Voltage-dependent calcium and calcium-activated potassium currents of a molluscan photoreceptor

Two-microelectrode voltage clamp studies were performed on the somata of Hermissenda Type B photoreceptors that had been isolated by axotomy from all synaptic interaction as well as any impulse-generating (i.e., active) membrane. In the presence of 2-10 mM 4-aminopyridine (4-AP) and 100 mM tetraethylammonium ion (TEA), which eliminated two previously described voltage-dependent potassium currents (IA and the delayed rectifier), a voltage-dependent outward current was apparent in the steady state responses to command voltage steps more positive than -40 mV (absolute). This current increased with increasing external Ca++. The magnitude of the outward current decreased and an inward current became apparent following EGTA injection. Substitution of external Ba++ for Ca++ also made the inward current more apparent. This inward current, which was almost eliminated after being exposed for approximately 5 min to a solution in which external Ca++ was replaced with Cd++, was maximally activated at approximately 0 mV. Elevation of external potassium allowed the calcium (ICa++) and calcium-dependent K+ (IC) currents to be substantially separated. Command pulses to 0 mV elicited maximal ICa++ but no IC because no K+ currents flowed at their new reversal potential (0 mV) in 300 mM K+. At a holding potential of -60 mV, which was now more negative than the potassium equilibrium potential, EK+, in 300 mM K+, IC appeared as an inward tail current after positive command steps. The voltage dependence of ICa++ was demonstrated with positive steps in 100 mM Ba++, 4-AP, and TEA. Other data indicated that in 10 mM Ca++, IC underwent pronounced and prolonged inactivation whereas ICa++ did not. When the photoreceptor was stimulated with a light step (with the membrane potential held at -60 mV), there was also a prolonged inactivation of IC. In elevated external Ca++, ICa++ also showed similar inactivation. These data suggest that IC may undergo prolonged inactivation due to a direct effect of elevated intracellular Ca++, as was previously shown for a voltage-dependent potassium current, IA. These results are discussed in relation to the production of training-induced changes of membrane currents on retention days of associative learning.     

Action of quinidine on ionic currents of molluscan pacemaker neurons

The effects of quinidine on the fast, the delayed, and the Ca2+- activated K+ outward currents, as well as on Na+ and Ca2+ inward currents, were studied at the soma membrane from neurons of the marine mollusk Aplysia californica. External quinidine blocks these current components but to different degrees. Its main effect is on the voltage- dependent, delayed K+ current, and it resembles the block produced by quaternary ammonium ions (Armstrong, C. M., 1975, Membranes, Lipid Bilayers and Biological Membranes: Dynamic Properties, 3:325-358). The apparent dissociation constant is 28 microM at V = +20 mV. The blocking action is voltage and time dependent and increases during maintained depolarization. The data are consistent with the block occurring approximately 70-80% through the membrane electric field. Internal injection of quinidine has an effect similar to that obtained after external application, but its time course of action is faster. External quinidine may therefore have to pass into or through the membrane to reach a blocking site. The Ca2+-activated K+ current is blocked by external quinidine at concentrations 20-50-fold higher compared with the delayed outward K+ current. In addition, it prolongs the time course of decay of the Ca2+-activated K+ current. Na+ and Ca2+ inward currents are also blocked by external quinidine, but again at higher concentrations. The effects of quinidine on membrane currents can be seen from its effect on action potentials and the conversion of repetitive "beating" discharge activity to "bursting" pacemaker activity.     

Effects of tetraethylammonium on potassium currents in a molluscan neurons

The effects of tetraethylammonium (TEA) on the delayed K+ current and on the Ca2+-activated K+ current of the Aplysia pacemaker neurons R-15 and L-6 were studied. The delayed outward K+ current was measured in Ca2+-free ASW containing tetrodotoxin (TTX), using brief depolarizing clamp pulses. External TEA blocks the delayed K+ current reversibly in a dose-dependent manner. The experimental results are well fitted with a Michaelis-Menten expression, assuming a one-to-one reaction between TEA and a receptor site, with an apparent dissociation constant of 6.0 mM. The block depends on membrane voltage and is reduced at positive membrane potentials. The Ca2+-activated K+ current was measured in Ca2+-free artificial seawater (ASW) containing TTX, using internal Ca2+ ion injection to directly activate the K+ conductance. External TEA and a number of other quaternary ammonium ions block the Ca2+-activated K+ current reversibly in a dose-dependent manner. TEA is the most effective blocker, with an apparent dissociation constant, for a one-to-one reaction with a receptor site, of 0.4 mM. The block decreases with depolarization. The Ca2+-activated K+ current was also measured after intracellular iontophoretic TEA injection. Internal TEA blocks the Ca2+-activated K+ current (but the block is only apparent at positive membrane potentials), is increased by depolarization, and is irreversible. The effects of external and internal TEA can be seen in measurements of the total outward K+ current at different membrane potentials in normal ASW.     

Inositol trisphosphate regulation of photoreceptor membrane currents.

In previous studies elevation of intracellular Ca2+ was shown to cause prolonged reduction of two voltage-dependent K+ currents (IA and ICa2+-K+) across the membrane of the isolated Hermissenda photoreceptor, the type B cell (Alkon et al., 1982b; Alkon and Sakakibara, 1985). Here we show that iontophoretic injection of inositol trisphosphate (IP3), but not inositol monophosphate, also caused prolonged reduction of IA and ICa2+-K+. IP3 injection also caused reduction of a light-induced K+ current (also ICa2+-K+) but did not affect the voltage-dependent Ca2+ current, ICa2+, or the light-induced inward current, INa+, of the type B cell. IP3 injection caused similar effects on the K+ currents of the other type of Hermissenda photoreceptor, the type A cell. INA+ of the type A cell, unlike that of the type B cell, was, however, markedly increased following IP3 injection. The differences of IP3 effects on the two types of photoreceptors may be related to differences in regulation of ionic currents by endogenous IP3 as reflected by clear differences (before injection) in the magnitude of IA, ICa2+-K+, and INa+ between the two cell types.     

Effects of 4-aminopyridine on potassium currents in a molluscan neuron

The effects of 4-aminopyridine (4-AP) on the delayed K+ current and on the Ca2+-activated K+ current of the Aplysia pacemaker neurons R-15 and L-6 were studied. The delayed outward K+ current was measured in Ca2+- free artificial seawater (ASW) containing tetrodotoxin (TTX), using brief depolarizing clamp pulses. External (and internal) 4-AP blocks the delayed K+ current in a dose-dependent manner but does not block the leakage current. Our results show that one 4-AP molecule combines with a single receptor site and that the block is voltage dependent with an apparent dissociation constant (K4-AP) of approximately 0.8 mM at 0 mV. K4-AP increases e-fold for a 32-mV change in potential, which is consistent with the block occurring approximately 0.8 of the distance through the membrane electrical field. The 4-AP block appears to depend upon stimulus frequency as well as upon voltage. The greater speed of onset of the block produced by internal 4-AP relative to when it is used externally suggests that 4-AP acts from inside the cell. The Ca2+-activated K+ current was measured in Ca2+-free ASW containing TTX, using internal Ca2+-ion injection to directly activate the K+ conductance. Low external 4-AP concentrations (less than 2 mM) have no effect on the Ca2+-activated K+ current, but concentrations of 5 mM or greater increase the K+ current. Internal 4-AP has the same effect. The opposing effects of 4-AP on the two components of the K+ current can be seen in measurements of the total outward K+ current at different membrane potentials in normal ASW and during the repolarizing phase of the action potential.     

pH titrations of molluscan paramyosin at two different ionic strengths.

Paramyosin extracted from the adductor muscle of Mercenaria mercenaria, the chowder clam, was titrated both in 0.3 M KCl and in 1 mM KCl. Both the presumed native form of the molecule, acid-R-paramyosin, and a slightly degraded form, beta-paramyosin, were studied. Titrations of both types of paramyosin were similar in 1 mM k+, except that the native paramyosin is more highly charged at pH 3.2 than beta-paramyosin, as postulated previously (DeLaney and Krause, 1976, Macromolecules, 9:455), and that more groups titrate on the native molecule than on beta-paramyosin, both between pH 3.2 and 3.3 and between pH 3.2 and 10. Titrations in 0.30 M KCl, unlike those in 1 mM K, depended on starting pH; long term exposure to alkali solutions during dialysis, previously shown to cause partial dephosphorylation of paramyosin (Cooley et al., 1979, J. Biol. Chem., 254:2195), apparently also leads to a change in intermolecular interactions sufficient to cause changes in the titration curves in 0.30 M KCl but not in 1 mM K+.     

Interaction of galantamine with ionic channels in molluscan neurons

Galantamine is widely used for the treatment of Alzheimer’s disease. According to the generally accepted viewpoint, its therapeutic effect is based on inhibition of acetylcholinesterase (AChE) and potentiation of nicotinic receptors. Alternative molecular targets for galanatamine, namely, voltage-gated Ca²⁺ and K⁺ channels of the neuronal membrane, are also widely discussed in the current literature. The present study is devoted to the analysis of effects of galantamine on high-threshold Ca²⁺ currents (I _Ca) and three different kinds of highthreshold K⁺ current, viz.: Ca²⁺-dependent K⁺ current (I _C), delayed rectifier (I _DR), and fast-inactivating K₊ current (I _Adepol). Experiments were conducted on molluscan neurons with the help of two-microelectrode voltageclamp technique. It was found that galantamine caused a fast, reversible and dose-dependent suppression of all types of high-threshold ionic currents. The maximal blocking effect of the alkaloid for I _Ca, I _C, and I _DR, was 100%, while for I Adepol the maximal suppression was only 60%. The mean values of IC ₅₀ for I _C, I _DR, I _Adepol, and I _Ca were 109, 237, 66, and 515 μ M, respectively, i.e., substantially higher than the corresponding values for the alkaloid-induced inhibition of AChE and potentiation of nicotinic receptors. It is concluded that the blockade of Ca₂₊ and K₊ channels has little or no contribution to the therapeutic activity of galantamine.     

Cadmium ions modulate GABA induced currents in molluscan neurons

The effect of Cd2+, as one of the most widespread toxic environmental pollutants, was studied on gamma-aminobutyric acid (GABA) evoked responses of identified neurons in the central nervous system of the pond snail, LYmnaea stagnalis L. (Gastropoda). In the experiments, the modulation of the action of GABA both on neuronal activity (current clamp recording) and on the a GABA activated membrane Cl- current (voltage clamp studies) has been shown. It was found that: 1. GABA could evoked three different various types of response in GABA sensitive neurons: i) hyperpolarization with strong inhibition of ongoing spike activity, ii) short depolarization with an increase of spike the activity, iii) biphasic respone with a short excitation followed by a more prolonged long inhibition. 2. In low-Cl- solution the inhibitory action of GABA was reduced or eliminated, but the excitatory one was not or only moderately affected. 3. CdCl2 inhibited the GABA evoked hyperpolarization, but left intact or only slightly reduced the excitation evoked by GABA. 4. The inward Cl- current evoked by GABA at a -75 mV holding potential was slightly augmented in the presence of I micromol/l Cd2+, but was reduced or blocked at higher cadmium concentrations. The effect of Cd2+ was concentration and time dependent. 5. Parallel with reducing the GABA evoked current, cadmium increased both the time to peak and the half inactivation time of the current. 6. CdCl2 alone, in 50 micromol/l concentration, induced a 1-2 nA inward current. The blocking effect of cadmium on GABA activated inhibitory processes can be an important component of the neuro-toxic effects of this heavy metal ion.     

Inactivation of calcium currents in the molluscan neuron somatic membrane

The time course of weakening of inward calcium currents (inactivation) during prolonged (of the order of 1 sec) depolarizing shifts of membrane potential was studied in isolated dialyzed neurons of snailHelix pomatia. This decay of the current recorded in this way can be approximated by two exponential functions with time constants of 20–70 and 250–350 msec, respectively. With an increase in pH of the intracellular solution to 8.5 the fast component of the decay disappeared completely; the kinetics of the slow component in this case was very slightly retarded. It is concluded that the fast component of decay of the recorded current does not reflect a change in the calcium current but is due to parallel activation of the nonspecific outward current; the slow component, however, is true in activation of the calcium current. The rate of inactivation of this current was shown to be determined by its maximal value and not by the level of the depolarizing potential shift and it depends on the conditions of accumulation of calcium ions near the inner surface of the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 525–531, September–October, 1982.     

The assembly of ionic currents in a thalamic neuron. III. The seven-dimensional model

We replace our earlier three-dimensional ha-model of a thalamic neuron (Rose & Hindmarsh, Proc. R. Soc. Lond. B 237, 289-312 (1989b)) by a seven-dimensional ha-model. The stability and state diagrams for this seven-dimensional model are shown to be similar to those of the three-dimensional system with which it is compared. Two examples that illustrate how the seven-dimensional model can be related to experimental recordings are then discussed in detail. In each case we show the state and stability diagrams and the time courses of the different ionic currents during the burst response. We discuss how the various components of the state diagram could be determined experimentally. These experiments are illustrated by using our model to simulate the results of actual experiments. Finally the state and stability diagrams are transferred to a current-voltage diagram. This shows the advantage of the state diagram compared with the current-voltage diagram for representing the dynamics of the firing of a thalamic neuron.     

The assembly of ionic currents in a thalamic neuron. I. The three-dimensional model

We have previously discussed qualitative models for bursting and thalamic neurons that were obtained by modifying a simple two-dimensional model for repetitive firing. In this paper we report the results of making a similar sequence of modifications to a more elaborate six-dimensional model of repetitive firing which is based on the Hodgkin-Huxley equations. To do this we first reduce the six-dimensional model to a two-dimensional model that resembles our original two-dimensional qualitative model. This is achieved by defining a new variable, which we call q. We then add a subthreshold inward current and a subthreshold outward current having a variable, z, that changes slowly. This gives a three-dimensional (v,q,z) model of the Hodgkin-Huxley type, which we refer to as the z-model. Depending on the choice of parameter values this model resembles our previous models of bursting and thalamic neurons. At each stage in the development of these models we return to the corresponding seven-dimensional model to confirm that we can obtain similar solutions by using the complete system of equations. The analysis of the three-dimensional model involves a state diagram and a stability diagram. The state diagram shows the projection of the phase path from v,q,z space into the v,z plane, together with the projections of the curves z = 0 and v = q = 0. The stability of the points on the curve v = q = 0, which we call the v, q nullcurve, is determined by the stability diagram. Taken together the state and stability diagrams show how to assemble the ionic currents to produce a given firing pattern.     

Zinc-induced changes in ionic currents of cardiomyocytes

The whole-cell voltage-clamp technique was applied to isolated ventricular myocytes to investigate the effects of extracellular and intracellular zinc application on L-type Ca²⁺ channel currents (I _Ca). Extracellular zinc exposure at micromolar concentration induced a reversible (with washout of ZnCl₂) reduction (30%) of I _Ca with no change in current-voltage relationship. On the other hand, an increase of intracellular free-zinc concentration, [Zn²⁺]_i, from normal (less than 1 nM) to approx 7 nM with 10 μM Zn-pyrithione exposure caused an inhibition of 33±6% in the peak of the I _Ca and altered the voltage dependency of L-type Ca²⁺ channels with a 10-mV left shift and a hump at around −40 mV in its current-voltage relation. In contrast, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) strongly inhibited the I _Ca (42±2%), with only a small but detectable outward shift of the holding current measured at the end of the pulses. Zn-pyrithione and TPEN caused a reproducible decrease of the I _Ca. Interestingly, TPEN application, without Zn-pyrithione pretreatment, inhibited the I _Ca (35±2%) with no change in voltage dependency. Taken together, the results suggest that both extracellular and intracellular zinc increases under pathological conditions in cardiomyocytes can alter the I _Ca, but their effects are not in the same order and same manner. One should consider these possible side effects when it is suggested to be vital to cardiovascular cell integrity and functions.     

Study of ionic currents across a model membrane channel using Brownian dynamics.

Brownian dynamics simulations have been carried out to study ionic currents flowing across a model membrane channel under various conditions. The model channel we use has a cylindrical transmembrane segment that is joined to a catenary vestibule at each side. Two cylindrical reservoirs connected to the channel contain a fixed number of sodium and chloride ions. Under a driving force of 100 mV, the channel is virtually impermeable to sodium ions, owing to the repulsive dielectric force presented to ions by the vestibular wall. When two rings of dipoles, with their negative poles facing the pore lumen, are placed just above and below the constricted channel segment, sodium ions cross the channel. The conductance increases with increasing dipole strength and reaches its maximum rapidly; a further increase in dipole strength does not increase the channel conductance further. When only those ions that acquire a kinetic energy large enough to surmount a barrier are allowed to enter the narrow transmembrane segment, the channel conductance decreases monotonically with the barrier height. This barrier represents those interactions between an ion, water molecules, and the protein wall in the transmembrane segment that are not treated explicitly in the simulation. The conductance obtained from simulations closely matches that obtained from ACh channels when a step potential barrier of 2-3 kTr is placed at the channel neck. The current-voltage relationship obtained with symmetrical solutions is ohmic in the absence of a barrier. The current-voltage curve becomes nonlinear when the 3 kTr barrier is in place. With asymmetrical solutions, the relationship approximates the Goldman equation, with the reversal potential close to that predicted by the Nernst equation. The conductance first increases linearly with concentration and then begins to rise at a slower rate with higher ionic concentration. We discuss the implications of these findings for the transport of ions across the membrane and the structure of ion channels.     

The ionic requirements for regulation by molluscan thin filaments

Initial studies on molluscan muscle regulation indicated that thin filaments do not confer Ca2+-dependence on vertebrate myosin ATPase, and hence that molluscan muscles do not possess thin filament-linked regulatory systems. Subsequently it was shown that molluscan thin filaments do, in fact, impart Ca2+-sensitivity but only at Mg2+ concentrations greater than those used in the earlier studies. In the present study it is shown that Mg2+ prevents significant dissociation of tropomyosin and troponin subunits from thin filaments at the low monovalent ion concentrations typically employed to assay actomyosin ATPase; as a result Mg2+ allows expression of the molluscan thin filament regulatory system under these conditions.     

The assembly of ionic currents in a thalamic neuron. II. The stability and state diagrams

In the previous model of a thalamic neuron (R.M. Rose & J.L. Hindmarsh, Proc. R. Soc. Lond. B237, 267-288 (1989], which we referred to as the z-model, the burst response was terminated by the slow activation of a subthreshold outward current. In this paper we show that similar results can be obtained if the burst response is terminated by slow inactivation of the subthreshold inward current, Isa. We illustrate the use of this new model, which we refer to as the ha-model, by using it to explain the response of a thalamic neuron to a double ramp current. The main aim of the paper is to show how the stability and state diagrams introduced previously can be used to explain various types of firing pattern of thalamic and other neurons. We show that increasing the threshold for the fast action potentials leads to low threshold spikes of increased amplitude. Also, addition of a second subthreshold inward current adds a new stability region, which enables us to explain the origin of plateau potentials. In addition, various types of subthreshold oscillation are produced by relocating a previously stable equilibrium point in an unstable region. Finally, we predict a sequence of responses to current steps from different levels of background current that extends the burst, rest, tonic sequence of thalamic neurons. The stability and state diagrams therefore provide us with a useful way of explaining further properties of thalamic neurons and appear to have further applications to other mammalian neurons.     

Guaiacol and vanilloid compounds modulate the A-type potassium currents in molluscan neurons

The actions of guaiacol (2-methoxy-phenol), vanillin (4-hydroxy-3-methoxy-benzaldehyd) and other vanilloid compounds such as zingerone (4-/4-hydroxy-3-methoxyphenyl/-2-butanon) and eugenol(2-methoxy4-/2-propenyl/phenol) were investigated on the fast outward potassium currents (A-type currents) in molluscan neurons. Guaiacol (0.01-0.1%, w/v) moderately decreased the peak amplitude but increased the rate of inactivation of the A-currents in dose-dependent way (Kd = 0.06% 4 mM, nH = 0.8). Vanillin (5 mM) slightly decreased the peak amplitude of the A-currents in Helix neurons but its action was more pronounced in dialysed Lymnaea nerve cells. However, vanillin similarly decreased the time-to-peak and the time constant of decay of the A-currents both in the faster and the slower inactivating Lymnaea and Helix neurons (Kd = 5 mM, nH = 0.6). The voltage-dependence of activation and inactivation of the A-currents were not significantly influenced by guaiacol and vanillin in Helix or Lymnaea neurons. Vanillin hardly influenced the delayed outward currents, but decreased the leak currents in the identified LPa and RPa 2,3 neurons. A structure-activity analysis clearly showed that increasing alkyl tail length from the aldehyde side of the vanillin molecule increased the efficacy of the various compounds on the amplitude of the A-currents and modified the kinetical influence on the A-current channel. Furthermore, an attenuation of the late outward currents and an increase of the leak conductance also developed in the presence of zingerone or eugenol. Excitatory actions of the studied vanilloids predominated on the various molluscan neurons.     

The effects of protons on 3'',5''-cGMP-activated currents in photoreceptor patches.

Macroscopic 3',5'-guanine cyclic monophosphate (cGMP)-activated currents from photoreceptor outer segment membranes were examined as the pH on the cytoplasmic face of inside/out patches was reduced. In the absence of divalent cations, protons reduced the current in both directions without affecting the shape of the current-voltage relation consistent with a voltage-independent block. When Ca2+ was added to the bath, increasing the [H+] relieved the Ca2+ block and eliminated the Ca(2+)-induced reversal potential shifts seen at pH 7.4. These results suggest that protons alter Na+/Ca2+ permeability of the channel and relieve Ca2+ block of the sodium transport.     

Role of ionic currents in the physiological response to angiotensin II.

Studies using conventional and patch-clamp microelectrode techniques demonstrate that in a number of cell types angiotensin II (AII) causes reversible changes in transmembrane ionic currents, and that these effects can be mimicked by various membrane-associated and cytosolic messengers. AII modulates the current amplitude of ion channels, as well as their activation threshold and their open/closed time probability. Stimulatory and inhibitory effects on ion channel activity are a fundamental feature of the development of AII actions on target organs.