Electrophysiological Studies on the Heart of the Bivalve Mollusc, Modiolus Demissus: I. Ionic Basis of the Membrane Potential

Intracellular electrical recordings from the isolated ventricles of three bivalves (Irisawa, Shigeto & Otani, 1967; Irisawa, Noma & Ueda, 1968; Ebara, 1969; Wilkens & Greenberg, 1970) have been reported in the literature. The bivalve heart features electrical waveforms similar to those of the vertebrate myocardial pacemaker tissues (Trautwein & Kassebaum, 1961), and behaves functionally as a unitary smooth muscle where impulses are transmitted from cell to cell by way of tight junctions or nexuses. These properties are ideal for extracellular recording by the sucrose-gap technique (Stämpfli, 1954), the method which has been used predominately in the present study.

Recordings from Modiolus heart suggest that the pacemaker mechanism is a diffuse property of the entire ventricle. The myogenic pacemaker of bivalves and other molluscs has been discussed in the reviews of Krijgsman & Divaris (1955) and of Hill & Welsh (1966). This paper deals with the ionic basis of the membrane potential in Modiolus heart in terms of its characteristic spontaneous nature. Of special significance to this preparation is the low membrane potential due, in part, to chloride and sodium conductances, and the rhythmical contributions of an electrogenic sodium pump. A preliminary account of some of these data has been given previously (Wilkens & Greenberg, 1970).

The ribbed mussel, Modiolus demissus, was collected from salt marshes along the Northern Gulf coast of Florida. This environment subjects the clam to extreme fluctuations in temperature (− 5 to 35 °C), salinity (5–35 ‰), and desiccation (Pierce, 1970). Specimens were maintained in salt water aquaria in the laboratory for 1–2 months prior to use. Animals collected during the summer were kept at room temperature (23 °C), while those collected during the winter were stored in a cold room at 10–12 °C. Unless otherwise noted, experiments were performed at room temperature.

Isolation of the bivalve heart has been previously described by Welsh & Taub (1948) and by Greenberg (1965). After the animal had been removed from its shell, the heart was exposed by opening the pericardial chamber. The rectum, which passes obliquely through the heart in Modiolus demissus, was removed by cutting transversely through the ventricle and rectum, at their anterior and posterior connexions, and teasing the freed rectal segment out of the heart. The ventricle was ligated just medial to the two auricular junctions, and isolated by cutting lateral to the knots.

Although electrical activity was recorded by intracellular and extracellular techniques, most of the experiments reported in this paper have used the extracellular sucrose-gap method (Stämpfli, 1954). This technique is advantageous since it permits continuous perfusion and simultaneous recording of electrical and mechanical responses of the heart to changes in the ionic environment.

The single-gap apparatus used in these experiments is illustrated schematically in Fig. 1. The central sucrose chamber lies at the centre of a small Plexiglas wheel. The hole (2·5 mm in diameter) drilled perpendicular to the plane of the wheel forms the actual perfusing chamber. Small holes through the rim of the wheel, and continuing along the length of the spokes, provide the incurrent and excurrent perfusing channels. Two thin rubber membranes, secured on both sides of the wheel by O-rings, are drawn inward by vacuum until they meet midway between the faces of the wheel and enclose the sucrose chamber. A small hole is burned at the centre of the membrane enclosing the sucrose compartment by means of a fine-tip soldering iron.

The two Plexiglas compartments on either side of the gap are mounted in adjustable clamps and pressed tightly against the hub of the sucrose gap, in line with the central compartment. The combination of these three components constitutes a continuous cylindrical perfusion chamber divided into three sections by two thin membranes.

The isolated heart is now drawn through the holes in the two membranes, from left to right, until a small piece enters the depolarizing (right) chamber. This end of the heart is secured, while the greater part of the heart remains in the test chamber and is attached to the tension monitor. The heart now seals off the three compartments and perfusion is begun. The apparatus can be checked for leakage between compartments by perfusing one chamber at a time. The direction of flow of the perfusion fluid is illustrated by arrows in Fig. 1. The tips of two calomel reference electrodes, fitted into each of the side compartments with small O-rings, make electrical contact with the bathing solution as it drains from the chamber; thereby contamination of the perfusate with the saturated KC1 in the electrodes is avoided.

The upper middle portion of the left compartment is left open to accommodate one arm of a bellcrank for transmitting tension to a mechano-electric transducer (Grass FT 03). A lightweight spring, interposed between the bellcrank and transducer, allows the heart to beat auxotonically. The transducer was mounted in a micro-manipulator.

The perfusing system is designed to permit rapid switching of solutions with minimal disturbance to the preparation. Flow is maintained by hydrostatic pressure, and controlled independently in each compartment of the apparatus. Care is taken to avoid short-circuiting the sucrose gap through the external plumbing.

The total volume of the test chamber and tubing immediately surrounding the heart is less than 0·1 ml, thereby permitting rapid and complete turnover of the perfusate. Solutions are perfused at a rate of 0·5–1·0 ml/min. A coil of stainless-steel tubing is connected into the flow system and immersed in an ice bath for lowering the termperature of the perfusate. Temperature is monitored by a thermistor embedded in the wall of the test chamber. A more detailed account of the perfusing network has been given elsewhere (Wilkens, 1970).

Electrical potentials are led from the calomel reference electrodes into a high-input-impedance cathode follower and d.c. pre-amplifier (Grass Model P6–12). The output of the pre-amplifier is fed directly into one channel of a dual beam oscilloscope (Tektronix Type 565) and into the second channel through a differentiating circuit (time constant, 1·5 msec). A Grass kymograph camera is used to record from the oscilloscope screen. Electrical signals are led simultaneously into the driver amplifier of a two-channel ink-writing polygraph (Grass Model 79). The mechanogram is recorded by the second channel of the polygraph.

The sucrose-gap recording method is illustrated in Fig. 2. Small biphasic potentials and the accompanying mechanical activity are recorded initially. Upon perfusion with isosmotic sucrose (0·72 M), the rise in external resistance of the centre compartment is indicated by an increase in spike amplitude. The increase in external resistance reduces conduction velocity across the gap approximately 50%, as estimated from the temporal separation of the peaks of the biphasic spike. The spike is blocked in the right compartment by perfusion with 0·54 M-KCI resulting in the recording of monophasic action potentials. The resulting depolarization (gap potential) is used as an indication of the actual membrane potential.

The biphasic nature of the spike indicates that the impulse is conducted across the high external resistance of the sucrose gap. The inability of sucrose to block conduction may be caused by the failure of sucrose to replace all of the electrolytes in the extracellular space and by the presence of low-resistance pathways between adjacent cells (Irisawa, Irisawa & Shigeto, 1969).

Intracellular recordings were made with glass pipettes having a resistance of 20–50 Mil measured in sea water. Microelectrode potentials were recorded with single-ended input into the same amplification and recording system as used with the sucrose-gap. The electrodes were suspended from a Ag-AgCl wire cemented, with silver paint, to a movable coil of 0·001 in. tungsten wire (Woodbury & Brady, 1956). The bathing solution was grounded with a large Ag-AgCl wire.

In all experiments the standard perfusing medium was artificial sea water (ASW) prepared from individual salt solutions, each made isosmotic to the sea water in Vineyard Sound, Massachusetts (M.B.L. formula, Marine Biological Laboratory, 1964). The ionic content of ASW, given in millimoles per litre, and of the other experimental salines (e.g. Cl-free SW) are given in Table 1.

High-potassium solutions, used in depolarization experiments, were made in multiples of normal sea-water potassium concentration by mixing K-free SW (see Table 1) with either 0·54 M-KCI or 0·44 M-K₂SO₄.

Na-free solutions were prepared by substituting for NaCl either tris(hydroxymethyl) aminomethane (Tris) or LiCl. The method for preparing Tris Cl (85 g Tris/1 H₂O + 33 ml cone. HC1) was determined empirically by adjusting the solution to 1040 m-osmoles at pH 7·9.

Propionate was used primarily to replace chloride; in a few experiments sulphate was the major anion. The propionate and sulphate results were indistinguishable. In preparing Cl-free solutions, calcium was added in crystalline form, since CaSO₄ is insoluble at concentrations isosmotic to sea water. To eliminate lesser constituents (K, Ca and Mg) a quantity of isosmotic NaCl, equal in volume to that of the deleted solution, was added.

Only large ASW reservoirs were aerated ; no attempt was made to oxygenate each test solution. However, no effect was observed in switching from aerated ASW to non-aerated test solutions. Presumably, either a slow rate of perfusion through the external tubing (Medical Grade Silastic) allowed sufficient time for gaseous equilibration, or the heart was insensitive to small differences in oxygen tension.

The isolated ventricle of M. demissus produces spontaneous, slow-rising pacemaker waves and action potentials (Fig. 3) arising from a relatively low level of membrane polarization. This potential, as in other spontaneous preparations, cannot be characterized as a resting potential and is measured at the level of maximum diastolic potential (Hutter & Trautwein, 1956).

The amplitudes of spike and membrane potentials are quite variable from one preparation to another. Measured intracellularly from the level of maximum diastolic potential, spike amplitude ranged from 26 to 56 mV, rarely overshooting zero potential, while values for membrane potentials varied from − 42 to − 64 mV. Table 2 lists the average value for membrane and action potentials, and compares data obtained by intracellular and extracellular recording techniques. Measurements of the membrane potential by KC1 (0·54 M) depolarization in the sucrose gap agreed well with data obtained intracellularly. The extracellular spike amplitude, however, was about 30 % less than intracellular values, although the other dimensions of the spike were faith-fully recorded by this technique (cf. Fig. 3 A, B; see also Wilkens, 1971).

As in many other excitable cells, the membrane potential of Modiolus hearts can be reduced (made less negative) by increasing the external potassium concentration (K_o). In 10–20 mM-K_o sea water depolarization produces an increase in tone and frequency. At concentrations above 40 mm membrane depolarization blocks the spike and puts the heart into contracture. For these experiments the right compartment (Fig. 1) served as the test chamber; the small amount of tissue in this compartment recovered more rapidly from high concentrations of potassium (2–3 min.).

In the following experiments hearts were depolarized twice with increasing concentrations of potassium – once in KC1 and once in K₂SO₄. Following each depolarization sufficient time was allowed for recovery in normal sea water. The data, averaged from three experiments, is shown in Fig. 4. Depolarization varies linearly with log K₀ above 40 HIM. In KC1 the slope of the line is 45·7 mV per tenfold increase of K₀; in K₂SO₄ it is 49·9 mV per tenfold increase. The difference between the slopes suggests that the membrane potential is also dependent upon the permeability of the membrane to anions.

Although the potential across the cell membrane, in the heart of Modiolus, depends primarily on the ratio of intracellular to extracellular potassium, the slopes of the depolarization curves are not indicative of a pure potassium equilibrium potential. The KC1 and K₂SO₄ depolarization curves indicate that, in addition to the effect of replacing sodium with potassium, the change in membrane potential reflects the replacement of chloride by sulphate or propionate anions.

To test the effect of anions on the membrane potential the heart was depolarized by K₂SO₄ following replacement of chloride in the normal perfusion fluid with propionate. Isosmotic K₂SO₄ and Cl-free (propionate) SW were mixed to achieve the desired potassium concentration. In Fig. 5 the heart was first depolarized by KC1 in sea water, as in the previous experiment (Fig. 4). The slope for KC1, estimated from the linear portion of the depolarization curve between 40 and 400 mM K₀, is 34 mV with a final membrane depolarization of 46 mV in 540 HIM-KQ. The heart was then perfused with Cl-free SW and, after establishing a new equilibrium potential, depolarized with K₂SO₄. The slope appears to have been decreased to 29 mV for a tenfold potassium increase and the membrane depolarization, at 540 mM-K₀, is only 37 mV.

On the basis of the previous depolarization experiments, in which a gradual chloride replacement increased the K-depolarization slope (Fig. 4), the reverse effect in Cl-free SW seemed questionable. To check the reliability of the experiment the heart was returned to normal sea water and depolarized with 540 mM-K₂SO₄. This procedure depolarized the membrane by 54 mV and the slope of the K-equilibrium potential was estimated, from this value, to be 42 mV per tenfold increase in K₀. Thus, as in Fig. 4, the slope again appeared to have been increased.

To explain the apparent decrease in slope, it is necessary to consider the effect due to Cl-free SW alone. When propionate or sulphate ions are substituted for chloride, hearts undergo a sustained depolarization of 9–22 mV (Table 3) and maintain this new potential for up to several hours. This response is reversible, as the membrane returns to near the original equilibrium potential in normal sea water.

Curve C must be interpreted in light of the sustained depolarization in Cl-free SW. The same experimental procedure for determining membrane depolarization, i.e. returning the heart to normal sea water between each successive step, was used in these experiments as the heart was returned to Cl-free SW containing potassium at 10 HIM between each test. As a result of this procedure, total membrane depolarization was achieved in two steps, by first removing chloride and then by increasing K₀. In the experiment shown in Fig. 5 the heart was depolarized by 22 mV by substituting propionate for chloride and, although the membrane was subsequently depolarized only 37 mV in 540 mM-K₀, the total membrane depolarization, the sum of each step, was 59 mV. This interpretation is illustrated by elevating the potassium depolarization curve to the base-line depolarization level maintained in Cl-free SW (Fig. 5, curve A). The same total depolarization would be predicted by an experimental procedure designed to reduce chloride and increase potassium simultaneously, i.e. resulting in a linear depolarization curve with an estimated slope of 48–50 mV per tenfold increase in Ko (see Fig. 11). By this interpretation, the slope of the K-depolarization curve would be, as expected (Kuriyama, 1968), increased by reducing the external chloride concentration. The difference in potential between K₂SO₄ depolarization in normal and Cl-free SW, 54 and 59 mV, respectively, is consistent with the fact that 540 mM-K₂SO₄ ASW still contains 214 mM-Cl, or approximately 40% of the original chloride content of sea water (540 mM-KCl is isosmotic to ASW whereas isosmotic K₂SO₄ must be diluted with ASW to have an equal potassium concentration).

The basis of the sustained depolarization in Cl-free SW is not clear. The membrane may be less permeable to chloride than are the membranes of other muscles where chloride is distributed passively (Hodgkin & Horowicz, 1959). Nevertheless, the membrane must be chloride permeable if the potential is sensitive to changes in Cl₀.

Sustained depolarizations have also been reported in visceral smooth muscle by Kuriyama (1968), and by Burnstock & Straub (1958), where replacement of chloride with sulphate produced a potential decrease of 41 mV per tenfold change in Cl₀. In Ascaris muscle the membrane potential depends on the equilibrium potential of chloride to a greater extent than on potassium (del Castillo, de Mello & Morales, 1964).

Between 10 and 40 mM-K₀ the membrane potential departs from the linear relation to log K₀ (Fig. 4). There was no difference, in this range, between K-depolarizations resulting from KC1 or K₂SO₄ ; nor was there a significant difference between depolarizations in normal or Cl-free SW (Fig. 5). This portion of the curve appeared to be independent of the anion.

In the absence of an anion effect, this departure from linearity of the K-depolarization curve is probably due to membrane short-circuiting caused by leakage of sodium ions. Experiments in Na-free SW support this notion. When sodium was replaced by a large impermeant ion, such as Tris, the membrane was hyperpolarized, and the heart stopped beating (Fig. 6). Hearts remained inactive for 10–15 min in Na-free SW, and then recovered spontaneous activity with a characteristic loss of the spike plateau (see inset). The magnitude of hyperpolarization, measured when the membrane potential reached equilibrium (15–20 min), varied predictably with the initial membrane potential of the preparation. Hearts with a less-negative initial membrane potential tended to be hyperpolarized by sodium removal to a greater extent than hearts having a high level of polarization (Fig. 7).

The heart of Modiolus is characterized by a low membrane potential and, presumably, by a high interspike sodium conductance which is reduced in Na-free SW. Predictably, therefore, the K-depolarization curve deviates from linearity at a relatively high level of extracellular potassium (40 mM). As a result, this heart has electrical properties similar to those of other pacemaker tissues, in particular, the pacemaker nodes in vertebrate hearts (Trautwein & Kassebaum, 1961).

Modiolus hearts were tested with ouabain and NaCN (Fig. 8). The results of these experiments are listed in Table 3. Depolarization was reversible in both ouabain (Fig. 8B) and, to a lesser extent, in NaCN (Fig. 8C). The actions of both were complete within several minutes, i.e. no additional effects were observed after as much as 20-30 min exposure. Mechanical activity was also sensitive to these substances. An increase in tone resulted from the depolarization in both ouabain and cyanide.

Ouabain is known to inhibit specifically the enzymes regulating active transport of sodium (Schatzmann, 1953). The metabolic inhibiting action of cyanide on oxidative metabolism is indirect. Nevertheless, both inhibit a mechanism contributing to the membrane potential, presumably an electrogenic metabolic pump. The increase in tone can only be explained, from lack of further evidence, if the coupling of electrical and mechanical events has a low threshold (unpublished results), i.e. reflecting the depolarization by a rise in diastolic tone.

Membrane depolarization and a rise in tone were also produced by substituting lithium for sodium in artificial sea water (Fig. 8 A). In skeletal muscle, lithium substitutes well for sodium in passive ionic fluxes; it does not substitute in the coupled metabolic transport across the cell membrane (Keynes & Swan, 1959). In this muscle, however, the effect of lithium is more complex. Substitution of lithium for sodium depolarizes the heart, while substitution of Tris for sodium results in membrane hyperpolarization. Both substitutes also reduce the plateau of the action potential (Wilkens, 1971). These differences suggest that, in addition to an effect on the electrogenic pump mechanism, the membrane permeabilities to lithium and sodium are dissimilar.

The membrane also becomes depolarized in K-free SW. Two examples of this response are shown in Fig. 9. The first heart (Fig. 9 A) was depolarized 4 mV in K-free SW and, at the end of a 10 min exposure, was subsequently repolarized 6 mV by potassium readmittance, undershooting the original level by 2 mV. After 45 min in K-free SW, another heart (Fig. 9B) was hyperpolarized by return to normal potassium, under-shooting the original level 10 mV and briefly arresting spontaneous activity. Both hearts became irregular in K-free SW and showed a variable increase in frequency. The irregular beat in the first example (see inset) resulted from a spike with a large plateau and after-hyperpolarization alternating with smaller potentials (Fig. 9A, b).

These observations are consistent with the idea that extracellular potassium participates in the mechanism of sodium extrusion from the cell and that, in the absence of K₀, sodium enters the cell, decreasing the membrane potential. On this hypothesis, once potassium is returned on the bathing medium, the pump is stimulated by the high internal sodium ; the resultant sodium efflux briefly hyperpolarizes the membrane.

The membrane potential in the Modiolus heart is sensitive to temperature. A 15–20 °C temperature reduction produced membrane depolarizations of 2–7 mV. This information, by itself, does not lead to the conclusion that the depolarization is due to a metabolism-dependent potential since the potential would be expected to decrease about 3·5 mV by the relation of the equilibrium potential to absolute temperature as predicted by the Nernst equation.

The effect of cooling on the membrane potential was more evident in K-depolarization experiments. In two different preparations the depolarization produced by 20–50 mM excess potassium was greater at room temperature than at 3–4 °C (cf. depolarization values, Fig. 10). In reference to the original membrane potential and the initial cooling depolarization, the depolarization curves at 3–4 °C not only have a reduced slope but also intersect with the depolarization curves at room temperature, the point being that the two curves are more early superimposable toward their linear ranges (above 40 mM-K₀). Thus, membrane depolarization observed in the cold tends to produce the greatest potential change in the portion of the curves (low 1%) where the effects of a high sodium conductance and offsetting action of the electrogenic, temperature-sensitive pump become important.

The ionic mechanism underlying the low membrane potential and diastolic pacemaker in Modiolus heart appear to be similar, in part, to those in other pacemaker tissues. The basis for the low membrane potential can be better understood by examining the various depolarization curves summarized in Fig. 11. By increasing potassium to 540 mM the membrane was depolarized 46 mV by KC1 in sea water with a slope of 34 mV per tenfold increase in ; 54 mV by K₂SO₄ in sea water with a slope of 42 mV per tenfold increase in K₀; and 59 mV by K₂SO₄ and Cl-free SW with an estimated slope of 48-50 mV per tenfold increase in 1%. Thus, by replacing chloride with a less rapidly penetrating anion, the increased magnitude of the final depolarization appears to be the result of complementing, and perhaps independent, potentials. This interpretation suggests that 15–30% of the membrane potential in the heart of Modiolus is dependent on a chloride equilibrium potential, although the primary membrane permeability is to potassium.

Additional experiments designed to establish the ionic conditions of the new equilibrium (in Cl-free SW) are needed. It would be interesting to pursue the relationship of some organic anions to the membrane potential and Donnan equilibrium. Pierce & Greenberg (1970) have recently demonstrated that the membrane of the heart is permeable to amino acids in dilute sea water and that these anions cross the membrane to maintain osmotic balance. Whether Cl-free SW would alter membrane permeability or whether the transfer of amino acids is sensitive only to a change in osmotic pressure has not been determined.

A second factor contributing to the low membrane potential is a Na-leakage current (I_Na), which in low Kg produces the shift of the observed potential from the linear or more theoretical electrochemical relationship. This sodium current appears to be independent of potassium and chloride conductances, as seen by the superimposability of the curves between 10 and 40 mM-K₀ in Fig. 5. It is possible, however, to increase the membrane potential and shift the curves closer to the K-equilibrium potential by removing Na₀. The linear relation between the membrane potential and log K₀ was also extended, in Mytilus heart, by removing Nao (Irisawa et al. 1967). In addition, the variability of the membrane potential in Modiolus can be attributed to the relative Na-leakage current from one preparation to another. A similar relation between low sodium hyperpolarization and resting potential was observed in Purkinje fibres of sheep with microelectrodes (fig. 1 of Trautwein & Kassebaum, 1961). Thus preparations which exhibit low membrane potentials appear to have a greater sodium permeability.

This continuous sodium current has been interpreted as being the ‘driving force’ in spontaneous muscles (Hutter & Trautwein, 1956; Noble, 1962). At maximum diastolic potential the membrane permeability to potassium (P_K) is high and the membrane potential is dependent primarily on potassium. As P_K decreases the membrane is depolarized by the contribution of I_Na to the overall potential leading, eventually, to the spike threshold. Changing either P_K or I_Na could therefore alter the inherent spontaneity of the heart as would be expected, for example, by increasing the inward I_Na. A net increase in I_Na, resulting from inhibition of the sodium pump by K-free SW, is the apparent explanation for the increased frequency in Fig. 9.

The direct effects of metabolic processes on the membrane potentials of muscles and nerves have been the topic of considerable recent research, particularly in the studies on molluscan giant neurones (Kerkut & Thomas, 1965; Carpenter & Alving, 1968; Carpenter, 1970; Moreton, 1969; Thomas, 1969; Gorman & Marmor, 1970). In these systems the exchange mechanism for active transport of sodium and potassium ions across the cell membrane creates electrogenic currents in addition to maintaining the long-term ionic imbalances required for continued regenerative activity. Sodium-pump electrogenesis is normally observed in relation to the membrane currents in the resting state. In Modiolus, where the membrane potential is not electrically analogous to a resting potential, the electrogenic pump appears to operate primarily in regenerative processes following each spike.

The evidence for the sodium pump is summarized in Table 3. The membrane potential in Modiolus heart is depolarized by 2–8 mV by inhibitors of the enzymes involved in active transport (ouabain and cyanide), by slowing the rates of these enzymatically coupled mechanisms (reduced temperatures), and by removing the substrates necessary for coupling of these transport mechanisms (K-free and Li-free SW).

A further observation concerning the membrane potential, a term used synonomously with the maximum diastolic potential, indicates a direct relationship between the electrical activity in the muscle, a high I_Na, and the opposing action of the sodium pump. In arrhythmical preparations (e.g. Fig. 12A), failure to produce a spike also fails to repolarize the membrane to the maximum diastolic potential. By superimposing expanded records of pacemaker depolarizations, with and without a spike, an after-hyperpolarizing phase of the action potential can be identified (Fig. 12B). The membrane potential or maximum diastolic potential is therefore dependent on the magnitude of the after-potential. The amplitude of the slow wave and after-hyperpolarization in this example were 7 and 5 mV, respectively. Action potentials with large plateaus also have a more negative after-hyperpolarization (positive after-potential) (Figs. 8A, 9A). Evidence that the plateau results from a prolonged sodium current will be presented in the following paper (Wilkens, 1971). Therefore the larger after-hyperpolarizing response appears to represent an increased electrogenic sodium current stimulated by a prolonged influx of sodium during the plateau. Artificial stimulation of the pump, by injecting sodium inside the cell, has been demonstrated in several instances (Kerkut & Thomas, 1965 ; Koike, Brown & Hagiwara, 1970 ; Moretón, 1969; Thomas, 1969).

The relationship between the plateau and after-hyperpolarization was dramatically illustrated (Fig. 8A) by the simultaneous reduction of both of these phases of the action potential in Li-SW. Here, the decrease in magnitude of the after-potential, hence the 8 mV depolarization, reflects the inability of lithium to substitute for sodium in the plateau, the gradual loss of internal sodium and, finally, the inability of lithium to substitute for sodium in the electrogenic pump. The reduction of the after-potential in Li-SW may, on the other hand, represent only the difference between sodium and lithium permeability in the formation of the plateau since the after-potential has already been shown to be dependent on the plateau. Lithium is also, reportedly, unable to generate currents across the membrane in the cultured chick heart (Pappano & Sperelakis, 1969).

Nevertheless, depolarization, or reduced after-hyperpolarization, can be directly attributed to the pump in the case of ouabain treatment, where the effect is present without a simultaneous attenuation of the plateau (Fig. 8 B). The plateau, in this slow-speed record, is identifiable as the lower, heavily shaded portion of the electrical trace.

These results, however, do not completely explain the basis for pacemaker rhythmicity in this preparation. One question raised by these experiments concerns the ability of the heart spontaneously to become depolarized in the absence of sodium. The ability to develop action potentials in the absence of sodium, and their dependence on calcium, will be discussed in the following paper (Wilkens, 1971).

The ionic basis for the membrane potential in the heart of Modiolus demissus, as the combination of the relative permeabilities of potassium, chloride, and sodium permeabilities, and the operation of a metabolic pump, tentatively explains the wide range in the observed membrane potential. Further study will be necessary to determine the interrelations of these factors quantitatively.

The author wishes to thank Dr M. J. Greenberg for his direction and support, Dr A. L. F. Gorman for helpful criticism of the manuscript, and Mr J. Webers for assistance in construction of the apparatus.

Contribution No. 8 from the Tallahassee, Sopchoppy and Gulf Coast Marine Biological Association.

Supported, in part, by U.S.P.H.S. Fellowship No. GM-43039 (General Medical Sciences) and by U.S.P.H.S. Grant No. HE-09283 (National Heart Institute).