O. A. Kryshtal

Separation of sodium and calcium channels in the surface membrane of molluscan nerve cells

By intracellular dialysis of isolated neurons of the mollusksHelix pomatia andLimnaea stagnalis and by a voltage clamp technique the characteristics of transmembrane ionic currents were studied during controlled changes in the ionic composition of the extracellular and intracellular medium. By replacing the intracellular potassium ions by Tris ions, functional blocking of the outward potassium currents was achieved and the inward current distinguished in a pure form. Replacement of Ringers solution in the extracellular medium with sodium-free or calcium-free solution enabled the inward current to be separated into two additive components, one carried by sodium ions, the other by calcium ions. Sodium and calcium inward currents were found to have different kinetics and different potential-dependence: τmNa=1±0.5 msec, τmCa=3±1 msec, τhNa=8±2 msec, τhCa=115±10 msec (Vm=0), GNa=0.5 (Vm=−21±2 mV), GCa=0.5 (Vm=−8±2 mV). Both currents remained unchanged by tetrodotoxin, but the calcium current was specifically blocked by cadmium ions (2·10−3 M), verapamil, and D=600, and also by fluorine ions if injected intracellularly. All these results are regarded as evidence that the soma membrane of the neurons tested possesses separate systems of sodium and calcium ion-conducting channels. Quantitative differences are observed in the relative importance of the systems of sodium and calcium channels in different species of mollusks.

Argiopine, argiopinines, and pseudoargiopinines as glutamate receptor blockers in hippocampal neurons

A homologous set of low-molecular weight compounds selectively blocking ionic currents were purified from venom from the spiderArgiope lobata with a selective blocking action on ionic currents activated by applying glutamate and its agonist kainic acid (KA) to the membrane of neurons isolated from the rat hippocampus. Three groups of these compounds — argiopine, argiopinines, and pseudoargiopinines, produced voltage-dependent glutamate- and KA-activated ionic currents at concentrations of 10−6-10−4 M, interacting primarily with agonist-activated ionic channels without affecting Kd values of the agonist. The blocking action could be partially reversed by argiopine application but only slightly when argiopinines and pseudoargiopinines were used. Kinetics of toxin effects on Ka-activated ionic currents showed at least two exponential components with different time constants. Simple and reversed rate constants of interaction between toxins and ionic channels were estimated from the plot of the kinetics of ionic current blockade and recovery against toxin concentration. Argiopine, argiopinines, and pseudoargiopinines lend themselves to further research into glutamate receptors of the mammalian CNS employing electrophysiological and biochemical techniques.

Action of calcium ions on channels of inward and outward currents in the molluscan neuron membrane

Changes in the characteristics of activity of sodium, calcium, and potassium channels in the surface membrane during variation of the calcium ion concentration in the extracellular and intracellular medium were investigated by the voltage clamp method during intracellular dialysis of isolated neurons of the mollusksLimnea stagnalis andHelix pomatia. Besides their direct role in passage of the current through the membrane, calcium ions were shown to have two actions, differing in their mechanism, on the functional properties of this membrane. The first was caused by the electrostatic action of calcium ions on the outer surface of the membrane and was manifested as a shift of the potential-dependent characteristics of the ion transport channels along the potential axis; the second is determined by closer interaction of calcium ions with the specific structures of the channels. During the action of calcium-chelating agents EGTA and EDTA on the inner side of the membrane the conductivity of the potassium channels is substantially reduced. With an increase in the intracellular free calcium concentration the conductivity is partially restored. The action of EGTA and EDTA on the outer side of the membrane causes a substantial decrease in the ion selectivity of the calcium channels and changes the kinetics of the portal mechanism. These changes are easily abolished by rinsing off the chelating agents or by returning calcium ions to the external medium. A specific blocking action of an increase in the intracellular free calcium concentration on conductivity of the calcium channels was found.

Antioxidant-caused changes in the permeability of proton-gated ion channels for sodium and calcium

Using a patch-clamp technique in the whole-cell configuration, we studied the effect of an exogenous antioxidant, dithiothreitol (DTT), on transmembrane currents in isolated cells obtained from the rat spinal ganglia. We demonstrated that this antioxidant (DTT) is capable of modulating the proton-gated current. In most neurons, proton-gated currents increased in the presence of the antioxidant. Since proton-gated receptor-channel complexes of sensory neurons are involved in different processes of signalling and transmission of sensory information in the peripheral nervous system, we hypothesize that the influences mediated by alterations of the concentrations of antioxidants participate in the formation of the state of algesia under normal physiological conditions and of that of hyperalgesia in pathological states. In addition, oxidative stress, which causes a shift in the balance of concentrations of antioxidants, accompanies numerous abnormal pathophysiological states, in particular diabetes, ischemia, and inflammation. Since proton-gated channels are permeable for calcium ions, an antioxidant-induced increase in calcium signalling can be significantly important for a number of biochemical processes occurring in tissues.

Blocking action of Nephila clavata spider toxin on ionic currents activated by glutamate and its agonists in isolated hippocampal neurons

The blocking action ofNephila clavata spider neurotoxin, or JSTX, on ionic currents activated by L-glutamate and its agonists when applied to the membrane of neurons isolated from the rat hippocampus was investigated using a concentration clamp technique. Crude JSTX venom was found to block L-glutamate-, quisqualate, and kainate-activated ionic currents induced by activating non-N-methyl-D-aspartate (non-NMDA) membrane receptors. Following the effects of JSTX, ionic currents activated by L-glutamate and its agonists declined to 34–36% of their initial value with no recovery during JSTX washout. An active fraction of JSTX at concentrations of 10−4–10−5 produced almost total but partially reversible blockade of ionic currents. The action of JSTX became less effective during depolarization. The concentration dependence of JSTX-induced blockade of kainate-activated ionic currents was investigated and the velocity constants of interaction between the toxin and glutamate receptors obtained. It is postulated that JSTX interacts with chemically-operated non-NMDA ionic channels, blocking their transition into a number of their possible open states.

Effects of Agonists of μ-Opioid Receptors on P-Type Calcium Channels in Rat Purkinje Neurons

Calcium channels of the P-type play an important role in synaptic transmission in the CNS of mammals; the major part of calcium ions entering the presynaptic terminal comes precisely via these channels. Using the whole-cell patch-clamp technique, we studied the effects of μ-opioids on P-type calcium channels in freshly isolated Purkinje neurons of the rat cerebellum. A selective agonist of μ-opioid receptors, DAMGO (10 nM), caused stable moderate (10 ± 1%, on average) but significant (Р < 0.001; n = 27) potentiation of P-type current in most units. This effect of DAMGO was rather appreciable already at a concentration of 1 nM and reached saturation at 100 nM. The effect developed rapidly (in about 10 sec); it was voltage-dependent and completely reversible. An endogenous selective agonist of μ-opioid receptors, endorphin-1, evoked nearly the same effect (increment 8 ± 1%, n = 6, P < 0.01). DAMGO-caused increase in the amplitude of P-type currents was completely removed after application of an antagonist of opioid receptors, naloxone (100 nM). These data indicate that agonists of μ-opioid receptors, even in nanomolar concentrations, can evoke appreciable potentiation of P-type calcium current mediated by interaction with opioid receptors of the corresponding type.

Mechanisms Underlying Positive Modulation of a Current through P-Type Calcium Channels in Purkinje Neurons by an Agonist of Opioid Receptors

In experiments on isolated rat Purkinje neurons using the whole-cell patch-clamp technique, the addition of 10 nM of an agonist of μ-opioid receptors (μ-ORs), DAMGO, to the bath solution led to moderate but highly significant intensification of the current through high-threshold calcium channels of the P type (increment 9-10%). We found that this effect was independent of the kind of cations (Ca2+ or Ba2+) coming via the plasma membrane. This effect (positive modulation of the current) was practically preserved in the case where a prepulse shifting the membrane potential to +50 mV preceded the test pulse, i.e., the effect was voltage-independent. The above-mentioned effect was almost unchanged under conditions where the intracellular solution contained 0.5 mM GTPβS (an irreversible blocker of G-proteins) or the same amount of GTPγS (a nonspecific activator of these proteins) instead of GTP. The addition of 0.5 mM cAMP to the intracellular solution also did not practically influence positive modulation of the P-current under the action of DAMGO. Preliminary 10-min-long incubation of the examined cells in a solution containing 0.5 μM calmidazolium (an antagonist of calmodulin-regulated enzymes) induced a twofold decrease in the DAMGO-evoked increment of the P-current. Based on the obtained data, we hypothesize that there is a high-affinity allosteric site of binding with agonists of μ-ORs in the molecule of the calcium P-channel, and that voltage-, calcium-, and G-protein-independent positive modulation of the current through these channels is realized by just such a mechanism.

Modulation by redox reagents of ATP-activated currents in neurons of the rat nodose ganglion

In in vitro experiments using a patch-clamp technique in the whole-cell configuration, we studied the effect of redox reagents on ATP-activated transmembrane currents in isolated cells of the rat nodose ganglion. It was demonstrated that endogenous and exogenous antioxidants, glutathione and dithiothreitol, respectively, are capable of modulating the ATP-activated current. In the presence of antioxidants, this current increased in most neurons, while upon the action of an oxidant this current was suppressed in some cells under study. Taking into account the fact that ATP receptors of sensory neurons are involved in nociception, we hypothesize that a certain level of antioxidants can determine the state of algesia under normal physiological conditions or of hyperalgesia in pathology. Since ATP receptor-operated channels possess a high conductance with respect to calcium ions, the enhancement of calcium signals upon the action of antioxidants can be an important factor for a number of biochemical processes in nerve tissues.

Responses Evoked in Afferent Fibers by Mechanostimulation of the Skin in vitro: Modulation by RFa-Like Peptides

In experiments in vitro on a skin patch-n. saphenous preparation, we studied the effects of RFa-like peptides on the responses of single slow-and fast-conducting afferent fibers evoked by mechanostimulation of the skin. RFa-like peptides were shown to exert modulatory influences on the mechanostimulation-evoked activity in a significant proportion of such units; a considerable proportion of the effects in the fibers of different types were of opposite direction. In most nociceptive thin C fibers, responses evoked by mechanostimulation of the skin were facilitated by the peptides, while those in A fibers were suppressed. It is supposed that RFa-like peptides are involved in the formation of the hyperalgesia state. The effect of these peptides on proton-activated ion channels (ASICs) is one of the possible mechanisms of an increase in the sensitivity of afferent fibers to mechanical stimuli. This modulatory effect was observed both in an acidified (pH 5.2) and in a normal (pH 7.4) media; this is why another mechanism exists that is not related to ASICs. A suppressory effect of RFa peptides on the mechanostimulation-evoked activity of primary afferents developed with a significant delay; probably, it is mediated by the effects of peptides on G proteins.

Potentiation of voltage-dependent calcium channel currents by NMDA receptor agonists

The effects of NMDA receptor agonists on voltage-dependent Ca2+ channels were studied in pyramidal neurons freshly dissociated from theCA3 region of the rat hippocampus. In a fraction of investigated cells (18 of 26), application of NMDA receptor agonists resulted in a rapid increase in the amplitude of whole-cell Ca2+ channel currents (Ca2+CC). This effect immediately disappeared on return to the control solution. The current-voltage relationship for the whole-cell Ca2+ channel currents was not shifted under this action of NMDA receptor agonists. It was shown that neither T-, nor L-type Ca2+CC were facilitated by NMDA receptor agonists. The experiments with specific blockers of various types (ω-CgTxGVIA, ω-Aga-IVA, and ω-CgTxMVIIC) showed that N-, P-, and Q-types of Ca2+ channels were not potentiated by NMDA receptor agonists. The involvement of other types of Ca2+ CC (R type, in particular) in the modulatory action of NMDA receptor agonists is considered.