INa difficult, if not impossible (Johnson & Lieberman, 1971; Beeler & McGuigan,

Transcription

1 J. Physiol. (1983), 340, pp With 5 text-figure8 Printed in Great Britain SODIUM CHANNELS IN CULTURED CARDIAC CELLS BY A. B. CACHELIN, J. E. DE PEYER, S. KOKUBUN AND H. REUTER* From the Department of Pharmacology, University of Berne, Friedbiihlstrasse 49, 3010 Berne, Switzerland (Received 9 December 1982) SUMMARY 1. Primary cardiac cell cultures were prepared from the hearts of neonatal rats. The patch-clamp method (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) was applied for studying whole-cell Na+ currents and single-channel Na+ currents, respectively. 2. Whole-cell recordings yielded voltage- and time-dependent Na+ currents which could be blocked by tetrodotoxin. 3. Single-channel Na+ currents were directly compared in cell-attached patches and in inside-out patches. 4. In cell-attached patches the elementary current was about -1 pa at -10 mv and the slope conductance over a 50 mv voltage range was ps (mean + S.D.). Inactivation during depolarization and after conditioning clamp steps, in the steady state, resulted from a reduced opening probability of Na+ channels. 5. In inside-out patches, with identical solutions at both membrane surfaces, there was a large (40-50 mv) shift of channel opening and inactivation kinetics towards more negative potentials. However, for levels of comparable opening probabilities, mean open times ofna+ channels were similar in cell-attached and inside-out patches. Tetrodotoxin (10-20 fsm) had no effect on Na+ channels when applied from the inside, but blocked them completely after application to the outside membrane surface. INTRODUCTION Until a few years ago, accurate measurements of Na+ currents ('Na) in heart muscle have not been available. The complexities of cardiac tissues as well as the inadequacies of available voltage-clamp techniques have made a reliable analysis of INa difficult, if not impossible (Johnson & Lieberman, 1971; Beeler & McGuigan, 1978; Reuter, 1979). Recent improvements of this situation have come from the use of more suitable multicellular (Colatsky & Tsien, 1979; Colatsky, 1980; Ebihara, Shigeto, Lieberman & Johnson, 1980) or single cell (Lee, Weeks, Kao, Akaike & Brown, 1979; Brown, Lee & Powell, 1981; Lee, Hume, Giles & Brown, 1981; Bodewei, Hering, Lemke, Rosenshtraukh, Undrovinas & Wollenberger, 1982) preparations. The results indicate that Na+ conductance (9Na) in cardiac muscle has voltagedependent kinetic properties very similar to those described by Hodgkin & Huxley * To whom reprint requests should be addressed.

2 390 A. B. CACHELIN AND OTHERS (1952) in their classical analysis of YNa in the squid axon. However, the unitary molecular events, i.e. the stochastic behaviour of single Na+ channel-conductance changes, underlying gna in heart muscle have not been investigated so far. Information from this type of investigation is important, because (i) it allows a direct comparison of molecular properties of cardiac Na+ channels with those in other excitable tissues (Sigworth & Neher, 1981; Horn, Patlak & Stevens, 1981; Fenwick, Marty & Neher, 1982b) and (ii) it may provide the basis for future pharmacological studies of drug interactions with Na+ channels in the heart. For example, a notable difference exists between equilibrium binding and binding kinetics of tetrodotoxin (TTX) with Na+ channels in nerve and muscle (for reviews see Ritchie & Rogart, 1977; Ulbricht, 1981) and heart (Reuter, Baer & Best, 1978; Cohen, Bean, Colatsky & Tsien, 1981). In the present study we have measured conductance properties of single Na+ channels in cultured cardiac cells from neonatal rat hearts. We have used the possibilities offered by the patch-clamp method as recently described by Hamill, Marty, Neher, Sakmann & Sigworth (1981). In our paper we compare INa measured in whole cells with Na+ channel behaviour in cell-attached and inside-out patches of membrane. METHODS Cultured cardiac cells. Primary cardiac cell cultures were prepared from hearts of neonatal (1-4-day-old) rats. The culture techniques used are a combination of various established methods (Mark & Strasser, 1966; Blondel, Roijeu & Cheneval, 1971; Paul 1975). Hearts were removed under sterile conditions and the ventricles were cut into small pieces. The tissue pieces were incubated at 0 0C for 20 hr in Ca-free Hanks solution containing 0-1 % trypsin (Boehringer Mannheim Corp.). After warming up to 37 TC for 30 min., the cell suspension was sedimented at 300 g and resuspended in culture medium (Dulbecco modified MEM containing 20% fetal calf serum). The cell suspension was filtered through cotton gauze, and myocardial cells were separated from fibroblasts by a differential attachment technique (Blondel et al. 1971). Myocardial cells were seeded at a density of about 1-3 x 105 cells/ml. on glass cover-slips which were put into medium-containing Petri dishes. The low density of seeding prevented the cell cultures from rapidly growing to confluency. The cultures were kept in an incubator (Forma) at 37 C in an H20-saturated, 5% C02-95 % air atmosphere. At the day of seeding (day 1) many cells were not yet attached to the cover-slip and were completely spherical. These cells were used for whole-cell recordings. At later stages (days 2-4) individual cells firmly adhered to the cover-slip and many of them had spindle-like shapes and showed striations. These cells were used for single channel recordings. Recording conditions. Electrophysiological recordings were made with the patch-clamp techniques as extensively described by Hamill et al. (1981) and by Fenwick, Marty & Neher (1982a). We used three options of this technique: (1) total membrane current was measured in whole spherical cells of um diameter; (2) single channel recordings were made in 'cell-attached' patches of membrane, or (3) in isolated 'inside-out' membrane patches where the membrane surface normally facing the cytoplasm was now facing the bathing medium. All conditions required a giga-ohm seal (usually GQl) between micropipette and cell membrane. For whole-cell recordings low resistance pipettes (2-5 MO) were used, while for patch-clamp measurements the pipette resistance was 8-15 MQ. In both conditions the pipettes were filled with isotonic salt solutions (see below). Cover-slips with cultured cardiac cells were placed into a water-jacketed Plexiglas chamber with a glass bottom. Solutions flowed into the chamber by gravity and were sucked off in a neighbouring trough connected to the recording chamber by means of a syphon. This reduced the electrical noise introduced by suction. The chamber was placed on a microscope stage (Diavert, Leitz) through which cooled water could be circulated in order to keep the temperature of the bathing solution between 16 and 18 'C. Micropipettes were prepared exactly as described by Hamill et al. (1981). For whole-cell recordings the pipettes were filled with nominally Ca-free, 140 mm-kci solution buffered with

3 Na+ CHANNELS IN CARDIAC CELLS 10 mm-hepes-koh to ph 7-2. Pipettes used for patch-clamp measurements were filled with the standard saline used in all experiments as bathing solution, which had the following composition (mm): NaCI, 137; KCO, 5-4; MgCl2, 2-0; CaCl2, 0-02; glucose, 10; HEPES-NaOH, 10; ph 7-4. Liquid junction potentials (< 5 mv) measured between standard saline and pipettes containing KCl saline were compensated (see Fenwick et al. 1982a). In some experiments TTX (10-5 M) or lidocaine (10-5 M) were added to the pipette solution or to the bathing solution. The low temperature was required in order to improve the time resolution of single channel openings. The low external Ca2+ concentration ([Ca2+]0) was necessary to reduce contraction of single cells, and the relatively high external Mg2+ concentration ([Mg2+]O) helped to avoid surface charge effects on kinetic parameters of gna* Data acquisition and analysis. All patch currents and pipette voltages were recorded on analogue tape (Racal 4DS tape-recorder). Current traces were low-pass filtered at 1 khz and digitized records were further analysed on a PDP 11/04 minicomputer. In a first step, records without channel openings (nulls) were averaged for each clamp potential and the averaged trace was subtracted from every single record at the same potential. After the subtraction of capacity and leakage current, the program calculated a base line for each record (heavy lines in current traces in Figs. 2, 3 and 4) and the mean open channel currents, i, were fitted to each record by eye (dotted lines in Figs. 2, 3 and 4). The steady-state probability, p, that a channel enters an open state was calculated from I PNit (1) where I is the mean current found by time-averaging the current flowing through open channels over the duration of the clamp pulse (50 or 100 msec) and N is the number of independent channels functioning in the patch during the pulse. Mean open times, I, that is the average time a channel spends in its open state, were calculated from t nit (2) p was obtained from eqn. (1), n is the number of opening transitions, and T is the clamp pulse duration (see Reuter, Stevens, Tsien & Yellen, 1982). Averages of multiple current traces provided times courses of mean currents at each potential. RESULTS Whole-cell Na+ currents Records of Na+ currents from a single spherical cardiac cell are shown in Fig. 1. Each trace is the average of four to six sweeps and is uncorrected for leakage current or capacity currents. Although hyperpolarizing voltage steps were applied in addition linear leak subtraction was not possible because of inward to depolarizing steps, rectification of the outward current during depolarization. In other cells, where the transient inward current was blocked with TTX (20 psm), the outward current traces were flat and showed no time dependence up to + 10 mv. Therefore, the time courses of the Na+ currents in Fig. 1 should not be distorted by outward currents. The time and voltage dependencies ofthe inward currents are typical for Hodgkin-Huxley-type Na+ currents and agree with those found in other single cells (cardiac cells: Brown et al ; Bodewei et al. 1982; chromaffin cells: Fenwick et al. 1982b; neuroblastoma cells: Moolenaar & Spector, 1978). In the experiment illustrated in Fig. 1, the resting potential was -70 mv. Holding the membrane potential at more negative levels (-90 mv) resulted in slightly larger peak inward currents upon depolarizing clamp steps, while no inward current could be recorded during clamp pulses from holding potentials (VH) positive to -50 mv. This indicates a rather steep voltage dependence of inactivation. However, a complete kinetic analysis has not been obtained from a 391

4 392 A. B. CACHELIN AND OTHERS single spherical cell, because stable current recordings were usually possible for only up to, 10 min. A plot of initial inward currents against voltage (I-V relationship, Fig. 1) shows that the current starts to be activated at potentials positive to -50 mv and reaches a peak at -15 mv. This agrees with I-V relations measured in multicellular cardiac preparations (Ebihara et al. 1980; Colatsky & Tsien, 1979; Vm (mv) PA pa bemv * -100/ 4-60 N-20 / VS _a L-300 Fig. 1. Whole-cell recordings of membrane currents in a speherical cultured cardiac cell (day 1). The pipette was filled with 140 mm-kci solution buffered with 10 mm-hepes- KOH to ph 7-2. The external solution was standard NaCl saline. After disruption of the membrane in the pipette the holding potential was set to -70 mv and depolarizing voltage-clamp steps (30 msec) were applied. Averages of four to six traces uncorrected for capacitative and leakage currents are shown at various potentials. Peak inward Na+ currents have been plotted against clamp potentials in the lower right-hand corner. Colatsky, 1980) and single cardiac cells (Brown et al ; but see Bodewei et al. 1982, for steeper negative resistance). Over the potential range -40 to 0 mv (Fig. 1) the time constants of activation, Tm' decreased from 0 9 to 0-3 and the time constants of inactivation, Th, from 9 to 2 msec. Because of the low [Ca2+]o (20 SUM) in the bathing solution Ca2+ currents could not be detected in our whole-cell recordings. Single Na+ channel currents Cell-attached patches. Single Na+ channel currents have been recorded and analysed in six cell-attached membrane patches in rat heart myocytes. Qualitatively similar, though incomplete results have been obtained in at least twenty additional patches. Typical single-channel currents are depicted in Fig. 2. In this patch there were at

5 Na+ CHANNELS IN CARDIAC CELLS 393 AV (mv) 40 L3 pa msec 50 ANfA- 0 WA#AV4*WQW-j'--' 60 1' 1....r... - T 100 IJi - "Ot... VH = r.p. -20 mv a V (mv) pa 0.1~ 0110,~ ~ - Fig. 2. Single Na+ channel currents recorded from a cell-attached membrane patch in a cultured cardiac cell (day 2). The pipette was filled with standard saline. Depolarizing voltage-clamp steps (A V; 50 msec duration) were applied from a holding potential (VH) which was 20 mv negative to the resting potential (r.p.). R.p. was estimated as being -60 to -70 mv. This estimate was obtained from occasional openings of non-selective, Ca-activated channels (Colquhoun et al. 1981) for which the current amplitude as a function of potential was known. Dotted lines on individual records indicate average single channel current levels at each potential. Data were low-pass filtered at 1 khz. The voltage dependence of single-channel currents (means of forty to sixty values each) is plotted in the lower part of the Figure. least six functional channels which opened upon depolarization. In other experiments we had between two and five functional channels in the patches. A 20 mv pipette potential was added to the resting potential (-60 to -70 mv) in order to obtain a holding potential, VH, between -80 and -90 mv. Fig. 2 shows single-channel currents recorded during depolarizing clamp steps of 50 msec duration and increasing J-2

6 394 A. B. CACHELIN AND OTHERS amplitude. With moderate depolarization (A V = 40 mv) channel openings were distributed throughout the clamp steps, while with increasing depolarization they occurred primarily at the beginning of the steps. As already shown by Sigworth & Neher (1980) and by Horn et al. (1981) inactivation can thus be accounted for by a reduced opening probability of the channels at later times during the clamp pulse. Single-channel currents decrease in amplitude with increasing depolarization. This is shown in the current-voltage (I-V) plot in the lower part of Fig. 2. Each value (open circle) gives the mean of single-current steps measured during twenty to forty identical depolarizations. As illustrated in the upper part of Fig. 2, current steps were of similar amplitude in this experiment ( pa at A V = 40 mv; mean+ S.D.). The I-V relationship was linear over a voltage range of 70 mv (slope conductance 14-3pS). In other experiments, at potentials negative to -50 mv, single-channel current amplitudes were little voltage-dependent, or even decreased. A similar result has been reported by Fenwick et at. (1982b) for single Na+-channel currents in chromaffin cells. Single-channel currents like those in Fig. 2 were observed in most of the other experiments yielding an average conductance of ps (mean+ S.D.). In two experiments, however, current steps were not equal at any given potential, but two different sizes could clearly be distinguished. Over a potential range of 50 mv the size of the smaller unitary currents was about 0-6 times that of the larger currents. Linear extrapolation of both I-V curves suggested very similar zero current potentials, and the voltage dependence of mean open times was also very similar. Unfortunately the smaller current steps occurred too infrequently for a meaningful statistical analysis. This result, however, could suggest that Na+ channels may not always be homogeneous, but that there could either be two distinct populations of Na+ channels, or that individual channels may have two or more conductance levels. Further experimental work is needed to clarify this question. Steady-state voltage dependence of inactivation of Na+ channels is shown in Fig. 3. The records in Fig. 3A were obtained from the same patch as in Fig. 2. In this experiment the holding potential was fixed at conditioning levels between 30 mv negative and 10 mv positive to the resting potential. Single-channel currents were always measured at potential steps 40 mv positive to the resting potential. The average single-channel current remained constant (-113 ± 0-1 pa, mean + S.D.) at this potential irrespective of the conditioning potential. However, the number of channel openings decreased as VH was made more positive. The voltage dependence of steady-state inactivation is plotted in Fig. 3B. For this plot forty to sixty current traces were averaged thus yielding the mean current I. The average number of open channels (pn) was found by dividing peak I by the single channel current, i, (eqn. (1)). The experimental results (Fig. 3B) could be fitted by the Hodgkin & Huxley (1952) h. function h 1+exp 1Vm }' ( ) with V0.5 =-8-5 mv negative to the resting potential (-60 to -70 mv) and k = 5.5 mv. The plot implies that inactivation results from a reduced average probability of the channels to open and that it follows a Boltzmann distribution. Virtually identical results have been obtained in two other experiments.

7 Na+ CHANNELS IN CARDIAC CELLS Mean open times, TO, of the channels were estimated according to eqn. (2) implying an exponential distribution of open times which could be verified by histogram plots. Mean open times of Na+ channels increased from 0 90 to 2-3 msec for clamp steps, A V, between 30 and 60 mv in the experiment illustrated in Fig. 2. The mean opening probability, p, increased from at A V = 30 mv to at 60 mv. In three other VH r.p w L6-0 o 1 0 A 2 pa.,... I.W_'~ y ~~~~~~~~~5 msec : ZCl1- B C 4*8 0*8 O O be A-- a ado o \0 a o..02 0L~~~~~~~ r.p FAA-*A +10 * Holding potential, VH (mv) Fig. 3. Steady-state inactivation of single Na+ channel currents (same cell-attached patch as in Fig. 2). The holding potential, VH, was set at different conditioning levels relative to the resting potential (r.p.). The command potential at which channel openings occurred was r.p. +40 mv. A, step sizes of single-channel currents were independent of the conditioning potentials while the number of channel openings decreased. B, average number of open channels (see text; left-hand ordinate) plotted against conditioning holding potentials (abscissa). The right-hand ordinate represents a relative scale (h.). The closed circle is a value repeated at the end of the series. The curve has been calculated from eqn. (3). experiments with identical A V and similar holding potentials, To ranged from 0 73 to 0-98 msec at A V = 30 mv and increased to at A V = 60 mv. Over the same potential range the probability of the channels entering an open state increased from 0-013X-0035 (A V = 30 mv) to at A V = 60 mv. Differences in resting potential and temperature between the different experiments could account for the variation of these values. In8ide-out patches. Single-channel currents could also be recorded in completely isolated inside-out membrane patches. Fig. 4 shows the results obtained in this configuration from the same patch documented in the cell-attached version in Figs. 2 and 3. In the experiment with the isolated patch we had identical standard NaCl saline (see Methods) on both sides of the membrane. The holding potential had to be set at -150 mv in order to be able to evoke Na+ channel openings during depolarizing clamp steps. In the experiment illustrated in Fig. 4, openings were regularly observed at clamp potentials positive to -110 mv. The pattern of singlechannel openings over the voltage range -100 to -20 mv in the inside-out patch was similar to that in the cell-attached configuration at clamp pulses A V = mv (Fig. 2) which corresponds to an absolute voltage range between C 0' 395

8 396 A. B. CACHELIN AND OTHERS about -60 to -50 and + 10 to + 20 mv. Despite the uncertainty of the absolute membrane potential in the cell-attached patch, it is clear that in inside-out patches the activation and inactivation ranges of Na+ channel kinetics were shifted by at least mv towards more negative potentials. Similar shifts were found in all other inside-out patches under the same experimental condition. The shift was not due to the relatively high Ca2+ concentration (20 /zm) at the inner surface of the membrane, since the addition of 0 5 mm-egta had no effect. Vm (mv) saw j -20.LL3A... 5 msec -60 ~~~~~~~~~~~~~~~~~~~~~~~ *. +50 pa mv Fig. 4. Single Na+ channel currents recorded from an inside-out membrane patch (same patch as in Fig. 2). Measurements were made with identical solutions (standard Na saline) at both membrane surfaces. Vm indicates absolute membrane potential. Mean singlechannel currents (means of forty to sixty records with single-channel openings) are plotted against Vm in the lower part of the Figure. Inspection of the I-V relationship in Fig. 4 shows additional differences between cell-attached and inside-out patch configurations. The reversal potential in the inside-out patch was 0 mv, as expected for symmetrical solutions on both sides of the membrane. The slope of the I-V relation showed marked curvatures at very negative and at positive potentials. Moreover, the steepest portion of the I- V relation between -50 and 0 mv in Fig. 4 yielded a conductance of 27-8 ps as compared to a constant slope conductance of 14X3 ps in Fig. 2. Mean open times increased from 0'85 to 2-37 msec with increasing depolarization of the isolated membrane patch in the potential range -100 to -70 mv. This corresponds to similar 10 values at levels of comparable open state probability, though at more positive membrane potentials, in the same cell-attached patch (cf. p. 395). Another experiment that we have performed with inside-out patches was to apply

9 Na+ CHANNELS IN CARDIAC CELLS TTX to the inner surface of the membrane. TTX is known to have a very low blocking potency and to show use-dependence on action potential upstroke velocities and Na+ currents in mammalian cardiac muscle (Reuter et al. 1978; Cohen et al. 1981; Brown et al. 1981). It was therefore of interest to see whether TTX could possibly act from the inside as well as from the outside of the membrane. If applied to the outside we have seen in many experiments that TTX, at concentrations between 10 and 20 /IM, completely blocked Na+ channel activity in cell-attached as well as in isolated patches. However, if the same concentration of TTX was added to the inner surface of an inside-out patch, it had no effect at all on Na+ channel activity. This excludes the possibility of TTX blocking the Na+ channel from the inside and agrees with similar results in squid axons (Narahashi, Anderson & Moore, 1966). On the other hand, lidocaine (10-5 M) blocked Na+ channel activity if applied from either side of the membrane patch. Averaged single-channel currents. As pointed out by Sigworth & Neher (1980), Na+ currents obtained from averaging single-channel currents and those recorded from whole cells should show the same properties, provided all channels are identical and function independently. Although it is not entirely clear that Na+ channels are identical in all patches (cf. p. 394), they did show identical properties in the patch illustrated in Figs. 2 and 4 as well as in other patches. Averages of the patch currents in Figs. 2 and 4 are compared in Fig. 5. Averaged currents obtained from up to sixty-four individual single channel records exhibit voltage and time dependencies typical for Na+ currents in whole cells. Although the main features of the averaged currents from the cell-attached and from the inside-out patch (Fig. 5 A, B) are similar, there are certain differences that are expected from the single channel recordings described above: (1) the negative resistance branch of the I- V relation is shifted along the voltage axis towards more negative potentials by about -40 to -50 mv in the inside-out patch configuration. There is no ready explanation for this shift which, to a smaller extent, was also observed when KCl instead of NaCl solution was applied to the inner membrane surface. Fenwick et al. (1982b) have observed similar, though less pronounced, shifts in isolated outside-out patches from chromaffin cells. They had asymmetric solutions (inside CsCl, outside NaCl) at both membrane surfaces. They considered the possibility that changes in membrane structure accompanying the formation of isolated patches could result in such shifts. On the other hand, Horn et al. (1981) with excised inside-out patches from myoballs and CsF solution at the inner surface apparently did not see such shifts. (2) Peak averaged currents are larger in inside-out patches which can be accounted for by the larger single channel currents in this potential range. (3) The time course of inactivation appears to be slower in inside-out patches which is most pronounced at potentials where the averaged currents reach their peak (A V = mv in Fig. 5B). This is due to a slightly less steep voltage dependence of the rates of inactivation. Time courses of inactivation deviated from single exponentials in both conditions. How do the averaged Na+ currents of cell-attached patches compare with the Na+ currents measured in whole cells? The voltage dependencies are very similar in both cases. Assuming a resting potential in the cell-attached patch of -60 to -70 mv, peak inward current is reached between -5 and -15 mv. At this potential p reaches a value of about In the whole-cell recording in Fig. 1, peak INa is obtained at -10 mv. In both instances the inward current is being activated at potentials 397

10 398 A. B. CACHELIN AND OTHERS positive to -60 mv. Also the time course of averaged and whole-cell Na+ currents are similar. Over the voltage range -30 to 0 mv, time constants of inactivation match very closely in both conditions. The degree of overlap between voltage-dependent activation and inactivation of 9Na can be directly estimated from the same cell-attached patch by comparing Figs. 3 and 5A. Steady-state inactivation is complete at VH = + 10 mv (Fig. 3), while at AV (mv) A VH + 40 = AV (mv) VH B L pa TZ... 5 msec VH =r.p. -20mV AWV (mv) A-. 00 A VH;-150 mv pa mv L Fig. 5. Averaged Na+ channel currents (means of forty-three to sixty-four single channel current traces). A, cell-attached patch, B, identical patch in inside-out configuration (same patch as in Figs. 2 and 4). A V indicates step potential displacements from the respective holding potentials (VH -80 to -90 mv in A, and -150 mv in B). Current-voltage plots of the corresponding averaged currents in the lower part of the Figure. Abscissa in B gives absolute membrane potential. the same voltage (AV = 30 mv in Fig. 5A) there is only a small opening probability (p = 0-016) of the channels and, correspondingly a small average current. This shows that there is only a minor overlap between the voltage dependencies of activation (mx) and inactivation (h.), and hence any steady-state Na+ current contribution resulting from this overlap will be small. This result is in essential agreement with that of Colatsky (1980), but it disagrees with Brown et at. (1981) who found a much flatter inactivation curve, and therefore a much larger range of overlap between h. and mo.

11 Na+ CHANNELS IN CARDIAC CELLS 399 DISCUSSION In this paper we describe properties of Na+ channels in cultured cardiac cells. Some pertinent discussion points have been dealt with in connexion with the results. It remains to compare briefly our findings with analogous ones obtained in other excitable cells. Although there is no direct evidence that Na+ channels are identical in intact cardiac muscle and in our cultured cells from neonatal rat hearts, at least the macroscopic Na+ currents appear to behave similarly (cf. Colatsky & Tsien, 1979; Colatsky, 1980). This applies also to INa measurements in disaggregated single cardiac cells from adult rats (Lee et al. 1979, 1981; Brown et al. 1981; Bodewei et al. 1982) and in spherical clusters of chick embryonic heart cells (Ebihara et al. 1980). Since, however, extensive kinetic analyses have not been performed in all studies, and since the experimental conditions were widely different, a detailed comparison of the results is hardly possible. Steady-state ranges of activation (mx) and inactivation (h.oo) seem to agree reasonably well in most studies, with the exception of that by Brown et al. (1981) who obtained a much flatter h,, curve. Their slope factor of h. (k = 11 mv) is about twice as large as that found by others, including ourselves (Fig. 4). An explanation for this discrepancy is that their conditioning pulses may not have been long enough to achieve a steady state. The main goal of our study, however, was to record single-channel Na+ currents by means of the 'giga-seal' patch-clamp method described by Sigworth & Neher (1981). Our findings in cultured rat cardiac cells are very similar to those reported by Sigworth & Neher (1981) for cultured rat skeletal muscle cells (myoballs). This applies to single-channel conductance as well as mean open times. In their Fig. 2 they show single-channel currents at 18 'C elicited by clamp steps 10 mv positive to the resting potential. This can be directly compared with our condition in Fig. 2 (17 'C). They found mean single-channel currents of pa while we measured pa, their mean open time was 0 7 msec while ours was 0 9 msec. Also the time courses and voltage dependencies of their averaged currents (their Fig. 3) are almost identical to ours (Fig. 5A). This suggests that basic single Na+ channel properties of cultured skeletal muscle and cardiac cells of the rat are the same (cf. also Horn et al. 1981). Na+ channels in cultured bovine chromaffin cells (Fenwick et al. 1982b) also appear to be very similar to those of cultured cardiac and skeletal muscle cells. It will be interesting to see whether a detailed kinetic analysis of the TTX action on single Na+ channels provides a clearer understanding for the differences seen with this toxin in cardiac muscle and other excitable tissues (Cohen et al. 1981). The maximum Na+ conductance, 9Na, estimated from our whole-cell recordings (Fig. 1) was about 10 ns/cell. With a single-channel conductance of about 15 ps this corresponds to ca. 670 channels/cell, or 1-2 channels/1smh2, as calculated from a membrane capacity, Cm, of 5 pf/cell and assuming a specific Cm of I #F/cm2. Taking the value of gna (25 ms/cm-2) obtained by Brown et al. (1981) for single adult rat heart cells and our single channel conductance value (15 ps), we calculate a density of approximately 16 channels/,um2. It is not clear whether this difference is a matter of cell differentiation during development, or whether it is due to differences in enzyme treatment during culturing and disaggregation procedures.

12 400 A. B. CACHELIN AND OTHERS Two other types of ion channels carrying inward current have previously been analysed in our cultured cardiac cells. One is a non-selective cation channel activated by the intracellular Ca2+ concentration (Colquhoun, Neher, Reuter & Stevens, 1981). Although this channel carries Na+ and K+ ions equally well, the main current carriers are Na+ ions if the membrane potential is negative. It may be involved in balancing the resting potential. This channel, however, can clearly be distinguished from the usual voltage-dependent Na+ channel described in this paper. The kinetics are very different, the non-selective, Ca2+-activated channel shows very little voltage dependence and is insensitive to TTX (Colquhoun et al. 1981). The other channel is a Ca2+ channel (Reuter et al. 1982). Although like Na+ channels it is activated by voltage, the kinetics are quite different. The Ca2+ channel opens frequently in bursts and inactivates slowly. It is insensitive to TTX, but can be modulated by isoproterenol (Reuter et al. 1982; Reuter, 1983). Ca2+ channels disappear rapidly in isolated patches (Fenwick et al. 1982b; A. B. Cachelin, J. E. de Peyer & H. Reuter, unpublished), while Na+ channels continue to function in this condition (Horn et al. 1981; Fenwick et al b; this paper). All this suggests fundamental differences in channel gating between both types of voltage-dependent inward current channels with need to be explored in greater detail. We wish to thank Miss C. Becker for providing us with tissue cultured cells and we are grateful to the Hoffmann-La Roche Research Department, Ffillinsdorf, for the gift of pregnant rats. Financial support by the Swiss National Science Foundation (grant Nr ) is gratefully acknowledged. REFERENCES BEELER, G. W. & MCGUIGAN, J. A. S. (1978). Voltage clamping of multicellular myocardial preparations: capabilities and limitations of existing methods. Prog. Biophys. molec. Biol. 34, BLONDEL, B., RoIJEU, I. & CHENEVAL, J. P. (1971). Heart cells in culture, a simple method for increasing the proportion of myoblasts. Experientia 27, BODEWEI, R., HERING, S., LEMKE, B., ROSENSHTRAUKH, L. V., UNDROVINAS, A. I. & WOLLEN- BERGER, A. (1982). Characterization of the fast sodium current in isolated rat myocardial cells: simulation of the clamped membrane potential. J. Physiol. 325, BROWN, A. M., LEE, K. S. & POWELL, T. (1981). Sodium current in single rat heart muscle cells. J. Physiol. 318, COHEN, C. J., BEAN, B. P., COLATSKY, T. J. & TSIEN, R. W. (1981). Tetrodotoxin block of sodium channels in rabbit Purkinje fibers. J. gen. Physiol. 78, COLATSKY, T. J. (1980). Voltage clamp measurements of sodium channel properties in rabbit cardiac Purkinje fibres. J. Phygiol. 305, COLATSKY, T. J. & TsIEN, R. W. (1979). Sodium channels in rabbit cardiac Purkinje fibres. Nature, Lond. 278, COLQUHOUN, D., NEHER, E., REUTER, H. & STEVENS, C. F. (1981). Inward current channels activated by intracellular Ca in cultured cardiac cells. Nature, Lond. 294, EBIHARA, L., SHIGETO, N., LIEBERMAN, M. & JOHNSON, E. A. (1980). The initial inward current in spherical clusters of chick embryonic heart cells. J. gen. Physiol. 75, FENWICK, E. M., MARTY, A. & NEHER, E. (1982a). A patch-clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine. J. Physiol. 331, FENWICK, E. M., MARTY, A. & NEHER, E. (1982b). Sodium and calcium channels in bovine chromaffin cells. J. Physiol. 331, HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F. J. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391,

13 Na+ CHANNELS IN CARDIAC CELLS HODGKIN, A. L. & HUXLEY, A. F. (1952). A quantitative description of membrane currents and its application to conduction and excitation in nerve. J. Physiol. 117, HORN, R., PATLAK, J. & STEVENS, C. F. (1981). Sodium channels need not open before they inactivate. Nature, Lond. 291, JOHNSON, E. A. & LIEBERMAN, M. (1971). Heart: excitation and contraction. A. Rev. Physiol. 33, LEE, K. S., HUME, J. R., GILES, W. & BROWN, A. M. (1981). Sodium current depression by lidocaine and quinidine in isolated ventricular cells. Nature, Loud. 291, LEE, K. S., WEEKS, T. A., KAO, R. L., AKAIKE, N. & BROWN, A. M. (1979). Sodium current in single heart muscle cells. Nature, Lond. 278, MARK, G. E. & STRASSER, F. F. (1966). Pacemaker activity and mitosis in cultures of newborn rat heart ventricle cells. Expl Cell Re8. 44, MOOLENAAR, W. H. & SPECTOR, I. (1978). Ionic currents in cultured mouse neuroblastoma cells under voltage-clamp conditions. J. Physiol. 278, NARAHASHI, T., ANDERSON, N. C. & MOORE, J. W. (1966). Tetrodotoxin does not block excitation from inside the nerve membrane. Science, N. Y. 153, PAUL, J. (1975). Cell and Tissue Culture. 5th edn., Edinburgh: Churchill Livingstone. REUTER, H. (1979). Properties of two inward membrane currents in the heart. A. Rev. Phyaiol. 41, REUTER, H. (1983). Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature, Lond. 301, REUTER, H., BAER, M. & BEST, P. M. (1978). Voltage dependence of tetrodotoxin action in mammalian cardiac muscle. In Biophy8ical A&pect8ofCardiac Mu8cle, ed. MORAD, M., pp London: Academic Press. REUTER, H., STEVENS, C. F., TsIEN, R. W. & YELLEN, G. (1982). Properties of single calcium channels in cardiac cell culture. Nature, Lond. 297, RITCHIE, M. J. & ROGART, R. B. (1977). The binding of saxitoxin and tetrodotoxin to excitable tissues. Rev. Physiol. Biochem. Pharmacol. 79, SIGWORTH, F. J. & NEHER, E. (1980). Single Na+ channel currents observed in cultured rat muscle cells. Nature, Lond. 287, ULBRICHT, W. (1981). Kinetics of drug action and equilibrium results at the node of Ranvier. Phy8iol. Rev. 61,