Calcium-dependent slow potassium conductance in rat skeletal myotubes

DEVELOPMENTAL

BIOLOGY

82,258-266 (1981)

Calcium-Dependent Slow Potassium Conductance in Rat Skeletal Myotubes JOHN Department

of Physiology

N. BARRETT, ELLEN F. BARRETT, AND LORI B. DRIBIN

and Biophysics,

University of Miami School of Medicine, P.O. Box 016430, Miami,

Received January

23, 1980; accepted in revised

fm

Florida

33101

August 4, 1980

A prolonged hyperpolarizing afterpotential (amplitude 5-20 mV, half decay time about 400 msec at 25°C) follows the action potential in myotubes and myosacs cultured from rat skeletal muscle. This slow hyperpolarizing afterpotential (hap) is mediated by an increase in membrane K conductance, because its reversal potential follows the Nernst potential for K and is not affected by other ions. The conductance increase measured during the hap (up to four times the resting input conductance) correctly predicts the time course of the slow hap. The slow hap is Ca dependent. Its amplitude decreases when bath [Cal is lowered, and both amplitude and duration increase when bath [Cal is raised. The slow hap is blocked by intracellular injection of the calcium chelator, EGTA. It is inhibited by solutions containing 2-4 mM manganese or l-5 mM barium, but is not blocked by 5-20 mM tetraethylammonium. Myotubes bathed in zero [Nab high [Cal solutions show calcium action potentials, which are inhibited by 2-10 mM manganese, nickel or cobalt. Myotubes bathed in isotonic Ca salts (or in 2 mM Ca plus 5 mM caffeine) show long-lasting (up to 10 see) spontaneous hyperpolarizations accompanied by prolonged contractions. These hyperpolarizations are associated with a large increase in input conductance, and they reverse in sign near the K equilibrium potential. They appear to reflect activation of the Ca-sensitive K conductance by Ca released from intracellular stores. The observation that spontaneous hyperpolarizations usually occur with no prior depolarization argues that at least a portion of the slow, Ca-sensitive K conductance system can be activated by internal Ca alone, with no requirement for plasma membrane depolarization. Cultured myotubes also have a faster K conductance system, which is inhibited by 5-20 mM tetraethylammonium or 1-5 mM barium, and is not dependent on Ca for its activation. INTRODUCTION

Myotubes of vertebrate skeletal muscle (and certain clonal muscle cell lines) grown in culture show spontaneous action potential discharge, with accompanying contractions. Several of the voltage-dependent ion permeability systems which contribute to this spontaneous discharge have already been described. They include a tetrodotoxin-sensitive fast Na current, a tetrodotoxininsensitive slow depolarizing current carried by both Ca and Na, a “delayed rectification” K current, and a slow Cl current (Kidokoro, 1973; Land et al., 1973; Fukuda, 1974; Fukuda et al., 1976a, b; Kano and Yamamoto, 1977). We find that the delayed rectification in rat skeletal muscle myotubes is actually attributable to two K conductance systems, which are distinguishable both kinetically and pharmacologically. We focus on the slower of these K conductance systems, demonstrating that it produces the prolonged hyperpolarizing afterpotential, and is Ca dependent. MATERIALS

AND METHODS

Preparation of Myotube Cultures Primary cultures of myoblasts were prepared from hindlimb skeletal muscles of late-term fetal or newborn 001%1606/81/040258-09$02.00/O Copyright All rights

0 1981 by Academic Press. Inc. of reproduction in any form reserved.

rats. The muscles were finely minced and incubated at 35°C in Hanks’ physiological salt solution containing 0.1% trypsin (bovine trypsin Type III, Sigma). After a ZO-min incubation the tissues were dissociated by trituration and the resulting cell suspension was filtered through a 200-mesh stainless-steel screen to remove cell clumps and debris. The filtered cell suspension was centrifuged, washed, and resuspended in fresh Hams F-12 medium supplemented with 10% fetal calf serum. This cell suspension was enriched for myogenic cells by preplating the rapidly adhering nonmuscle cells for 60-90 min in loo-mm glass petri dishes (Yaffe, 1968). The resulting myoblast-enriched cell suspension was plated onto collagen-coated glass coverslips placed in 35-mm plastic culture dishes. Cultures were maintained at 37°C in a 5% COz-95% air, water-saturated atmosphere. The myoblasts proliferated and within 3-6 days fused to form myotubes. In these cultures the majority of myotubes are elongated, but a small percentage round up into structures called myosacs or myoballs. Myosacs are advantageous for our electrophysiological studies because their spherical geometry makes them virtually isopotential, and because their contractions rarely dislodge the recording electrodes. In order to increase the percentage of myotubes that round up into myosacs, some of the cultures 258

BARRETT, BARRETT, AND DRIBIN

were treated for 48 hr with medium containing vinblastine (10e7 M). Comparison of recordings from normal myotubes and vinblastine-induced myosacs indicated that this vinblastine treatment did not significantly alter any of the membrane conductance systems studied here. Fukuda et al. (1976a, b) enhanced myosac formation in chick skeletal muscle with a related drug, colchicine, and likewise found that this drug treatment produced no detectable alteration in membrane electrical properties. Electrophysiological

Recording Techniques

After l-3 weeks in culture mature myotubes and myosacs were perfused with Tyrode’s solution at 2530%. In most experiments the muscle cell was penetrated with two separate glass micropipets, one to pass current, the other to record transmembrane potential. Both micropipets were filled with 0.5 M K sulfate, and had resistances of 30-70 MO (in experiments involving intracellular EGTA injection, one micropipet contained 1 M K EGTA). A Picometric preamplifier was used with the voltage recording electrode. Injected current was measured as the voltage drop across a lOlO- resistor between the current electrode and the amplifier current source. When a single electrode was used to pass current and record voltage, a standard Wheatstone bridge was used to correct for the current-induced voltage drop across the electrode tip. RESULTS

Characteristics of the Slow H~perpolarixing Afterpotential Myotubes and myosacs selected for these experiments had resting potentials of at least -50 mV (range 51-75 mV) and overshooting action potentials. Many cells were spontaneously active, and in all cells in normal Tyrode’s solution the threshold for action potential initiation was close to the resting potential. A single, brief depolarizing pulse often set off a long train of action potentials (e.g., Fig. 5B). All action potentials were followed by a prolonged hyperpolarizing afterpotential (hap), examples of which are shown in Fig. 1 and in the upper trace of Fig. 2. In our cell sample the peak amplitude of this hap in normal Tyrode’s solution averaged 10 mV (range 4-22 mV in 1’7 cells), and the potential change decayed monotonically to half its peak value in about 400 msec (range 200-700 msec in 40 cells). The slow hap overlaps in time with myotube contraction, but the hap is not merely an artifact due to movement of the cell with respect to the recording electrodes, because the slow hap could be recorded in mus-

Ca-Dependent

K Current

in Myotubes

259

cles where contraction was blocked. For example, myotubes stimulated in a modified Tyrode’s solution containing 2 mM Mn and less than 0.5 mM Ca quickly lost both the slow hap and their ability to contract. When these myotubes were returned to normal Tyrode’s solution (2 mM Ca, 0 mM Mn), the slow hap returned much more rapidly than contractions, indicating that the slow hap is independent of myotube contraction. Conductance Increase during the Slow hap Conductance changes during the slow hap were measured by injecting a small depolarizing or hyperpolarizing current pulse into the myotube at various times following the action potential. Figure 1 shows the results of one such experiment. Current pulses applied during the peak of the hap produced a smaller voltage change than identical current pulses applied before the action potential or after the hap, indicating that membrane conductance is increased during the hap. This conductance increase was not due to the hyperpolarization per se, because the steady-state current-voltage relationship in resting myotubes is linear over the range -60 to -95 mV. When the voltage change reached a steady state during the current pulse, input conductance was measured by dividing the injected current amplitude by the magnitude of the final voltage change. When a steady state was not reached, we used instead the time integral (area) of the voltage change, which is proportional to the input resistance of the cell (Barrett and Barrett, 1976). In a sample of 14 cells, the input conductance at the peak of the hap in 2 mM Ca averaged 2.1 (range 1.2-4.7) times the resting value. Figure 4A plots the time course of the slow conductance change measured in one myosac. The Slow Conductance Is SpecQic for K Figure 2 shows superimposed hap’s recorded during passage of various levels of steady hyperpolarizing cur-

FIG. 1. Increase in myosac input conductance during the slow hap. An action potential was evoked by intracellular current injection, and a single, constant-amplitude, hyperpolarizing current pulse was injected at varying times before, during, or after the subsequent hap. Five superimposed current (upper) and voltage (lower) traces are illustrated. Measurements of the areas and final amplitudes of the current-induced voltage changes indicate that the input resistance of this myosac dropped from a resting value of 27 M Q to only 5.8 M Q during the peak of the hap.

260

DEVELOPMENTALBIOLOGY -_ __+ -----------------20 nPfL 100 msec

de+

FIG. 2. Reversal of the slow hap. The five superimposed voltage traces show action potentials and afterpotentials evoked in one myosac during passage of five levels of steady hyperpolarizing current. Membrane potential was recorded with a separate electrode. Dashed line indicates 0 mV. The hap reverses at -90 mV (arrow).

rent. As the magnitude of the hyperpolarizing current increased, the slow hap became smaller and then reversed in sign to become a prolonged depolarizing afterpotential. In normal Tyrode’s solution (5 mMK) this reversal usually occurred in the range -80 to -90 mV. The reversal potential varied with extracellular [K] in a manner very close to that expected if the conductance increase associated with the hap were specific for K. Figure 3 plots the relationship between reversal potential and log [K]o measured in 3 myosacs (10 additional myosacs gave similar results). Data points from a given myosac are connected by a line. For [KJJ between 2.5 and 20 mM the average slope of these lines is very similar to that expected for a perfectly K-selective conductance (dashed line, 59 mV per decade). The relationship between reversal potential and log [K10 was similar whether the myosacs were grown in 2.6 or 5 mM K. The results of Figs. l-3 suggest that the slow hap is mediated by a K-specific increase in membrane conductance. Consistent with this hypothesis, large variations in extracellular [Cl] (0 to 155 mM, Cl replaced by sulfate or proprionate) and in extracellular [HCOS] (0 to 40 mM, pH held constant by varying pCOz) pro-

'0.

w+1, WI FIG. 3. Slow hap reversal potential as a function of extracellular [K]. Data from three myosacs (each represented by a different symbol) are plotted, points from a given myosac are connected by lines. For [Kb between 2.5 and 20 mM the slopes of the lines fall near the slope expected for a perfectly K-selective conductance change (dashed line), suggesting that the slow hap is due mainly to an increased membrane permeability to K.

VOLUME82, 1981

duced little change in the slow hap. Thus the slow hap is not mediated by the Cl conductance described in chick myotubes by Fukuda (1974) and Fukuda et al. (1976b). The voltage trajectory of the slow hap can be predicted from measurements of the membrane conductance change (Figs. 1, 4A) and the reversal potential (Fig. 2), as follows: The two major currents flowing during the decay of the slow hap are the leak current, IL (current through the resting membrane conductance), and the current through the slow conductance channels, Is (mostly carried by K). Capacitative currents are negligible because the membrane potential is changing so slowly. Since the steady-state current-voltage relationship between the resting and reversal potentials is linear, IL(~)

= ai

- EL)

and

IS(~)

= QS(MVQ

- GJ,

where V(t) is the time course of membrane voltage, go is the leak conductance, gs(t) is the time course of the slow conductance change, and E,,, is the reversal potential of the slow conductance. Since the net current must equal zero, IL(t) + Is(t) = 0. Expressing all voltages with respect to a resting potential of zero, this becomes (fJs(Q(I/'(G

- &"))

+ (QLVQ)

= 0.

Rearranging, V(t) = ss(wre” gs(t>

+ QL *

(1)

The dots in Fig. 4B show predicted V(t) values for one myosac in which gs(t) and E,,, were measured as in Figs. 1 and 2, respectively. The line traces the hap actually recorded in this myosac. The reasonably good agreement of predicted and measured voltage trajectories indicates that the measured slow conductance change can account for most of the hap. The discrepancy at very early times is probably due in part to residual inward (Na, Ca) currents activated by the action potential. The small discrepancy at later times may be measurement error, or may reflect the presence of an additional small hyperpolarizing current, such as an electrogenic pump current. Myotube membranes appear to possess an electrogenic Na pump, because following several minutes of lo-Hz stimulation we recorded a prolonged hyperpolarization lasting several seconds, whose later components were blocked by ouabain. Ca Dependence of the Slow K Conductance To test whether the slow K conductance in myotubes might, like some neuronal slow K conductances, be Ca dependent (Meech, 1978), we lowered bath [Cal from the

BARRETT, BARRETT, AND DRIBIN

-G

-74 -------lz.Lz

261

Ca-Dependent K Current in Myotubes

B B

-04

>

5 -94

.

0

200 400 600 Time (msec)

10 mv

800

FIG. 4. (A) Time course of the slow conductance change in 4 m&Z Ca, measured from records like those of Fig. 1 as described in the text. In this myosac the conductance increase decayed approximately exponentially with a time constant of 280 msec. (B) Comparison of predicted (dots) and recorded (solid line) hap’s in 4 mM Ca. Voltages were predicted from the conductance data of A using Eq. (1). The reversal potential was -96 mV, measured as in Fig. 2. The dashed line marks the resting potential.

L 200 msec

L

20 mV

normal 2 mM to only 0.1-0.2 mlM. Myotubes in these low [Cal solutions frequently had depolarized resting potentials and low input resistances, probably because Ca ions help establish high-resistance electrical sealing between the shaft of the electrode and the cell membrane.l Figure 5A shows spontaneous activity recorded from a myosac bathed in 0.2 mM Ca. The membrane potential displays pronounced oscillations, some of which trigger short bursts of action potentials. The slow hap is absent, and accordingly the minimal interspike interval is short. The small, brief hap that remains probably reflects the decay of a separate, faster K conductance (see below). Figure 5B shows the end of a train of action potentials recorded in the same cell in the normal 2 mM Ca. The slow hap is pronounced, and the interspike interval is longer. There is a small membrane potential oscillation during recovery from the last hap but the oscillation is smaller than that seen in low [Cal. In supranormal(20 mM) [Cal the slow hap was larger and often more prolonged than in normal [Cal (Fig. 5C). Even more prolonged hap’s were observed in isotonic Ca salt solutions (see Fig. 9). The hap’s recorded in high [Cal often showed a prolonged plateau, sometimes at i When myotubes bathed in low [Cal solutions were locally microperfused with normal [Cal over a small membrane area (diameter -20 pm) including either of the two electrode entry zones, the resting potential and input resistance increased toward their normal values. Similar microperfusion of membrane areas remote from the electrode entry zones had little or no effect on these electrical properties. 2 mM Mn, Co, or Ba were at least as effective as 2 m&f Ca in this respect, but even 20 mM Mg was less effective than 1 mAf Ca.

500 msec

FIG. 5. Effect of bath [Cal on the slow hap. (A) Spontaneous discharge in low Ca (0.2 m&f). The slow hap is nearly undetectable; the brief hap that remains is due to a faster K conductance system. Note the prominent membrane potential oscillations between bursts. (B) The end of a train of 12 action potentials recorded in normal Ca (2 mM) in the same myosac. Note the prominent slow hap, the longer interspike interval, and the longer duration of the burst compared to the low [Cal data of A. Same calibrations in A and B. (C) Superimposed action potentials evoked in high Ca (20 mM) in a different myosac. (Two of the current pulses were subthreshold.) The hap has a large amplitude and shows a prolonged plateau before decaying back to the resting potential. The hap’s became progressively shorter during stimulation at 0.3 Hz. pH approximately 8.

the reversal potential, but usually at a more depolarized potential. The hap recorded in 20 mM Ca became progressively shorter during repetitive stimulation at 0.3 Hz (Fig. 5C). This progressive shortening was even more evident in isotonic CaClz (Fig. 9C). Progressive hap shortening was not observed in normal 2 mM Ca. Possible mechanisms are considered in the Discussion. The hap tended to be longer at alkaline than at acid pH (bath pH changed by varying pC0,). The disappearance of the slow hap in low [Cal solutions and its augmentation in high [Cal solutions suggest that this potential is Ca dependent. Consistent with this hypothesis, divalent manganese, which inhibits voltage-dependent Ca fluxes in many tissues (e.g., Baker et al., 19’73), also blocked the slow hap (not shown).

262

DEVELOPMENTALBIOLOGY

Action potentials evoked in 2-4 miI! Mn (with or without Ca) had only a brief hyperpolarizing afterpotential, often followed by rapidly damped oscillations similar to those seen in low [Cal. Myotubes bathed in Mn had higher than normal thresholds, did not discharge spontaneously, and did not contract following action potentials. The blockage of the slow hap was readily reversible upon washout of Mn and readdition of Ca, but the blockage of contraction was more persistent. To test whether the slow K conductance is sensitive to extracellular or intracellular [Cal, we injected the calcium chelator EGTA into myosacs. Depending on the size of the myosac, injection protocols ranged from 1 to 10 nA for l-5 min. These EGTA injections usually increased the cellular input resistance (by up to three times), but had little or no persisting effect on the resting membrane potential (at most + 5-mV change). Figure 6A shows an action potential evoked in an EGTAfilled myosac bathed in normal Tyrode’s solution (2 mM Ca). The slow hap is absent, and remained absent over a wide range of membrane potentials. Myosac contraction was also reduced or abolished. The brief hap and oscillations that remain are similar to those recorded in low [Cal (Fig. 5A) or in Mn. Similar results were seen in more than 10 other EGTA-injected myosacs, but not in myosacs injected with similar current magnitudes through standard KeSOd-filled micropipets. These results indicate that the slow K conductance is activated by a rise in intracellular [Ca”‘], since EGTA injection should not reduce (and may even increase) the Ca influx associated with the action potential, but should prevent or reduce the resultant rise in intracellular [Ca”‘]. The observation that EGTA injection frequently increased cellular input resistance suggests some resting activation of the Ca-sensitive K conductance in impaled (and perhaps in normal) myosacs. Fast K Conductance The observation that myosacs in which the slow K conductance has been blocked (by exposure to low [Cal or 2-4 mM Mn, or by injection of EGTA) still show a rapidly decaying action potential and a brief (
VOLUME82, 1981 A

0

c

FIG. 6. (A, B) Action potentials and subthreshold responses recorded in a myosac injected with EGTA (A) and subsequently bathed in 5 mM TEA (B). In both A and B the upper traces record injected current pulses (one of which was subthreshold), the lower traces transmembrane voltage. EGTA was injected by passing a MO-nA current for 5 min through an intracellular electrode filled with 1 M K-EGTA. (Subsequent experiments showed that similar effects could be achieved using much smaller current intensities, l-10 nA.) EGTA injection abolished the slow hap, and subsequent exposure to TEA prolonged action potential repolarization and eliminated the fast hap seen in A. Same calibrations in A and B. (C) Action potential and slow hap in another myosac bathed in Tyrode’s solution plus 20 mM TEA.

abolished the fast hap (Fig. 6B). These results support the hypothesis that myosacs have a separate, voltagesensitive fast K conductance system that is selectively inhibited by external TEA. The actual decay of the fast K conductance after an action potential is probably much faster than the decay of the fast hap (Figs. 5A, 6A), because these myosacs have a long (20-120 msec) membrane time constant. Calcium Action Potentials The existence of a Ca-sensitive K conductance suggests that primary rat myotubes have a voltage-dependent Ca conductance system. To demonstrate this Ca conductance system, we abolished voltage-dependent Na currents by adding 10e6 to 10e5 M tetrodotoxin (TTX), and/or by using solutions in which bath Na was replaced by tetramethylammonium (TMA) and Ca. The

BARRETT,BARRETT,

ANDDRIBIN

Ca-Dependent

263

K Current in Myotubes

:7-T

FIG. 7. (A) Superimposed calcium action potentials evoked by depolarizing current steps of varying magnitude in a myosac bathed in a solution containing zero Na (TMA substituted), 20 mM Ca, 10 mM TEA, and 10m5TTX. (B) Inhibition of calcium action potentials in the same myosac following addition of 10 m&f Ni to the bath. FIG. 8. Action

potentials

B n.A L

0.5 20 mv

1set

evoked in a myosac following

infusion

of

fast K conductance was also inhibited with 10 mMTEA a solution containing 5 mM Ba and 0.1 mM Ca into the bath. (A) or occasionally with 1 mM 4-aminopyridine. Figure 7A Three superimposed traces show the early effects of Ba: a slowing of the repolarizing phase of the action potential and the disappearance shows calcium action potentials evoked by depolarizing current pulses in a myosac bathed in 10e5 M TTX, 10 of the slow hap and its accompanying conductance change. (B) Superimposed action potential and subthreshold response recorded later mM TEA, and 20 mM Ca, with TMA substituted for and with a slower time base. The action potential (whose rising phase bath Na. The calcium action potential is smaller and was retouched for clarity) now exhibits a prolonged plateau, during slower than the sodium action potential, and its latency which membrane conductance is much increased (note the reduced and amplitude vary with the amplitude of the depolar- voltage change when the hyperpolarizing current pulse was applied izing current step. Addition of 10 mM nickel to this during the plateau). The upper trace in B records injected current. myoball reversibly abolished the regenerative Ca cur- Same myosac as Fig. 1. rent (Fig. 7B); divalent manganese (10 mM) and cobalt (20 mM) also abolished the Ca currents; Co was less Spontaneous and Evoked Hyperpolarizing Potentials effective than Mn or Ni. in Isotonic CaC12 Another way to accentuate the depolarization proMyosacs bathed in isotonic (110 mM) CaC12had restduced by current flow through Ca channels is to add barium to normal Tyrode’s solution, since in other ex- ing potentials of -65 to -80 mV, and most showed spontaneous hyperpolarizing potentials which lasted up to citable cells Ba has been shown to pass readily through Ca channels, but to inhibit K conductances (Sperelakis 10 set (Figs. 9A, B). Some spontaneous hyperpolarizaet al., 1967; Hagiwara et al., 1974; Heyer and Lux, 1976). tions (S in Fig. 9C) were preceded by a depolarizing Figure 8A shows action potentials evoked in one myosac (Ca) current, but most showed no prior depolarization. shortly after exposure to a solution containing 5 mM The hyperpolarizations probably reflect activation of Ba and only 0.1 mM CA: the falling phase of the action the Ca-sensitive K conductance because they were acpotential is prolonged, and the slow hap and its asso- companied by a prolonged contraction (indicating inciated conductance change disappear. Following longer creased intracellular [Ca2’]) and a large increase in input conductance (up to four times the resting input exposure to Ba the action potential became extremely long, often lasting several seconds (Fig. 8B; see also conductance), and because they reversed at membrane Kidokoro, 1973). Records similar to those in Figs. 8A potentials near the K equilibrium potential (average and B were also seen in solutions containing 2 mM Ca -85 mV, range -80 to -95 mV in 10 cells). Spontaneous and only 1 mM Ba. The action potential and afterpo- transient conductance increases persisted at all memtential changes were reversible following brief exposure brane potentials tested (range -110 to -30 mV, Fig. to Ba. The prolongation of the action potential in Ba 9B). Membrane hyperpolarization increased the frequency of the spontaneous conductance increases (from is probably due both to enhanced depolarizing current 0,8/min at -65 mV to 2.7/min at -100 mV in the myosac flow through Ca (and Na) channels, and to inhibition of hyperpolarizing current flow through fast and slow of Fig. 9). Hyperpolarization occasionally altered the K conductance channels. Thus the effects of Ba on myo- magnitude and duration of the conductance increase, sacs are similar to its effects on other excitable tissues but this effect was not consistent, and was not studied systematically. Spontaneous behavior like that of Figs. which contain voltage-sensitive Ca channels.

264

DEVELOPMENTALBIOLOGY A

-27

mV 1 IlA 10 mV

L5oec

B

VOLUME82,198l

500-msec duration) probably due to Ca influx, followed by a much longer hyperpolarizing phase dominated by K efflux (e.g., El in Fig. 9C). When biphasic potentials were evoked at a very low rate, the amplitude and duration of the hyperpolarizing phase resembled those of the spontaneous hyperpolarizations. However, when biphasic potentials occurred at shorter intervals, the later potentials (S and E2 in Fig. 9C) showed a larger depolarizing phase and a smaller (or absent) hyperpolarizing phase. DISCUSSION

-66

mv

-89

mV

-98

mV

c

El

a

E2

-w”

FIG. 9. Spontaneous and evoked hyperpolarizations in a myosac bathed in isotonic CaClz (110 mM, plus 6 m&f KCL and 1 mM PIPES buffer). In each part the upper traces record injected current, the lower traces membrane voltage. (A) Spontaneous hyperpolarization at the resting potential (-65 mV) and inversion of the spontaneous potential when the membrane was hyperpolarized to -97 mV. (B) Spontaneous potentials during passage of brief depolarizing current pulses at three different levels of membrane potential (-65, -63, and -98 mV), showing that spontaneous conductance increases occur whether the membrane potential is more depolarized than, at, or more hyperpolarized than the reversal potential. (C) Evoked (E) and spontaneous (S) biphasic (depolarizing to hyperpolarizing) potentials. Potentials occurring shortly after a previous biphasic sequence have a larger depolarizing and a smaller hyperpolarizing phase. The timedependent depression of the hyperpolarizing phase may reflect the time needed to refill intracellular Ca stores (see Discussion). Same calibrations in A-C.

9A and B was observed in more than 30 myosacs bathed in isotonic CaClz. Five myosacs bathed in 110 mM Ca proprionate also showed spontaneous hyperpolarizations, indicating that these potentials are related more to high bath [Cal than to high bath [Cl]. We also observed spontaneous hyperpolarizations in a few myosacs in normal 2 mM Ca during a brief period following addition of 5 mM caffeine, a drug known to enhance Ca release from the sarcoplasmic reticulum of adult skeletal muscle (reviewed in Endo, 19’77).Caffeine also markedly prolonged the evoked slow hap. Application of depolarizing current pulses in myosacs bathed in isotonic CaCl, evoked prolonged hyperpolarizations and contractions. Most evoked potentials were biphasic, consisting of a depolarizing phase (5-10 mV,

The experiments described here demonstrate that cultured rat skeletal myotubes and myosacs, like many neurons (Meech, 1978), have at least two K-selective conductance systems, which are distinguishable both kinetically and pharmacologically. The fast, voltagesensitive K conductance system is inhibited by TEA and insensitive to Ca. It contributes to the falling phase of the action potential and the early phase of the hyperpolarizing afterpotential (hap). The slower K conductance is Ca sensitive, and (like the voltage-sensitive Ca conductance and myotube contraction) is inhibited by the divalent cations Mn, Ni, and Co. The slow K conductance is also inhibited by intracellular injection of EGTA, but is not blocked by extracellular application of 20 mM TEA. This slow K conductance mediates the slow hap, and the spontaneous and evoked prolonged hyperpolarizations seen in myosacs bathed in very high [Cal (or in normal [Cal plus 5 mM caffeine). Possible Mechanic for Spontaneous Hyperpolarizations The spontaneous and evoked hyperpolarizations recorded in myosacs bathed in isotonic Ca salts (Fig. 9) are similar to those seen in caffeine-treated (2-10 mM) bullfrog sympathetic ganglion cells (Kuba and Nishi, 1976; Kuba, 1980). These authors suggest that the rhythmic spontaneous hyperpolarizations are due to alternating accumulation and release of Ca by some intracellular sequestering system, with internal Ca release signaled by activation of a Ca-sensitive K conductance system in the neuronal plasma membrane. A similar mechanism probably underlies the spontaneous myosac hyperpolarizations, since myotubes have a Casensitive K conductance, and develop a specialized sarcoplasmic reticular system for internal Ca accumulation and release. Apparently both caffeine and high bath [Cal enhance release of this internal Ca store in myotubes. If this model for generation of spontaneous myotube hyperpolarizations is correct, then internal Ca stores can be released, and the Ca-sensitive K conductance can be activated, without detectable depolariza-

BARRETT,BARRE'IT,ANDDRIBIN

tion of the plasma membrane. The finding that the plasma membrane potential affects the frequency of spontaneous hyperpolarizations suggests some direct or indirect link between the plasma membrane and the Ca-releasing system. Because the hyperpolarizations evoked in isotonic CaC12(Fig. 9C, El) resemble the spontaneous hyperpolarizations, it is likely that hyperpolarizations evoked in high bath [Cal also involve release of internal Ca stores. If so, then the progressive decline in the amplitude and/or duration of the evoked hyperpolarization observed during repetitive stimulation in 20-110 mM Ca (Figs. 5C, 9C) may be caused by depletion of certain intracellular Ca stores, and/or by enhanced activity of Ca-extruding or Ca-sequestering pumps. Another possible mechanism for the decline is a desensitization of the Ca-sensitive K conductance at high intracellular [Ca”‘] (see Fig. 3 in Simons, 1976), but this is unlikely to be the sole explanation, because the decline of the hyperpolarization was associated with a weakening of myosac contraction, suggesting that the decline is associated with a reduced intracellular [Ca2+].The decline in the evoked hyperpolarization is probably not due to decreased Ca entry into the myosacs, because the depolarizing (Ca influx) phase of the response in isotonic CaC12increased during repetitive stimulation (Fig. 9C). Do Adult Skeletal Muscles Possess a Ca-Sensitive Slow K Conductance? Like rat myotubes, adult frog skeletal muscle also has a voltage-sensitive Ca conductance system (Sanchez and Stefani, 1978) and at least two K conductance systems (Adrian et al., 1970) in the plasma membrane. As in rat myotubes (Fig. 6), the faster K current in adult frog skeletal muscle is more sensitive to TEA than the slower K current (Stanfield, 1970). A portion of the K conductance of adult muscle is Ca sensitive. Sanchez and Stefani (1978) report a Ca-linked slow outward current in adult frog muscle, and Fink and Luttgau (1976) report a large increase in K conductance in metabolically exhausted frog muscle fibers, where intracellular [Ca”‘] is presumably much higher than normal. However, it is not yet clear whether a Ca-dependent slow K conductance contributes substantially to the afterpotentials or functioning of adult skeletal muscle under physiological conditions, Action potentials in adult frog skeletal muscle fibers are followed by early and late depolarizing afterpotentials (Freygang et al., 1964). These afterpotentials involve the transverse tubules, because they disappear when muscles are functionally detubulated with glycerol (Gage and Eisenberg, 1969). The late depolarizing afterpotential is thought to be influenced by K accumulation within the transverse tu-

Ca-Dependent K Current

in Myotubes

265

bules (Freygang et al., 1964; Almers, 1980), but it might also be affected by a slow K conductance, because Adrian et al. (1970) report a slow K conductance in adult frog skeletal muscle whose time course and reversal potential (around -80 mV) resemble those of the late afterpotential. (Since the resting membrane potential in adult muscle is around -90 mV, this reversal potential is unlikely to be the K equilibrium potential across the sarcolemma, but it could easily represent the average K equilibrium potential across transverse tubular membranes following the K efflux associated with an action potential.) This slow K conductance may be Ca sensitive, since Freygang et al. (1964) report that 2 mM Ni (a potent antagonist of Ca currents, see Fig. 7) abolishes the late afterpotential. Activation of a slow K conductance in transverse tubular membranes would enhance the depolarizing afterpotential produced by K accumulation in the transverse tubules, but at the same time would (along with the muscle membrane’s high Cl conductance) oppose repetitive discharge during the depolarizing afterpotential. Thus at present it seems reasonable to suggest that the Ca-sensitive slow K conductance seen in rat myotubes also exists in the transverse tubules of adult skeletal muscle, and may influence the late depolarizing afterpotential. There are marked similarities between the electrical properties of cultured myotubes and adult denervated muscle (see also Li et al., 1959; Powell and Fambrough, 1973). In both tissues the average resting potential is -60 to -70 mV (see Albuquerque et al., 1971, for denervated rat skeletal muscle), significantly more depolarized than in adult innervated muscle. In both myotubes and denervated adult muscle the action potential threshold is near the resting potential, and there is a pronounced slow hap (ionic mechanism in adult muscle not yet determined). Both tissues exhibit spontaneous action potential discharge and contractions. Purves and Sakmann (1974) observed both regular and irregular discharge patterns in denervated rat muscle, and we have observed similar patterns in myotubes. In both muscles the regular activity is preceded by membrane potential oscillations that wax and wane between trains of action potentials (e.g., Fig. 5A). These similarities in electrical behavior between cultured myotubes and adult denervated muscle may be related to the absence of innervation. Available evidence, then, suggests that mature myotubes and both innervated and denervated adult skeletal muscle all possess a slow K+ conductance system. Activation of this slow conductance system would slow the rate at which the muscle cell could be depolarized to threshold. It would help prevent the cell from discharging repetitively in response to the mechanical deformation associated with its own contraction, or in

266

DEVELOPMENTALBIOLOGY

response to the depolarizing afterpotentials associated with action potentials, capacitative currents, and K accumulation in the transverse tubular system. It is likely that the slow K conductance acts as a pacemaker in noninnervated muscle, regulating the rate of spontaneous discharge and helping to sustain that discharge by insuring sufficient time between spikes to allow recovery from Na inactivation (see also Thesleff and Ward, 1975). The finding that myotubes cultured in medium containing 2.6 mM K show more spontaneous activity than those cultured in 5 mM K (Friedman and Powell, 1977, for mouse; and our observation for rat) is consistent with the idea that the slow K conductance helps maintain spontaneous discharge, since the slow afterhyperpolarization is larger in 2.6 mMK than in 5 mMK (Fig. 3). Since contractile activity helps to maintain muscle in a healthy state (reviewed in Drachman, 1976), the postulated pacemaker function of the slow K conductance might be important in maintaining myotubes and denervated muscle cells until innervation is (re)established. We thank A. Toro and Y. Valdes for typing the manuscript. This work was supported by Grants NS 12207 and NS 12404 from the National Institutes of Health. REFERENCES ADRIAN, R. H., CHANDLER,W. K., and HODGKIN, A. L. (1970). Slow changes in potassium permeability in skeletal muscle. J. PhysioL 208,645-668. ALBUQUERQUE, E. X., SCHUH,F. T., and KAUFFMAN,F. C. (1971). Early membrane depolarization of the fast mammalian muscle after denervation. Pfluegers Arch. 328,36-50. ALMERS, W. (1980). Potassium concentration changes in the transverse tubules of vertebrate skeletal muscle. Fed. Proc. 39, 15271532. BAKER, P. F., MEVES, H., and RIDGWAY,E. B. (1973). Effects of manganese and other agents on the calcium uptake that follows depolarization of squid axons. J. PhysioL 231, 511-526. BARRETT,E. F., and BARRETT,J. N. (1976). Separation of two voltagesensitive potassium currents, and demonstration of a tetrodotoxinresistant calcium current in frog motoneurones. J. PhysioL 255, 737-774. DRACHMAN,D. B. (1976). Trophic interactions between nerves and muscles: The role of cholinergic transmission (including usage) and other factors. In Biology of Cholinergic Function (A. M. Goldberg and I. Hanin, eds.), pp. 161-186. Raven Press, New York. ENDO, M. (1977). Calcium release from the sarcoplasmic reticulum. PhysioL Rev. 57, 71-108. FINK, R., and LUTTGAU,H. C. (1976). An evaluation of the membrane constants and the potassium conductance in metabolically exhausted muscle fibres. J. Phl/sioL 263,215~238. FREYGANG,W. H., JR., GOLDSTEIN,D. A., and HELLAM, D. C. (1964). The afterpotential that follows trains of impulses in frog muscle fibers. J. Gen. PhysioL 47,929-952. FRIEDMAN,B. A., and POWELL,J. A. (1977). Method for maintenance of spontaneous membrane activity of cultured muscle. J. CeU BioL 75, 323a.

VOLUME82, 1981

FUKUDA, J. (1974). Chloride spike: A third type of action potential in tissue-cultured skeletal muscle cells from the chick. Science 185, 76-78. FUKUDA, J., HENKART, M. P., FISCHBACH,G. D., and SMITH, T. G., JR. (1976a). Physiological and structural properties of colchicine-treated chick skeletal muscle grown in tissue culture. Develop. Biol. 49,395411. FUKUDA, J., FISCHBACH,G. D., and SMITH, T. G., JR. (1976b). A voltage clamp study of the sodium, calcium and chloride spikes of chick skeletal muscle cells grown in tissue culture. Develop. Biol. 49,412424. GAGE, P. W., and EISENBERG,R. S. (1969). Action potentials, afterpotentials and excitation-contraction coupling in frog sartorius muscles without transverse tubules. J. Gen. PhysioL 53, 298-310. HAGIWARA, S., FUKUDA, J., and EATON, D. C. (1974). Membrane currents carried by Ca, Sr and Ba in barnacle muscle fiber during voltage clamp. J. Gen. PhysioL 63, 564-578. HEYER, C. B., and Lux, H. D. (1976). Control of the delayed outward potassium currents in bursting pace-maker neurones of the snail, Helix pomatia. J. PhysioL 262,349-382. KANO, M., and YAMAMOTO,M. (1977). Development of spike potentials in skeletal muscle cells differentiated in vitro from chick embryo. J. CelL PhysioL SO,439-444. KIDOKORO,Y. (1973). Development of action potentials in a clonal rat skeletal muscle cell line. Nature New Biol. 241,158-159. KUBA, K. (1980). Release of calcium ions linked to the activation of potassium conductance in a caffeine-treated sympathetic neurone. J. PhysioL 298,251-269. KUBA, K., and NISHI, S. (1976). Rhythmic hyperpolarizations and depolarization of sympathetic ganglion cells induced by caffeine. J. NeurophysioL 39,547-563. LAND, B. R., SASTRE,A., and PODLESKI,T. R. (1973). Tetrodotoxinsensitive and -insensitive action potentials in myotubes. J. CeU. Physiol. 82,497-510. LI, C.-L., ENGEL,K., and KLATZO,I. (1959). Some properties of cultured chick skeletal muscle with particular reference to fibrillation potential. J. Cell. Camp. PhysioL 53,421-444. MEECH,R. W. (1978). Calcium-dependent potassium activation in nervous tissues. Annu. Rev. Biophys. Bioeng. 7,1-18. POWELL,J. A., and FAMBROUGH,D. M. (1973). Electrical properties of normal and dysgenic mouse skeletal muscle in culture. J. CeU. Physiol. 82, 21-38. PURVES,D., and SAKMANN,B. (1974). Membrane properties underlying spontaneous activity of denervated muscle fibres. J. PhysioL 239, 125-153. SANCHEZ,J. A., and STEFANI, E. (1978). Inward calcium current in twitch muscle fibres of the frog. J. PhysioL 283, 197-209. SIMONS,T. J. B. (1976). The preparation of human red cell ghosts containing calcium buffers. J. PhysioL 256.209-225. SPERELAKIS,N., SCHNEIDER,M. F., and HARRIS, E. J. (1967). Decreased K+ conductance produced by Ba++ in frog sartorius fibers. J. Gen. PhysioL 50,1565-1583. STANFIELD, P. R. (1970). The effect of the tetraethylammonium ion on the delayed currents of frog skeletal muscle. J. PhysioL 209,X)9229. THESLEFF,S., and WARD, M. R. (1975). Studies on the mechanism of fibrillation potentials in denervated muscle. J. PhysioL 244, 313323. YAFFE, D. (1968). Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc. Nat. Acad Sci. USA 61,477-483.