Single channel activity associated with the calcium dependent outward current inHelix pomatia

A recently improved version of the extracellular patch clamp technique (9, 13) was used to record currents from microscopic membrane areas of Helix neurons with predominant Ca²⁺ dependent outward currents. Current fluctuations in the patches consisted mainly of frequently interrupted, one-sided steps indicating discrete open-closed state changes of single channels with an ohmic conductance of approximately 19 pS. Frequency of occurrence of the elementary events compares with amplitudes of macroscopic currents during depolarizing voltage steps of varied amplitude. Average delays in appearance of the events vary in line with delayed time courses of the cell's outward current.     

The effects of capsaicin on the early outward current in identified snail neurones

An investigation of the action of capsaicin (CAP) on the early outward current (IA) in identified neurones of Helix pomatia was performed using the single electrode clamp. CAP (60-300 microM) caused a dose-dependent reduction of the IA-currents in LPa3 and RPa3 neurones both in normal and Na-free solutions. There was also a marked shortening of the time constant for the inactivation of IA, and a 5-10 mV shift in the hyperpolarizing direction of the curve relating the steady-state inactivation of IA to membrane potential. The equilibrium potential for IA was not changed in up to 300 microM CAP solutions.     

Contribution of restricted extracellular space to the inactivation of calcium current in the snail neuron

The mechanisms underlying the inactivation of calcium current (ICa) were investigated in isolated nerve cell bodies of Helix aspersa using a suction pipette technique that allowed voltage clamp and internal perfusion at the same time. ICa was recorded after eliminating the Na and K currents by removing Na+ and K+ both in external and internal solutions, and ICa inactivation due to intracellular Ca2+ accumulation was blocked by 5-25 mM EGTA. The inactivation rates of ICa, IBa and ISr corresponded to two exponential processes. The inactivation rates of the inward currents (IMn, ICd and IZn) less than 1/5 of ICa fitted a single exponential. However, when neurons were superfused with hypertonic external solution by adding 100 mM sucrose together with internal EGTA, the steady-state inactivation of ICa, IBa and ISr was reduced, and the inactivation processes changed to a single exponential similar to that of IMn, ICd and IZn. In contrast, internal perfusion with the hypertonic solution had no effect on the inactivation of ICa, IBa and ISr. Therefore, it was concluded that the inactivation process of ICa is dependent not only on the membrane voltage and the intracellular Ca2+ accumulation as described previously, but is also affected by the rapid fall in the concentration of Ca2+ in the restricted extracellular spaces (RES) which gets enlarged by the hypertonic external solution. The same is also true for IBa and ISr.     

Lithium slows neuronal calcium regulation in the snail Helix pomatia

Steady-state and transient changes in intracellular calcium concentrations ([Ca2+]i) of snail neurons (Helix pomatia) were measured by the Ca2+ indicator Arsenazo(III) following manipulation of the extracellular concentration of lithium chloride (LiCl). Application of LiCl in concentrations equivalent to those used in the treatment of manic-depressive illness produces slowing in Ca2+ reequilibration after Ca2+-influx during depolarization, concomitantly with steady-state elevation of [Ca2+]i of about 100 nM, suggesting a change in Ca2+ reequilibration as a prominent action of LiCl. This mechanism may be relevant to the therapeutic effects of LiCl.     

Effects of calcium and calcium-chelating agents on the inward and outward current in the membrane of mollusc neurones.

1. Effects of internal and external Ca and Ca-chelating agents, EGTA and EDTA on transmembrane ionic currents were studied in isolated, internally dialysed neurones from the molluscs, Helix pomatia and Limnea stagnalis.2. The possible pharmacological effect of internally applied EGTA was investigated on the background of constant free Ca concentration (5.3 x 10(-9)M). EGTA had no effect on Ca and Na inward currents but considerably depressed the delayed K outward current. No effective removal of this action could be achieved by the elevation of intracellular free Ca.3. In the absence of divalent cations in the external medium, EGTA (as well as EDTA) applied either intra- or extracellularly caused the appearance of a very large Na inward current with kinetics similar to those of Ca inward current and with the reversal potential around 10 mV. Effective concentrations of chelating agents were 0.1 mM (extracellular) and 1.0 mM (intracellular).4. Increase in intracellular Ca in the absence of EGTA (by dialysis of the cell with Ca-saturated solutions) did not produce any significant effect on the delayed K outward current. The small change observed in this current could be evaluated as a depression of maximum slope conductance and a shift to more negative membrane potential.5. Ca inward current has been found extremely sensitive to internal Ca. 5.8 x 10(-8)M of internal free Ca produced its complete depression. This effect was reversible. Na inward current could be inhibited with 3.5 x 10(-7)M intracellular Ca.     

Effects of extracellular pH on neuronal calcium channel activation

Previous studies have shown that extracellular pH (pHo) alters gating and permeation properties of cardiac L- and T-type channels. However, a comprehensive study investigating the effects of pHo on all other voltage-gated calcium channels is lacking. Here, we report the effects of pHo on activation parameters slope factor (S), half-activation potential (Va), reversal potential (Erev), and maximum slope conductance (Gmax) of the nine known neuronal voltage-gated calcium channels transiently expressed in tsA-201 cells. In all cases, acidification of the extracellular bathing solution results in a depolarizing shift in the activation curve and reduction in peak current amplitudes. Relative to a physiological pHo of 7.25, statistically significant depolarizing shifts in Va were observed for all channels at pHo 7.00 except Cav1.3 and 3.2, which showed significant shifts at pHo 6.75 and below. All channels displayed significant reductions in Gmax relative to pHo 7.25 at pHo 7.00 except Cav1.2, 2.1, and 3.1 which required acidification to pHo 6.75. Upon acidification Cav3 channels displayed the largest changes in Vas and exhibited the largest reduction in Gmax compared with other channel subtypes. Taken together, these results suggest that significant modulation of calcium channel currents can occur with changes in pHo. Acidification of the external solution did not produce significant shifts in observed Erevs or blockade of outward currents for any of the nine channel subtypes. Finally, we tested a simple Woodhull-type model of current block by assuming blockade of the pore by a single proton. In all cases, the amount of blockade observed could not be explained in these simple terms, suggesting that proton modulation is more complicated, involving more than one site or gating modification as has been previously described for cardiac L- and T-type channels.     

Evidence for a transient potassium membrane current dependent on calcium influx in crab muscle fibre.

1. Voltage-clamp experiments were achieved on crab muscle fibre with the double sucrose-gap technique. 2. The accuracy of the imposed voltage has been controlled with an impaled micro-electrode connected to an external circuit. 3. Step depolarizations elicit two kinds of records. In type I fibres, the initial current exhibits only an inward calcium component. In type II fibres, the initial current exhibits a hump, transient outward current, mixed with the calcium current; these fibres exhibit always action potentials with fast repolarization. 4. A potassium origin is suggested for this outward current, due to its dependence on [K]o and its inhibition by TEA. 5. In fibres with a composite initial current, the voltage dependence of the availability of the measured inward current appears complex. It can be shown to be the sum of a simple calcium inactivation (which is observed alone in TEA solution) and a fast potassium inactivation. This potassium conductance is nearly half-available at the resting membrane potential. 6. The origin of the transient outward current is tentatively described. Consecutive to a transient internal increase of calcium ions (due to the calcium current) its activation curve is shifted in an hyperpolarizing direction resulting in an increased activation for an apparent identical depolarization. 7. This fast outward current which overlaps the calcium inward current can account for the low amplitude and the variability of the electrical activity of crab muscle fibres.     

External K+ ions increase rate of opening of outward current channels in snail neurons

Outward currents in isolated, internally-perfused snail neurons were slowed and reduced in amplitude by replacement of external K with Na, Cs, or tris. This behavior is not predicted by electrodiffusion theory, but can be described by a second-order kinetic model. The effect of potassium replacement is to reduce the rate constant for channel opening by aout 25% without affecting the rate constant for channel closing. The open channels may be stabilized by external potassium ions, thus increasing the mean channel open time.     

Voltage clamp studies of a transient outward membrane current in gastropod neural somata

1. Outward directed membrane currents have been studied in voltage clamp experiments on isolated neural somata of the marine gastropod Anisodoris.2. Stepping the membrane potential from a hyperpolarized level to a value in the neighbourhood of resting potential (-35 to -50 mV at 5 degrees C) results in an outward current transient, I(A), which is apparently carried by potassium ions.3. The peak amplitude of I(A) is dependent upon both the holding voltage level and the test step voltage while the time courses of development and decay are independent of, or only slightly dependent on, these parameters.4. The developing and decaying phases of I(A) are approximated by exponentials, leading to time constants for development of 10-25 msec and for decay of 220-600 msec over the aggregate of cells studied (data at 5 degrees C). Q(10) for the processes is approximately 3.5. It is concluded that the transport mechanism for I(A) is at least operationally distinct from the mechanism underlying delayed outward current, I(K).     

Glutamate-induced oxidative stress, but not cell death, is largely dependent upon extracellular calcium in mouse neuronal HT22 cells

Elucidating the relationship of glutamate-induced Ca2+ flux and oxidative death of neuronal cells may be of great relevance for neurodegenerative diseases in human beings. Mouse hippocampal HT22 cells provide a model system to study this relationship at the molecular level. Here we show that stimulation of HT22 cells with 5 mM glutamate is cytotoxic. Glutamate-induced cytotoxicity was associated with the generation of reactive oxygen species (ROS) and activation of the death executioner caspases 1 and 3. Treatment of HT22 cells with the calcium chelator, EGTA, and the calcium channel blocker, CoCl2, revealed that glutamate-induced cell death was dependent, in part, on glutamate-induced Ca2+ influx from extracellular stores. However, activation of caspases 1 and 3 and death of HT22 cells were also observed when Ca2+ was lacking in the extracellular milieu and ROS production abrogated. These findings led us to conclude that glutamate-induced death of mouse HT22 cells utilizes a complex mechanism that relies only in part on Ca2+ influx and ROS production. Additional studies are warranted to evaluate glutamate-induced death mechanisms that operate independently of Ca2+ influx and generation of ROS.     

Potential-dependent membrane current during the active transport of ions in snail neurones

1. The membrane current caused by the iontophoretic injection of sodium into giant neurones of the snail Helix pomatia was investigated under a long lasting voltage clamp. The inhibition of this current by ouabain (10(-4) M) and by cooling to + 7 degrees C confirmed its link with the active transport of ions. Therefore this current is called the pump current.2. Over the range of membrane potential -40 to -100 mV the changes in the steady current-voltage curves caused by the pump current development were investigated. The pump current was found to be potential-dependent. It decreased with increasing hyperpolarization of the neurone.3. With large hyperpolarizations the current-voltage curves obtained before the sodium injection and after eliciting the pump current coincided with each other. An increase in the membrane conductance was observed over the range of membrane potential corresponding to the pump current display.4. The applied sodium injections did not cause any marked changes in the passive permeability of the membrane. This fact made it possible to measure the charge transferred across the membrane during operation of the pump current. Unlike previous data, the ratio of this value to the charge used to inject sodium into the neurone appeared to be a variable.5. When the preparation was cooled to + 11 degrees C, and also during the first few minutes after the application of a potassium-free solution, both the pump current and the membrane potential at which it disappeared could increase.6. The pump current measurements during a number of transitions from one fixed level of the membrane potential to another showed that the current did not depend upon the potential at which it developed before each transition.7. The data presented allow the suggestion that the potential dependence of the pump current is determined by the changes in the rate of active transport of potassium, while the rate of active transport of sodium remains constant.     

Contraction of epithelial (MDCK) cells in response to low extracellular calcium is dependent on extracellular sodium

Like other cells of epithelial origin, MDCK cells respond with a reversible structural transformation to a diminution in the concentration of extracellular Ca²⁺. Upon deprivation of Ca²⁺ in the medium the cells undergo an active contraction mediated by the actin-myosin cytoskeleton, in parallel to detachment of the intercellular contacts and appearance of free spaces in the epithelium or monolayer (Castillo et al., 1998). We now present results indicating that the decrease of external Ca²⁺ plays an indirect and non-specific role in activating contraction, probably by allowing an influx of Na⁺. The omission of external Ca²⁺ had no effect when it was replaced by Mg²⁺, Ba²⁺ or Hg²⁺, and the addition of any of these divalent cations induced relaxation of cells previously contracted by exposure to low Ca²⁺. A null or weak response was observed also when Ca²⁺ was lowered in a solution where Na⁺ was replaced by choline or in the presence of amiloride (30 M), which reduces the permeability of the plasma membrane to Na⁺. Restitution of Na⁺ or removal of amiloride were followed by contraction in the same cultures. Li⁺ proved an able substitute of Na⁺ as requisite for cell contraction in response to Ca²⁺ depletion. Monensin (0.1 mM) –an ionophore selective for Na⁺– and to a lesser extent ouabain (0.1 mM) –an inhibitor of Na⁺ extrusion across the plasma membrane– , both stimulated contraction in the presence of the normal level of external Ca²⁺. Decreasing by half the normal concentration of external K⁺ facilitated cell contraction, but typical responses were observed when K⁺ was increased to 40 mM by partial substitution for Na⁺. These findings attest that cell contraction in response to low Ca²⁺ is likely due to an increase in the permeability of the plasma membrane to Na⁺, though not to membrane depolarization as such. Evidences from other motile systems suggest that Na⁺ influx might in turn cause an elevation of cytoplasmic Ca²⁺, which activates the actin-myosin cytoskeleton.     

A calcium dependent inward current in frog skeletal muscle fibres

Summary Slow inward currents are here reported in frog skeletal muscle. The currents are turned on by depolarising the membrane beyound about –45 mV, and are blocked by replacement of Ca²⁺ by Mg²⁺ and in the presence of Ca²⁺ by Co²⁺ at 20mM.     

Inhibition of transient outward current by intracellular ion substitution unmasks slow inward calcium current in cardiac Purkinje fibers

The ionophore nystatin was used to replace intracellular K⁺ with Cs⁺ in calf Purkinje fibers. Such replacement inhibited the transient outward current, and unmasked a large slow inward current (I_si). These results support the involvement of K⁺ as a charge carrier of the transient outward current, and suggest a method for better analysis of I_si.     

Nitric oxide increases excitability by depressing a calcium activated potassium current in snail neurons

In gastropods, the interneuronal messenger, nitric oxide (NO), modulates spike frequency and synaptic transmission. We have characterized the effect of NO on ion currents underlying neuronal excitability, using current-clamp and two-electrode voltage-clamp techniques. Identified neurons of the pulmonate snail, Helix pomatia, respond to the NO donor sodium nitroprusside (SNP) by increasing the firing frequency and decreasing the latency. Voltage-clamp experiments revealed that SNP or S-nitro-N-acetylpenicillamine (SNAP) depressed the macroscopic outward current, while the control compound N-acetylpenicillamine (NAP) had no effect. Current voltage curves generated from voltage steps to different membrane potentials ranging from -40 to +180 mV showed an N-shaped outward current. Superfusion of ganglia with Ca(2+) free Helix solution abolished the N-shape, indicating the contribution of a Ca(2+) activated K(+) current (I(K,Ca)). Exposure of neurons to SNP or SNAP diminished the N-shape, indicating that NO affects I(K,Ca). The depressing effect of SNP on the outward current was slow and reached steady state in about 5 min. In conclusion, our findings indicate that NO enhances excitability in Helix nervous system by decreasing I(K,Ca).     

Dihydropyridine inhibition of neuronal calcium current and substance P release

Dihydropyridine (DHP) calcium channel antagonists, which inhibit the slowly inactivating or L-type cardiac calcium (Ca) current, have been shown to be ineffective in blocking⁴⁵Ca influx and Ca-dependent secretion in a number of neuronal preparations. In the studies reported here, however, the antagonist DHP nifedipine inhibited both the L-type Ca current and potassium-evoked substance P (SP) release from embryonic chick dorsal root ganglion (DRG) neurons. These results suggest that, in DRG neurons. Ca entry through L-type channels is critical to the control of secretion. The inhibition of Ca current by nifedipine was both voltage and time-dependent, significant effects being observed only on currents evoked from relatively positive holding potentials maintained for several seconds. As expected from these results, nifedipine failed to inhibit L-type Ca current underlying the brief plateau phase of the action potential generated from the cell's normal resting potential; likewise, no significant effect of the drug was observed on action potential-stimulated SP release evoked by electrical field stimulation. The results of this work are discussed in terms of an assessment of the role of L-type Ca channels in neurosecretion.This work was supported by United States Public Health Service Grant NS16483 (KD) and by a USPHS Postdoctoral Fellowship (SGR)     

Further studies of the potential-dependence of the sodium-induced membrane current in snail neurones.

1. The potential-dependence of the membrane current induced by intracellular injections of sodium ions was studied on giant neurones of the snail Helix pomatia. This current decreases with membrane hyperpolarization at room temperature and can be reversed at sufficiently negative holding potentials. The same injections at 7 degrees C, as well as injections of lithium or potassium ions do not induce membrane currents and do not increase membrane conductance. 2. An increase in the amount of injected sodium changes the potential-dependence of the induced membrane currents. Small injections (about 1 muC) induce a current that does not depend upon the membrane potential. Further increase in the injection size not only increases the induced current but also enhances its potential-dependence and often reveals the existence of a reversal potential. The latter reaches -60 to -65 mV with large sodium injections. 3. An increase in extracellular potassium concentration from 4 to 8 mM shifts the reversal potential 17 mV in the depolarizing direction, and a decrease from 4 to 2 mM shifts it 14 mV in the hyperpolarizing direction. Replacement of potassium by rubidium or elimination of sodium ions from the outside solution, does not affect the induced current or its potential-dependence. 4. The coefficient of electrogenicity (the ratio between the amount of charge transferred by the sodium-induced membrane current and the amount brought into the cell during the injection) increases with an increase in the injection size if the membrane potential is clamped near the resting potential level. This relation is reversed when the holding potential is -80 mV. The reversal takes place at holding potentials near -60 mV. 5. 10 mM TEA does not affect the induced current and its potential-dependence. 6. It is suggested that the potential-dependence of the sodium-induced membrane current is a result of a specific increase in the membrane potassium conductance that is coupled with high activity of the sodium pump.     

Potassium current kinetics in bursting secretory neurons: effects of intracellular calcium.

1. The kinetics of delayed rectifier (IK) and transient potassium (IA) currents and their modification by intracellular calcium ions in bursting X-organ neurons of the crayfish were studied with whole-cell patch-clamp technique. Activation and inactivation kinetics were analyzed according to Hodgkin and Huxley-type equations. 2. IK activates with sigmoidal time course at membrane potentials more positive than -38.4 +/- 3.5 (SD) mV (n = 5), and does not inactivate. The conductance through delayed rectifier channels (gK) is described by the equation gK = GKn2. 3. IA activates at membrane potentials close to the resting potential (-52.2 +/- 4.3 mV, n = 5) and, after a peak, inactivates completely. The conductance through A-channels (gA) can be described by the product of independent activation and inactivation parameters: gA = GAa4b. Both activation and inactivation processes are voltage and time dependent. 4. Steady-state activation of IK and IA as well as inactivation of IA can be described by Boltzmann distributions for single particles with valencies of 2.55 +/- 0.01 (n = 5), 1.60 +/- 0.25 (n = 5), and 3.87 +/- 0.39 (n = 3), respectively. 5. Increasing [Ca2+]i, we observed the following: 1) a considerable inactivation of IK during test pulses, 2) an increase of maximal conductance for IA, 3) a reduction of the valency of IA inactivation gating particle (from 3.87 to 2.27), 4) a reduction of the inactivation time constants of IA, and 5) a shift of the inactivation steady-state curve to more positive membrane potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

A transient outward current in rat sympathetic neurones

Voltage-clamped rat sympathetic neurones exhibited a transient outward current similar to that previously described in invertebrate neurones and marine egg cells. It had an activation threshold of about -60 mV and displayed both voltage and time-dependent inactivation. In current clamp, this current manifested as a pronounced outward rectification at the onset of electrotonic potentials, increasing the latency to directly-evoked action potentials. The amplitude of the current was dependent on the external K+ concentration and was reduced by 4-aminopyridine.     

Voltage-clamp studies of the calcium inward current in an identified snail neurone: comparison with the sodium inward current.

1. Membrane currents were recorded under voltage clamp from cell A of the snail Helix aspersa. 2. In sodium-free saline the inward current was reduced to 75% of that in normal saline (containing both sodium and calcium). 3. The inward current in sodium-free saline was dependent on the external calcium concentration. 4. Calcium-free saline reduced the inward current to 30% of that in normal saline. (Na, Ca)-free saline abolished the inward current. 5. Changes in calcium concentration shifted the curve relating calcium conductance to membrane potential along the voltage axis. 6. Inactivation of inward current in both normal saline and sodium-free saline developed exponentially with time. 7. Steady-state inactivation of calcium inward current was similar to that for sodium current and it is concluded that the conductance mechanisms for these two ions bear a close resemblance.