The Electrophysiology of Electric Organs of Marine Electric Fishes : III. The electroplaques of the stargazer, Astroscopus y-graecum

The electroplaques of Astroscopus y-graecum were studied in situ with microelectrode recordings. Despite the distant taxonomic relations and the different origins of the organs, their properties in the teleost and torpedine marine electric fishes are remarkably similar. Only the innervated membrane (the dorsal) is electrogenically reactive in Astroscopus, and it, too, does not respond to electrical stimuli. As in the torpedine fishes, the uninnervated membrane of the electroplaques offers a very low resistance to the discharge of the innervated membrane. Additional direct evidence for electrical inexcitability of the reactive surface was obtained by denervating one of the bilateral organs. The denervated one did not respond to strong electrical stimuli which evoked responses in the opposite, innervated organ. The denervated electroplaques had a normal resting potential and were depolarized by acetylcholine and carbamylcholine similarly to normal cells. Other properties related to electrical inexcitability were also demonstrated. A pharmacological finding of considerable theoretical significance is that desensitization occurred on depolarizing cells with acetylcholine but was absent on depolarizing them with carbamylcholine.     

The Electrophysiology of Electric Organs of Marine Electric Fishes : II. The electroplaques of main and acccessory organs of Narcine brasiliensis

Studies on the electric organs of Narcine brasiliensis and particularly of the responses of the electroplaques of the accessory organ confirm and amplify data obtained on the electroplaques of Torpedo nobiliana. Only the innervated surface is electrogenically reactive and the uninnervated surface has a low resistance, as in Torpedo electroplaques. However, in the accessory organ of Narcine the innervated surface is the dorsal, rather than the ventral, and it has a different pattern of innervation. The responses of single cells of the accessory organ exhibit marked facilitation on repetitive stimulation. The facilitated responses, like the individual responses of Torpedo and of the main organ of Narcine, are electrochemically graded on changing the membrane potential with applied currents, and are inverted in sign when outward currents through the innervated face are very strong.     

The Mechanism of Dual Responsiveness in Muscle Fibers of the Grasshopper Romalea microptera

Dually innervated Romalea muscle fibers which respond differently to stimulation of their fast and slow axons are excited by intracellularly applied depolarizing stimuli. The responses, though spike-like in appearance, are graded in amplitude depending upon the strength of the stimuli and do not exceed about 30 mv. in height. In other respects, however, these graded responses possess properties that are characteristic of electrically excitable activity: vanishingly brief latency; refractoriness; a post-spike undershoot. They are blocked by hyperpolarizing the fiber membrane; respond repetitively to prolonged depolarization, and are subject to depolarizing inactivation. As graded activity, these responses propagate decrementally. The fast and slow axons of the dually responsive muscle fibers initiate respectively large and small postsynaptic potentials (p.s.p.'s) in the muscle fiber. These responses possess properties that characterize electrically inexcitable depolarizing activity. They are augmented by hyperpolarization and diminished by depolarization. Their latency is independent of the membrane potential. They have no refractory period, thus being capable of summation. The fast p.s.p. evokes a considerable or maximal electrically excitable response. The combination, which resembles a spike, leads to a twitch-like contraction of the muscle fiber. The individual slow p.s.p.'s elicit no or only little electrically excitable responses, and they evoke slower smaller contractile responses. The functional aspects of dual responsiveness and the several aspects of the theoretical importance of the gradedly responsive, electrically excitable component are discussed.     

Electrophysiology of electric organ in Gymnotus carapo

The electric organ of G. carapo is formed by linearly arrayed electroplaques which lie in four tubes on each side of the fish. In one tube the electroplaques are innervated on their rostral surfaces, in the others on the caudal. Both surfaces of each electroplaque produce spikes, and either can be excited alone by a suitably oriented externally applied stimulating current. The innervated surface, however, has a lower threshold, and in the normal organ activity, which is a continuous discharge at 35 to 60/sec., it is always fired first by the large neurally evoked postsynaptic potential. The spike of the innervated face then fires the opposite face. The potential recorded external to the innervated face is initially negative and becomes positive when the other face fires. The potential outside the other face is inverted. The p.s.p.'s are electrically inexcitable, have short duration, and are augmented by hyperpolarization. A single electroplaque is innervated by several nerve fibers, which produce summative p.s.p.'s. Homosynaptic facilitation of p.s.p.'s is common. The synapses are cholinoceptive. The organ discharge begins with synchronized activity in the rostrally innervated electroplaques. After a brief interval, the electroplaques in the other three tubes fire. The organ discharge therefore is triphasic, resulting from the summation of the two diphasic components that are oppositely directed and asynchronous. Observations on the sensory role of the organ are included.     

The Efflux of Potassium from Electroplaques of Electric Eels

1. The movement of labeled potassium ions has been measured across the innervated membranes of single isolated electroplaques, obtained from the organ of Sachs of Electrophorus electricus, mounted in an apparatus which allowed a separate washing of the two membranes. 2. Equations have been derived for a 3 compartment system in series in which tracer from a large pool in one outer compartment is collected in the other outer compartment. The amount of unlabeled ion in the middle compartment may be calculated and also the fluxes across the two membranes. 3. The flux of potassium across the innervated membranes of resting cells in a steady state was between 700 to 1000 µµmoles/cm.²/sec. and was unaffected by d-tubocurarine. 4. Direct stimulation of electroplaques with external electrodes caused an increase in the efflux of potassium from the innervated membrane of 5 to 8 µµmoles/cm.²/impulse, which was unaffected by d-tubocurarine; no change occurred in the efflux across the non-innervated membrane. 5. It is concluded that the discharge of electroplaques is accompanied by a small outward movement of potassium ions across the innervated membrane of the same order of magnitude as that found on excitation of squid giant axons. The data show a basic similarity of potassium movements across these two entirely different types of conducting membranes and suggest that this phenomenon may be a general feature of bioelectric currents propagating an action potential.     

Tetrodotoxin--a sensitive component of the depolarizing response to GABA administration to the pyramidal neuron dendrites of the CA1 field of the hippocampus

The intracellular recording of CA1 neurons in mouse hippocampal slice preparation was used to study the properties of depolarizing responses to iontophoretically applied GABA to their apical dendrites. Reversal potential of depolarizing responses was dependent on parameters of injecting current. It was about -60 mV and - (45-55) mV when iontophoretic currents 40-60 nA and 8-20 nA were used respectively. Application of tetrodotoxin (0.1-0.5 microM) resulted in decrease in amplitude of depolarizing responses evoked by weak currents, increase in slope of plot, reflecting relationship between response amplitude and membrane potential, and hyperpolarizing shift of reversal potential. Blocking++ of synaptic transmission with low calcium solution did not produce such changes. These results suggest that GABA depolarizing responses have a potential-sensitive component due to activation of sodium channels.     

Voltage-activation of high-conductance K+ channel in the insulin-secreting cell line RINm5F is dependent on local extracellular Ca2+ concentration

Patch-clamp single-channel current recording experiments have been carried out on intact insulin-secreting RINm5F cells. Voltage-activation of high-conductance K+ channels were studied by selectively depolarizing the electrically isolated patch membrane under conditions with normal Ca2+ concentration in the bath solution but with or without Ca2+ in the patch pipette solution. When Ca2+ was present in the pipette, 40 mV to 120 mV depolarizing pulses (100 ms) from the normal resting potential (-70 mV) regularly evoked tetraethylammonium-sensitive large outward single-channel currents and the average open state probability during the pulses varied from about 0.015 (40 mV pulses) to 0.1 (120 mV pulses). In the absence of Ca2+ in the pipette solution the same protocol resulted in fewer and shorter K+ channel openings and the open-state probability varied from about 0.0015 (40 mV pulses) to about 0.03 (120 mV pulses). It is concluded that Ca2+ entering voltage-gated channels raises [Ca2+]i locally and thereby markedly enhances the open-state probability of tetraethylammonium-sensitive voltage-gated high-conductance K+ channels.     

Analysis of Depolarizing and Hyperpolarizing Inactivation Responses in Gymnotid Electroplaques

In electroplaques of several gymnotid fishes hyperpolarizing or depolarizing currents can evoke all-or-none responses that are due to increase in membrane resistance as much as 10- to 12-fold. During a response the emf of the membrane shifts little, if at all, when the cell either is at its normal resting potential, or is depolarized by increasing external K, and in the case of depolarizing responses when either Cl or an impermeant anion is present. Thus, the increase in resistance is due mainly, or perhaps entirely, to decrease in K permeability, termed depolarizing or hyperpolarizing K inactivation, respectively. In voltage clamp measurements the current-voltage relation shows a negative resistance region. This characteristic accounts for the all-or-none initiation and termination of the responses demonstrable in current clamp experiments. Depolarizing inactivation is initiated and reversed too rapidly to measure with present techniques in cells in high K. Both time courses are slowed in cells studied in normal Ringer's. Once established, the high resistance state is maintained as long as an outward current is applied. Hyperpolarizing inactivation occurs in normal Ringer's or with moderate excess K. Its onset is more rapid with stronger stimuli. During prolonged currents it is not maintained; i.e., there is a secondary increase in conductance. Hyperpolarizing inactivation responses exhibit a long refractory period, presumably because of persistence of this secondary increase in conductance.     

Calcium entry induced by acetylcholine action on snail neurons

A study was made of excitatory and inhibitory responses elicited by acetylcholine (ACh) in neurons of the snail Eobania vermiculata. At resting potential, ACh evoked a depolarizing inward current in some neurons (D-cells) and a hyperpolarizing current in others (H-cells). The currents elicited by ACh were nonlinearly dependent on membrane potential. After either D- or H-cells were equilibrated in chloride-free isotonic calcium, ACh evoked a depolarizing inward current which reversed sign at about -55 mV. These results suggest that ACh causes an influx of Ca2+ in both types of neurons.     

Electrophysiological Evidence for Intrinsic Pacemaker Currents in Crayfish Parasol Cells

I used sharp intracellular electrodes to record from parasol cells in the semi-isolated crayfish brain to investigate pacemaker currents. Evidence for the presence of the hyperpolarization-activated inward rectifier potassium current was obtained in about half of the parasol cells examined, where strong, prolonged hyperpolarizing currents generated a slowly-rising voltage sag, and a post-hyperpolarization rebound. The amplitudes of both the sag voltage and the depolarizing rebound were dependent upon the strength of the hyperpolarizing current. The voltage sag showed a definite threshold and was non-inactivating. The voltage sag and rebound depolarization evoked by hyperpolarization were blocked by the presence of 5–10 mM Cs²⁺ ions, 10 mM tetraethyl ammonium chloride, and 10 mM cobalt chloride in the bathing medium, but not by the drug ZD 7288. Cs⁺ ions in normal saline in some cells caused a slight increase in mean resting potential and a reduction in spontaneous burst frequency. Many of the neurons expressing the hyperpolarization-activated inward potassium current also provided evidence for the presence of the transient potassium current I_A, which was inferred from experimental observations of an increased latency of post-hyperpolarization response to a depolarizing step, compared to the response latency to the depolarization alone. The latency increase was reduced in the presence of 4-aminopyridine (4-AP), a specific blocker of I_A. The presence of 4-AP in normal saline also induced spontaneous bursting in parasol cells. It is conjectured that, under normal physiological conditions, these two potassium currents help to regulate burst generation in parasol cells, respectively, by helping to maintain the resting membrane potential near a threshold level for burst generation, and by regulating the rate of rise of membrane depolarizing events leading to burst generation. The presence of post-burst hyperpolarization may depend upon I_A channels in parasol cells.     

Mechanism of the Schultz-Dale Reaction in the Denervated Diaphragmatic Muscle of the Guinea Pig

The mechanism of the contractions elicited by specific antigens in immunologically sensitized muscle tissue (Schultz-Dale responses) has been investigated on single fibers of denervated guinea pig hemidiaphragms. This preparation can be either actively or passively allergized, showing Schultz-Dale responses similar to those of visceral muscle. Specific antigens were applied with an electrically operated microtap to discrete areas of the cell surface while recording the electrical activity with intracellular microelectrodes. In this manner, a depolarizing action of the antigens on the muscle membrane was demonstrated. Brief applications of antigen gave rise to phasic potential changes (antigen potentials) similar to those elicited in the same fibers with acetylcholine-filled microtaps. However, antigen potentials occur only in denervated fibers sensitized to the specific antigen or closely related proteins; they are not seen in either innervated fibers of allergized animals or in denervated, nonallergized fibers. Repeated antigen application to the same area of the fiber causes a local irreversible desensitization. The antigen potentials are associated with a reduction in the resistance of the muscle membrane, similar to that caused by acetylcholine. It is concluded that besides causing the liberation of biogenic amines from the mast cells, antigens exert a direct action on the permeability of the muscle membrane; the molecules of antibody adsorbed to the cells appear to act as specific chemoreceptors for the antigen.     

Mechanisms of direct and neural excitability in electroplaques of electric eel

1. Current flow outward through the caudal, reactive membrane of the cell causes direct stimulation of the electroplaque. The electrical response in denervated as well as in normal preparations recorded with internal microelectrodes is first local and graded with the intensity of the stimulus. When membrane depolarization reaches about 40 mv. a propagated, all-or-nothing spike develops. 2. Measured with internal microelectrodes the resting potential is 73 mv. and the spike 126 mv. The latter lasts about 2 msec. and is propagated at approximately 1 M.P.S. 3. The latency of the response decreases nearly to zero with strong direct stimulation and the entire cell may be activated nearly synchronously. 4. Current flow inward through the caudal membrane of the cell does not excite the latter directly, but activation of the innervated cell takes place through stimulation of the nerve terminals. This causes a response which has a latency of not less than 1.0 msec. and up to 2.4 msec. 5. The activity evoked by indirect stimulation or by a neural volley includes a prefatory potential which has properties different from the local response. This is a postsynaptic potential since it also develops in the excitable membrane which produces the local response and spike. 6. On stimulation of a nerve trunk the postsynaptic potential is produced everywhere in the caudal membrane, but is largest at the outer (skin) end of the cell. The spike is initiated in this region and is propagated at a slightly higher rate than is the directly elicited response. Strong neural stimulation can excite the entire cell to simultaneous discharge. 7. The postsynaptic potential caused by neural or indirect stimulation may be elicited while the cell is absolutely refractory to direct excitation. 8. The postsynaptic potential is not depressed by anodal, or enhanced by cathodal polarization. 9. It is therefore concluded that the postsynaptic potential represents a membrane response which is not electrically excitable. Neural activation of this therefore probably involves a chemical transmitter. 10. The nature of the transmitter is discussed and it is concluded that this is not closely related to acetylcholine. 11. Paired homosynaptic excitation discloses facilitation which is not present when the conditioning stimulus is direct or through a different nerve trunk. These results may be interpreted in the light of the existence of a neurally caused chemical transmitter or alternatively as due to presynaptic potentiation. 12. The electrically excitable system of the electroplaque has two components. In the normal cell a graded reaction of the membrane develops with increasing strength of stimulation until a critical level of depolarization, which is about 40 mv. 13. At this stage a regenerative explosive reaction of the membrane takes place which produces the all-or-nothing spike and propagation. 14. During early relative refractoriness or after poisoning with some drugs (eserine, etc.) the regenerative process is lost. The membrane response then may continue as a graded process, increasing proportionally to the stimulus strength. Although this pathway is capable of producing the full membrane potential the response is not propagated. 15. Propagation returns when the cell recovers its regenerative reaction and the all-or-nothing response is elicited. 16. Excitable tissues may be classified into three categories. The axon is everywhere electrically excitable. The skeletal muscle fiber is electrically excitable everywhere except at a restricted region (the end plate) which is only neurally or chemically excitable. The electroplaque of the eel, and probably also cells of the nervous system have neurally and electrically excitable membrane components intermingled. The electroplaques of Raia and probably also of Torpedo as well as frog muscle fibers of the "slow" system have membranes which are primarily neurally and chemically excitable. Existence of a category of invertebrate muscle fibers with graded electrical excitability is also considered. 17. In the eel electroplaque and also probably in the cells of neurons, tests of the mode of neural activation carried out by direct or antidromic stimulation cannot reveal the neurally and chemically activated component. The data of such tests though they appear to prove electrical transmission are therefore inadequate for the detection and study of the chemically initiated process.     

Methods for intracellular recording from hippocampal brain slices under high helium pressure

A method for intracellular recording from rat hippocampal brain slices under helium pressure is described. The preparation is mounted on a horizontal mobile platform that is rolled into the pressure chamber and can be viewed at pressure. Remote manipulation of the glass microelectrodes is achieved by a high-resolution electrically driven commercially available system. The slice is superfused continuously from a closed system within the chamber. Temperature is maintained at 37 degrees C and PO2 at 0.5 atm within the pressure chamber. A pressure of 200 ATA can be obtained, although thus far recordings have been made up to only 130 ATA. The experiments demand that a number of sample recordings be made from the same slice at both ambient and high pressure, and tests have proved that, although difficult, this can be achieved. The resting membrane potential, the current-voltage relationship, and the action potential responses to short (8 ms), medium (80 ms), and long (800 ms) depolarizing current pulses have all been measured in CA1 pyramidal neurons.     

The inhibitory effects of dantrolene on action potential-induced calcium transients in cultured rat dorsal root ganglion neurons

We investigated the actions of dantrolene Ca(2+)-induced on Ca(2+)-release (CICR) evoked by action potentials in cultured rat sensory neurons. The effect of dantrolene on action potential after-depolarization and voltage-activated calcium currents was studied in cultured neonatal rat dorsal root ganglion cells (DRG) using the whole-cell patch-clamp technique. Depolarizing current injection evoked action potentials and depolarizing after-potentials, which are activated as a result of CICR following a single action potential in some cells. The type of after-potentials was determined by inducing action potentials from the resting membrane potential. Extracellular application of dantrolene (10 microM) abolished after-depolarizations without affecting action potential properties. Furthermore, dantrolene significantly reduced repetitive action potentials after depolarizing current injection into these neurons, but had no significant effect on the steady-state current voltage relationship of calcium currents in these neurons. We conclude that dantrolene inhibits the induction of action potential after depolarizations by inhibiting CICR in cultured rat sensory neurons.     

The membrane components of crustacean neuromuscular systems. I. Immunity of different electrogenic components to tetrodotoxin and saxitoxin

Axon spikes in crayfish and lobster neuromuscular preparations were blocked by tetrodotoxin or saxitoxin (concentration 10⁻⁹ to 10⁻⁸ g/ml). Responses evoked in the excitatory synaptic membrane by ionophoretically applied glutamate, or in the inhibitory by GABA were unaffected by concentrations of the poisons up to 10⁻⁵ g/ml. These confirm other findings that the poisons do not affect electrically inexcitable membrane components. “Miniature” p.s.p.’s, which indicate local secretory activity in the presynaptic terminals were unaffected by the poisons. Electrical stimuli applied to the axon terminals elicited localized p.s.p.’s after spike electrogenesis of the axons was blocked. Thus, persistence of secretory activity may be linked to persistence of depolarizing K activation in the axons. Spikes induced in the muscle fibers by procaine were not affected by the poisons. In correlation with other data this finding indicates that the depolarizing electrogenic element, which does not depend upon Na activation in the normally gradedly responsive muscles, differs chemically from the Na activation component which is present in the conductile membrane of various cells. Three other varieties of electrically excitable response which are present in crayfish muscle fibers (hyperpolarizing Cl activation, depolarizing K inactivation, and K activation) were, likewise, immune to the toxin.     

Spontaneous action potentials and resting potential shifts in fertilized eggs of the tunicate Clavelina

Electrical activity in the fertilized egg of the tunicate Clavelina was studied with microelectrode recording and voltage clamp techniques. The resting potential could assume either of two stable values (approximately ?70 or ?30 mV) and could be shifted between these values by direct current stimulation. Spontaneous shifts between two stable resting potentials were also seen. Egg cells produced action potentials spontaneously and in response to depolarizing stimuli. Inward currents were carried by both Na and Ca ions and a prominent outward potassium current was seen with depolarization to voltages above ?15 mV. The steady-state current-voltage relationship (I–V curve) of the membrane showed two voltages where the net membrane current equaled zero: approximately ?35 and ?70 mV. Between these two voltages, membrane current was inward and carried by noninactivating Na and Ca currents. Inward rectification, which was blocked by external Rb, occurred at voltages below ?70 mV. The voltage dependence of inward rectification is thought by the authors to be important for establishing the more negative resting potential; it is also thought the presence of inward current which does not inactivate completely at voltages more negative than about ?20 mV is an important determinant of the more depolarized resting potential.     

Analysis of Spike Electrogenesis and Depolarizing K Inactivation in Electroplaques of Electrophorus electricus, L

Voltage clamp analyses, combined with pharmacological tools demonstrate the independence of reactive Na and K channels in electrically excitable membrane of eel electroplaques. Spike electrogenesis is due to Na activation and is eliminated by tetrodotoxin or mussel poison, or by substituting choline, K, Cs, or Rb for Na in the medium. The K channels remain reactive, but K activation is always absent, the electroplaques responding only with K inactivation. This is indicated by an increased resistance when the membrane is depolarized by more than about 30 mv. The resting resistance (1 to 5 ohm cm²) is dependent upon the ionic conditions, but when K inactivation occurs the resistance becomes about 10 ohm cm² in all conditions. K inactivation does not change the EMF significantly. The transition from low to high resistance may give rise to a negative-slope voltage current characteristic, and to regenerative inactivation responses under current clamp. The further demonstration that pharmacological K inactivation (by Cs or Rb) leaves Na activation and spike electrogenesis unaffected emphasizes the independence of the reactive processes and suggests different chemical compositions for the membrane structures through which they operate.     

The potassium current activated by 2-nicotinamidoethyl nitrate (nicorandil) in single ventricular cells of guinea pigs

Membrane currents through potassium channels activated by nicorandil, which has a potent coronary vasodilating action, have been studied in ventricular cells of guinea pigs by using the single pipette whole-cell clamp technique. In the presence of 0.1 mM nicorandil, the duration of the action potential was shortened from 196 to 145 ms. Nicorandil markedly increased outward currents at potentials positive to the resting potential. When the difference in the currents before and after the application of nicorandil were plotted against the membrane potential, the current-voltage relation reversed close to the potassium equilibrium potential. The difference current during depolarizing pulses showed no time-dependent relaxation. These results indicate that the current evoked by nicorandil is carried by K+ ions and has voltage-independent kinetics. Power-density spectra obtained in the presence of nicorandil were fitted well by a single Lorentzian curve with a corner frequency of 4.4 Hz. The amplitude of the single-channel unit current was estimated from the relation between the variance and the mean current, and was 0.27 +/- 0.1 pA (n = 7) at -35 mV. The estimated slope conductance was 4.6 +/- 1.7 pS. Nicorandil did not affect Ca2+ currents. It is concluded that nicorandil activates a small-conductance K+ channel without affecting the Ca2+ channel.     

Voltage-activated Ca2+ currents in insulin-secreting cells

Membrane voltage and voltage-clamped membrane currents have been investigated with the whole-cell patch clamp method in the insulin-secreting cell line RINm5F. The mean resting membrane potential of RINm5F cells was found to be -52 mV. Overshooting spike potentials could be evoked by depolarising voltage steps in the absence of a secretagogue. Inward membrane currents evoked by depolarising voltage steps were dependent upon extracellular Ca2+ and blocked by Co2+, nifedipine and verapamil. Outward membrane currents which were evoked by depolarising voltage steps to positive membrane potentials were reduced when Ca2+ entry was prevented. It is concluded that the voltage-activated Ca2+ currents underlie the voltage-activated spike potentials recorded from insulin-secreting cells.     

Electrophysiological Control of Reversed Ciliary Beating in Paramecium

Quantitative relations between ciliary reversal and membrane responses were examined in electrically stimulated paramecia. Specimens bathed in 1 mM CaCl₂, 1 mM KCl, and 1 mM Tris-HCl, pH 7.2, were filmed at 250 frames per second while depolarizing current pulses were injected. At current intensities producing only electrotonic shifts the cilia failed to respond. Stimuli which elicited a regenerative response were followed by a period of reversed ciliary beating. With increasing stimulus intensities the latency of ciliary reversal dropped from 30 to 4 ms or less, and the duration of reversal increased from 50 ms to 2.4 s or more; the corresponding regenerative responses increased in amplitude and rate of rise. With progressively larger intracellular positive pulses, electric stimulation became less effective, producing responses with a progressive increase in latency and decrease in duration of reversed beating of the cilia. When 100-ms pulses shifted the membrane potential to +70 mV or more, ciliary reversal was suppressed until the end of the pulse. "Off" responses then occurred with a latency of 2–4 ms independent of further increases in positive potential displacement. These results suggest that ciliary reversal is coupled to membrane depolarization by the influx of ions which produces the regenerative depolarization of the surface membrane. According to this view suppression of the ciliary response during stimulation occurs when the membrane potential approaches the equilibrium potential of the coupling ion, thereby retarding its influx. Previous data together with the present findings suggest that this ion is Ca²⁺.