of human cardiac Na + channels represents a first step in identifying improved therapeutic agents to correct such aberrant function. Blockade of cardiac Na + channel activity has been a longstanding pharmacological approach to correct rhythm disturbances. Class I antiarrhythmic drugs, typified by lidocaine, quinidine and flecainide are examples of agents which decrease Na ÷ current (for reviews see Vaughn Williams, 1991; Woosley, 1991). While these drugs block cardiac Na + channels, they can be differentiated based upon their rates of association a n d / o r dissociation from the channel. In addition, another set of pharmacological agents, typified by DPI 201-106, has been used to slow or remove inactivation of cardiac Na ÷ channels (Scholtysik, 1989). These latter agents prolong action potential duration and increase cardiac contractility, potentially making them useful for inotropic and antiarrhythmic therapy in patients with depressed cardiac contractility.

246 Recent advances at the molecular level have facillitated structure/function studies of neuronal Na ÷ channels and following the identification of several neuronal and skeletal muscle Na ÷ channel genes (Noda et al., 1986; Kayano et al., 1988; Auld et al., 1988; Trimmer et al., 1989; Kallen et al., 1990), cardiac Na ÷ channel genes were also identified (Gellens et al., 1992; Rogart et al., 1989; George et al., 1992). Gellens et al. (1992) reported the cloning and expression of a human heart Na ÷ channel cDNA which gives rise to voltage-dependent, tetrodotoxin-resistant channels when expressed in Xenopus oocytes. The cloning and expression of this cDNA now allows detailed electrophysiological and pharmacological characterization of human cardiac Na ÷ channels. In this study we have investigated the interaction of both blockers and inactivation modifiers with human heart Na ÷ channels. With the exception of lidocaine, the effects of these agents on cRNA-derived Na + channels have not been previously reported. Expression of Na ÷ channels from cRNA, which encodes the ~-subunit of the channel complex, allows two questions to be addressed: (1) will selected pharmacological agents interact with the human heart a-subunit when expressed in the absence of other cardiac proteins, and (2) are the observed interactions similar to those previously reported for Na ÷ channels in native cardiac membranes? A prelimary report of these results has been presented in abstract form (Krafte et al., 1992).

2. Materials and Methods

2.1. Oocyte preparation and injection Female Xenopus laevis were purchased from NASCO (Ft. Atkinson, WI). All animal care and use procedures were approved by an Institutional Animal Care and Use Committee. Frogs were anesthetized in 0.17% tricaine and oocytes were surgically removed. Surrounding follicular cells were removed by treating the oocytes with 0.2% collagenase (Boehringer-Mannheim type A or B) for 2-3 h in OR-2 which consisted of (in mM): NaCI 82.5, KC1 2, MgCI 2 1, HEPES 5, pH 7.5. Stage V-VI (Dumont, 1972) oocytes were selected and placed in a standard buffer consisting of (in mM): NaCI 96, KC1 2, CaC12 1.8, MgC12 1, HEPES 5, pH 7.5 which was supplemented with 0.5 mM theophylline, 2.5 mM sodium pyruvate, and 5 0 / z g / m l gentamicin. RNA injections were performed with a Drummond microdispensor (Drummond Scientific Co., Broomall, PA) mounted on a manual micromanipulator. Approximately 50 nl of cRNA solution (see in vitro transcription below) was injected per oocyte. Oocytes were incubated in standard buffer for 24-72 h prior to

recording and the incubation buffer was exchanged daily (see standard buffer above for composition).

2.2. In vitro transcription Human heart Na + channel cRNA was prepared by in vitro transcription of a plasmid previously described by Gellens et al. (1992). Plasmid DNA (10 /xg) was linearized with the restriction nuclease, XbaI, and transcribed utilizing the Promega in vitro transcription system (Promega Corp., Madison, WI) and SP6 RNA polymerase per the directions of the manufacturer. cRNA was initially resuspended in 20 tzl of RNase-free distilled water. Transcription reactions yielded approximately 6 /xg of cRNA when measured spectrophotometrically (A = 260 nM). Current amplitudes upon expression, however, did not correlate well with the amount of transcribed RNA. We, therefore, routinely diluted the initial transcript (50X to 400X) to ensure current amplitudes following oocyte expression would be < 5/xA.

2.3. Electrophysiological recording Two-microelectrode voltage clamp was performed at 21°-23°C using a Dagan 8500 Intracellular PreampClamp (Dagan Corp., Minneapolis, MN). Current and voltage electrodes were filled with 3 M KC1 and had resistances < 1 MD. Data were digitized at 10 kHz and filtered at 3 kHz. Data acquisition and analysis were performed with the aid of the pCLAMP software package (Axon Instruments, Foster City, CA) and a laboratory computer equipped with a LabMaster 125 kHz DMA board. Oocytes were perfused continuously with standard buffer where the NaC1 concentration was increased to 140 mM (see Oocyte preparation and injection) during recording. To eliminate the small resistive component of membrane current, amplitudes were measured at a series of voltages more negative than - 6 0 mV. A linear regression was performed on these values to determine membrane resistance. Linear resistive components of membrane current were then calculated based upon test potential and subtracted from raw data records. Peak INa+was measured from these subtracted records as the most negative value following a step depolarization to potentials > - 5 0 mV with allowances for the settling time of the capacity transients. No endogenous voltagedependent Na ÷ currents were observed in the batches of oocytes used for this study (Krafte and Volberg, 1992).

2.4. Curve fitting All curve fitting was performed using nonlinear regression analysis and commerically available software

247

each drug in 1 N HCI and then diluting with standard buffer to give a 1 mM solution. The pH of these solutions was adjusted to 7.4. Flecainide was dissolved in DMSO to give a 120 mM stock. DPI 201-106 and RWJ 24517 were synthesized in the Medicinal Chemistry Department at Sterling Winthrop Pharmaceuticals Research Division (Rensselaer, NY). BDF 9148 was a gift from Beiersdorf AG (Hamburg, Germany). DPI 201-106, RWJ 24517, and BDF 9148 were prepared as 10 mM stocks in DMSO and 1 N HC1. Dilutions of stock solutions were made with 140 mM NaC1 standard buffer to achieve the desired test concentration. In experiments using DMSO as a vehicle the final concentration was < 0.1% which had no effect on control

(GraphPad Software, Inc., San Diego, CA). Equations are given in the appropriate figure legends. Parameters obtained from these fits were used to quantify concentration-response relationships, steady-state inactivation, and the rates of drug block and recovery from block.

2.5. Drugs/chemicals Unless otherwise specified all reagents were obtained from Sigma Chemical Co. (St. Louis, MO). Flecainide was a gift from 3M Pharmaceuticals (St. Paul, MN). Stock solutions of lidocaine and quinidine were prepared by dissolving appropriate amounts of

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Fig. 1. General Properties of h u m a n heart Na Currents Expressed in Oocytes. (A) Selected m e m b r a n e currents elicited by 30 ms voltage steps from - 100 m V to - 5 0 , - 4 0 , - 3 0 and - 2 0 mV. (B) Typical current-voltage relationship for h u m a n heart Na currents in oocytes. (C) Representative h~ curve for h u m a n heart currents. Curves were defined by fitting the equation:

Xenopus

l=(l+exp((Vm-Vl/2)//k) -1) V1/2 111/2

to the data where Vm is the m e m b r a n e potential (mV); the potential where half of the available current is inactivated (mV); and k is a slope factor (mV). T h e representative experiment in (C) has a value of - 5 7 mV and a k value of 7.2 mV. Current amplitudes were measured at - 2 0 inV. (D) Tetrodotoxin concentration-response curve. Data were obtained from 25 cells exposed to a single concentration of tetrodotoxin and presented as m e a n s + SEM. Current amplitudes were measured for voltage steps to - 2 0 mV. The smooth curve is a fit to the data of the equation: I = (1 + ( [ t e t r o d o t o x i n ] / I C s o ) n) - ' where [tetrodotoxin] is the concentration in /zM; ICso is the tetrodotoxin concentration where the current was reduced by 50%; and n is a Hill coefficient. The data were well described by this equation with an ICs0 value of 1.8 /~M and a Hill coefficient of 1.24. The 95% confidence intervals for the fitted equation were 1.5-2.1 /xM for the ICs0 value and 1.0-1.49 for the Hill coefficient. Data were obtained following voltage steps from - 100 m V to - 20 inV.

248

currents. All stock solutions were prepared on the day of use. Tetrodotoxin was purchased as the citrate salt from CalBioChem (San Diego, CA).

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Fig. 2. Tonic block by class I antiarrhythmic agents. Tonic block was assessed by exposing oocytes to a single concentration of flecainide (©), quinidine (e), or lidocaine ( • ) for 10 minutes and then measuring peak current amplitude. Holding potential was - 1 0 0 mV and currents were elicited by 30 ms test depolarizations. Data are plotted as the mean _+SEM for five values at each concentration. The smooth curves are derived from fitting the same equation as Fig. 1D to the data with test agents substituted for tetrodotoxin. Fitted values for flecainide, quinidine and lidocaine with 95% confidence intervals in parenthesis were: IC50-41 /~M (33-50), 198/~M (146-270) and 521 /~M (397-684); Hill coefficient-0.83 (0.69-0.97), 0.80 (0.57-1.0) and 0.89 (0.68-1.1), respectively.

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Fig. 3. U s e - D e p e n d e n t Block of h u m a n heart Na + Currents by Class I Antiarrhythmic Agents. Following m e a s u r e m e n t of tonic block (see Fig. 2 legend) current amplitudes were measured during a 10 Hz train of test depolarizations (30 ms) from - 1 0 0 mV to - 2 0 mV. Reductions in current amplitude in the absence of drug ( < 10%) have been subtracted. Data are plotted as the relative current during the train ( I , th pulse/Ilst p,lsc) vs. pulse number. Values are the m e a n s + S E M (n = 5). Data are shown for the concentrations noted in the figure for flecainide (A), quinidine (B) and lidocaine (C). (D) Summary of parameters obtained by fitting the equation: I=A

*exp(-t/r)+(1-A) to the normalized data, where t is time (s); A is a relative amplitude term for the exponential; and ~- is a time constant (s).

249

3.2. Resting state block of human heart Na + channels

representative inactivation curve is illustrated in Fig. 1C. The mean half-inactivation voltage (V~/2) and slope factor (k) were - 5 7 + 0.5 mV and 7.3 +_ 0.3 mV, respectively (n = 16). Prepulses of longer durations ( > 1 s) resulted in inactivation curves with Vl/2 values more negative than - 5 7 mV indicating the presence of a slow inactivation process (Rudy, 1978). Slow inactivation was not quantitatively investigated in this study, however. Fig. 1D illustrates that relatively high concentrations (/~M) of tetrodotoxin were necessary to block Na + currents. The effects of tetrodotoxin were assessed on current amplitude measured at - 2 0 mV following steps from - 1 0 0 mV. The concentration-response relationship for tetrodotoxin block of human heart Na + current was well defined by an ICs0 value of 1.8 /zM and a Hill coefficient of 1.24 (n = 25, see figure legend).

Fig. 2 illustrates the concentration-response relationships for lidocaine, quinidine, and flecainide obtained using experimental protocols to define drug interactions with resting Na ÷ channels (tonic block). Flecainide was the most potent blocker of human heart Na ÷ currents with an IC50 value of 41 /xM and a Hill coefficient of 0.83. Na ÷ current block by quinidine and lidocaine was characterized by IC50 values of 198 /~M and 521 /zM, respectively. The Hill coefficient for quindine was 0.8 and for lidocaine 0.89.

3.2. Use-dependent block of human heart Na + channels Use-dependent block was assessed following 10 rain exposures to test agent by applying a 10 Hz train of

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Fig. 4. Recovery from U s e - D e p e n d e n t Block. Panels represent: (A) lidocaine (100 p~M), (B) quinidine (300 tzM) and (C) flecainide (60 ~zM) at concentrations which produced 3 0 - 7 0 % u s e - d e p e n d e n t block. U s e - d e p e n d e n t block was induced by trains of 30-60 pulses (30 ms) to - 20 mV at 10 Hz followed by a single test pulse at various intervals. Sufficient time was allowed between trains for current amplitudes to return to pretrain values. Current amplitude was normalized to the pretrain value and plotted vs. the interval between the train and the test pulse. Values are m e a n s + SEM of three experiments in each panel. Holding potential was - 120 mV to promote recovery. Panel D illustrates the rate of recovery on the same time scale for each test agent. Symbols: ( o ) lidocaine, (o) flecainide, and (zx) quinidine. Rates of recovery for lidocaine and quinidine were quantified by fitting the equation: I= Al(1-exp(-

t/,cl) ) + A 2 ( 1 - e x p ( -

t/r2))+

C

to the data where 1 is the normalized current; A 1 and A 2 the amplitude terms; r 1 and r 2 the time constants (s); and C is an initial value.

250

depolarizations from - 1 0 0 mV to - 2 0 mV. Current amplitude as a function of pulse number is illustrated in Fig. 3A, B and C for flecainide, quinidine, and lidocaine, respectively. All three compounds showed use-dependent block of Na + current. Fig. 3D gives a summary of the mean time constant values (~') and relative amplitude of the exponential component at each concentration where use-dependent block could be measured. At 100 /zM concentrations, the relative rates of use-dependent block are lidocaine > flecainide = quinidine. When tested at 300 /xM the relative order from fastest blocker to slowest is lidocaine > flecainide > quinidine.

3.3. Recovery from use-dependent block Following a 10 Hz train of stimuli, test pulses were given at various intervals to determine the rate at which Na ÷ current recovered from use-dependent block induced by lidocaine, quinidine, or flecainide. These experiments were performed at a holding potential of - 1 2 0 mV to maximize recovery. When experiments were performed at - 8 0 mV it was often impossible to achieve recovery of pretrain amplitudes, particularly with flecainide. Fig. 4 illustrates recovery from use-dependent block by lidocaine (A), quinidine (B) and flecainide (C). Na ÷ current block by lidocaine recovered the most rapidly and current amplitudes had returned to greater than 90% of pretrain values in < 1 sec. The recovery rate was best described by a double exponential process with time-constants of 0.12 s and 0.59 s. A double exponential fit was significantly better than a single exponential (F-test, P < 0.0001). Quinidine took longer to recover from use-dependent block and intervals of > 30 s had to be used before current amplitudes reached > 90% of pretrain values. The recovery rate for quinidine was best described by a single exponential process with a time constant of 34 s. Flecainide recovered from use-dependent block with the slowest rate as illustrated in Fig. 4C; current amplitudes returned to 90% of pretrain values after ~ 80 s. The data we obtained for recovery with flecainide were not well described by an exponential process and we, therefore, have not quantified this rate. Fig. 4D presents the data from the previous three panels on the same time scale to illustrate the relative rates of recovery among these three antiarrhythmic agents. While the resolution of the lidocaine data is lost on this scale, it illustrates the rank order of recovery rates which was lidocaine > quinidine > flecainide.

3.4. Modulation of human heart Na + current inactivation DPI 201-106 is an agent which slows a n d / o r removes inactivation of cardiac Na ÷ channels (Buggisch

et al., 1985). We examined the effects of a 10 min exposure to DPI 201-106 (10 /xM) on Na ÷ current inactivation. Fig. 5A and B illustrate control current recordings (A) and those following exposure to DPI 201-106 (B) at the same test potentials. DPI 201-106 removed the majority of channel inactivation over the 30 ms test pulse duration resulting in large maintained inward currents at the end of the pulse. The peak current-voltage curve as well as the current at the end of the test depolarization are illustrated in Fig. 5C. Exposure to DPI 201-106 resulted in an attentuation of peak inward current and a large increase in maintained inward current. The relative voltage-dependence of channel activation was not affected. Fig. 6 illustrates the concentration dependence for the effects of DPI 201-106 and two structurely related compounds, RWJ 24517 and BDF 9148, on inactivation. Changes in current integrals were used to assess the concentration dependence of the effect. All compounds showed statistically significant (grouped t-test, P < 0.05) increases in current integrals at 1, 3 and 10 /zM. Table 1 summarizes the effects of these agents on current integrals, peak amplitudes, end current amplitudes, and use-dependent block. The latter parameter

A

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Fig. 5. Effects of DPI 201-106 on human heart Na + Currents. (A) Control current records elicited by test depolarization from -100 mV to - 60 mV, - 40 mV, - 20 mV, and 0 mV. (B) Currents elicited by the same test depolarizations as panel A following a 10 min exposure to 10 /~M DPI 201-106. (C) Current-voltage relationships for peak (©,o) and end ( zx,zx) currents, where end current is defined as the current value at the end of a 30 ms test depolarization, in control (open symbols)and followingexposure to 10/xM DPI 201-106 (closed symbols).

251

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Fig. 6. Concentration Dependence of Inactivation Modification by DPI 201-106, RWJ 24517, and BDF 9148. Inactivation modification was assessed by measuring current integrals in control and in the presence of test agent and plotting % increase in integral vs. concentration. Values presented are means _+SEM (n = 5).

was assessed as described for Fig. 3. In general, these three agents had similar effects upon human cardiac Na + channels. All slowed channel inactivation, reduced peak current amplitudes at 10 ~ M and caused significant use-dependent block during a 10 Hz train of depolarizations. At concentrations where inactivation was not completely slowed a n d / o r removed, current records at the end of a train of stimulation showed predominantly normal inactivation. These results imply that the modified channel may be more susceptible to use-dependent block than unmodified channels.

4. Discussion

Tetrodotoxin-resistant mammalian cardiac Na + channels have been expressed in Xenopus oocytes from both tissue R N A (Krafte et al., 1991) and cRNAs (Gellens et al., 1992; Cribbs et al., 1990). In addition, a tetrodotoxin resistant Na + channel isoform derived TABLE 1 Effects of DPI 201-106, RWJ 24517 and BDF 9148 on human heart Na + currents

DPI201-106

RWJ 24517

BDF 9148

conc. (~M)

current peak end use-depenintegral current current dent block (% change) (% change) (% of (%) peak)

1 3 10 1 3 10 1 3 10

8_+ 4 68_+ 8 174-+47 32_+17 77-+11 139-+32 51-+14 70-+15 142_+15

-14_+1 -10-+2 -18-+5 0_+7 0_+5 -11-+6 -8+2 -10-+1 -15_+3

12+_1 24_+1 47-+3 17+3 27+4 45_+4 19-+2 25-+3 47_+3

-15_+1 -23+1 -48_+2 -15+3 -28-+5 -37+5 -20+3 -21-+2 -46_+3

from denervated skeletal muscle, SkM2, has also been expressed in oocytes (White et al., 1991). In all cases, Na + channels translated from these RNAs show voltage-dependent activation and inactivation as well as tetrodotoxin sensitivities which are characteristic of channels observed in mammalian myocardial cells. In this study we report the effects of Class I antiarrhythmic agents and inactivation modifiers on human heart Na + channels expressed in Xenopus oocytes. Since both classes of agent are active against human heart channels, our data support the view that the a-subunit, which is encoded by the Na channel gene used in these studies (Gellens et al., 1992), is the primary site of interation between Class I antiarrhythimcs and inactivation modifiers such as DPI 201106. These data are consistent with previously reported results describing the effects of local anesthetics on neuronal Na + channels stably expressed in Chinese hamster ovarian cells (Ragsdale et al., 1991). Chinese hamster ovarian cells express endogenous Na + channels (Lalik et al., 1993) and in this previous study it is difficult to completely rule out contributions from endogenous channel subunits with respect to the site of drug interaction. Taken in conjunction with our data, however, where batches of oocytes were used where endogenous Na + channels were not expressed, one can reasonably conclude that local anesthetic/Class I antiarrhythmic drugs interact with the a-subunit of the channel complex. In addition, we have also found differences in the characteristics of block by Class I antiarrhythmic agents when measured in Xenopus oocytes compared to data reported for mammalian cells. These results along with data regarding the general properties of the human heart Na + channel are discussed below. 4.1. Properties o f human heart Na + channels - comparison to previous results

The electrophysiological properties we observed for expressed human heart Na + channels are consistent with those previously reported by Gellens et al. (1992). Current activation was near - 5 0 mV and peak currents were observed at - 2 0 mV which are comparable to the activation properties of human heart (Gellens et al., 1992), rat heart (Cribbs et al., 1990), and skeletal muscle type-2 (White et al., 1991) Na ÷ currents. We observed V1/2 and k values for steady-state inactivation of - 5 7 mV and 7.3 mV consistent with those previously reported for human heart Na + currents of - 6 2 mV and 7.7 mV. Tetrodotoxin block was well described by a single-site binding curve with an ICs0 value of 1.8/~M. Gellens et al. (1992) reported an ICs0 value of 5.7 ~ M and observed that human heart Na + channels were approximately 3-fold less sensitive to block compared to SkM2 where an IC50 value of 1.9

252 /xM has been reported (White et al., 1991). Given the scatter in the earlier concentration-response data, however, and the results reported in this study, it appears unlikely that there is any difference in the tetrodotoxin sensitivity between human heart and skeletal muscle type-2 Na ÷ channels. A rat heart Na channel has also been reported to have comparable sensitivity to block by tetrodotoxin with an IC50 value of 1.52/zM (Cribbs et al., 1990).

4.2. Block by class I antiarrhythmic drugs Lidocaine, quinidine, and flecainide all blocked human heart Na ÷ currents indicating that the a-subunit encoded by a human heart Na ÷ channel gene is the primary site for interaction between these agents and cardiac Na ÷ channels. These three antiarrhythmic drugs are representative of different subclasses of Na + channel blockers based primarily on their relative rates of recovery from block. This is true whether one ascribes to the classification of Vaughn Williams (1984) where these agents would be Class IA, IB, and IC antiarrhythmic drugs or to the Sicilian Gambit (Task Force of the Working Group on Arrhythmias of the European Society of Cardiology, 1991) where they would represent fast, medium, and slow rates of recovery. To a first approximation, block of human cardiac Na + channels expressed in oocytes is consistent with these classification schemes. The rank order of recovery rates from fastest to slowest was lidocaine > quinidine > flecainide. Other investigators have, however, reported that recovery from block is better described by a two exponential process for quinidine and flecainide, particularly when protocols are utilized which induce use-dependent unblocking (Snyders and Hondeghem, 1990; Anno and Hondeghem, 1990). We did not observe two exponentials in the recovery process for quinidine under our experimental conditions. The relative order of potency for resting state interaction with cardiac Na ÷ channels was flecainide > quinidine > lidocaine. All three of these agents have previously been classified as post-activation state blockers (i.e., not resting state blockers) and most studies have utilized concentrations optimized to produce use-dependent block (Snyders and Hondeghem, 1990; Anno and Hondeghem, 1990; Matsubara et al., 1987; Grant et al., 1989; Konzen et al., 1990; Nitta et al., 1992). Our data for resting state or tonic block are consistent with these previous reports, in that the agents tested are not potent resting state blockers. There is, however, one notable difference apparent in Class I antiarrhythmic agent block of human cardiac Na ÷ channels expressed in oocytes when compared to nonhuman, mammalian cardiac cells; particularly with respect to quinidine and flecainide. Both of these agents have been reported to show use-dependent block

of Na ÷ channels at lower concentrations than that necessary for tonic block (Snyders and Hondeghem, 1990; Anno and Hondeghem, 1990). While we observed such use-dependent block at lower concentrations of lidocaine than those necessary for tonic block, the opposite relationship was found for quinidine and flecainide. It is possible that this difference is a function of measuring Na ÷ currents arising from channels produced by the a-subunit alone and that other subunits or proteins are necessary for normal use-dependent block. This is unlikely, however, since we have previously been able to demonstrate a similar relationship for quinidine block of Na ÷ channels expressed from guinea pig heart R N A where presumably all subunits are expressed (Volberg et al., 1991). Another explanation is that the Xenopus oocyte membrane environment may differ from that of a mammalian cell and that this environment affects channel properties. Consequently, one should be cautious not to over-extrapolate the data with respect to use-dependent block until the human heart Na ÷ channel gene can be expressed in mammalian cells and block assessed in this system. Finally, it remains possible that human cardiac Na ÷ channels are different than those of other mammalian species which have been studied with respect to use-dependent block. This last point, although only one of several possibilities, merits further consideration since tonic block could lead to greater cardiac depression and deleterious side-effects for quinidine and flecainide.

4.3. Agents which slow inactivation We have previously reported that DPI 201-106 selectively modifies inactivation of cardiac Na ÷ channels as opposed to neuronal Na ÷ channels expressed in the Xenopus oocyte system (Krafte et al., 1991). It should be noted, however, that DPI 201-106 will modify certain neuronal-type Na ÷ channels expressed in cultured cells (Romey et al., 1987). DPI 201-106 had significant effects upon inactivation of human heart Na ÷ channels expressed in oocytes suggesting an interaction of this agent with the a-subunit of the cardiac Na + channel. Two other related compounds, RWJ 24517 and BDF 9148, reported to have similar effects in the heart (Brasch and Iven, 1991; Ravens et al., 1991; Salata et al., 1991) produced similar results. In addition, each of these agents also blocked Na ÷ channels during a train of stimuli. Other investigators have suggested that slowing of inactivation and block of Na ÷ channels are a function of DPI 201-106 binding to two different sites on the Na ÷ channel (Wang et al., 1990). Since DPI 201-106 has been shown to displace batrachotoxinin (Romey et al., 1987) as reported for Class I antiarrhythmic agents (Sheldon et al., 1987), the block may arise at the same site as that accessed by lidocaine,

253

flecainide, and quinidine. Notably, use-dependent block by DPI 201-106, BDF 9148, and RWJ 24517 occurred at lower concentrations than that necessary for lidocaine, flecainide and quinidine during an identical stimulation protocol. This is consistent with the reported ICs0 values for batrachotoxin displacement by DPI 201-106 being lower than those for local anesthetics (Romey et al., 1987; McNeal et al., 1985). In conclusion, the ability to express cardiac Na + channels from cRNAs, in conjunction with molecular biological techniques, should allow one to determine molecular sites of action for various pharmacological agents. As a first step in this process we have defined the properties of human heart Na + channels expressed in Xenopus oocytes and obtained results consistent with those reported by Gellens et al. (1992). We have also characterized the interaction of representative Class I antiarrhythmic agents which block Na + channels and found that while these agents affect human heart channels, we note differences in potency with respect to use-dependent block compared to previously reported data from mammalian cells. These differences may reflect a true property of the human heart Na + channel protein or, equally possible, may be a function of expression in the Xenopus oocyte. In addition, we have also characterized a series of inactivation modifiers and found these agents are able to slow a n d / o r remove inactivation of Na + channels expressed from human heart a-subunits. Expression of human heart Na + channels in a mammalian cell system will further elucidate the functional and pharmacological properties of human cardiac channels.

Acknowledgments The authors wish to thank C.C. Chadwick and W.A. Volberg for critically reviewing this manuscript and providing constructive suggestions for revisions.

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