Role of intracellular calcium channels of nerve terminals in the regulation of mediator secretion

The review is presented, analysing the modern state of knowledge about the role of intracellularly stored calcium of nerve terminals in regulation of quantal mediator secretion in synapses. The data are considered, concerning the properties of two Ca(2+)-channels superfamilies, i.e. the ryanodine receptors (RyR) and IP3-receptors, which are incorporated into the membrane of endoplasmic reticulum fragments. The localization of cisternae, containing RyR and IP3-receptors in neurons and nerve terminals are described. The data, demonstrating the pattern of calcium signalization in neurons and terminals after their interaction with specific blockers or activators of RyRs or IP3-receptors are presented. The facts, demonstrating that calcium induced calcium release via RyRs or IP3-receptors takes part in controlling spontaneous secretion of different types of vesicles in synaptic terminals and supports the slow and fast types of regulated exocytosis of synaptic vesicles, in the course of single or repetitive activity of central or peripheral synapses are analysed.     

Hypoosmotic shock activates Ca channels in isolated nerve terminals

Influence of hypotonic swelling on Ca²⁺ (⁴⁵Ca²⁺) uptake in rat brain synaptosomes was studied. A decrease in medium osmolality from 310 to 260-180 mOsm led to a progressive stimulation of ⁴⁵Ca²⁺ accumulation. The effect was blocked by verapamil (IC₅₀ = 5 μM), CoCl₅₀ = 58 μM) and retained at a fixed concentration of external sodium indicating the involvement of Ca²⁺ channels rather than Na⁺/Ca²⁺ exchange in swelling-induced Ca²⁺ influx. The populations of calcium channels observed in hypoosmotic and depolarizing conditions are different in three aspects: (i) kinetics of ⁴⁵Ca²⁺ entry; (ii) insensitivity to dihydropyridines and ω-conotoxin GVIA; (iii) insensitivity to preliminary depolarization by high potassium. The effects of swelling and depolarization on Ca²⁺ uptake were additive. No change in membrane potential monitored with diS-C₃-(5) was recorded during synaptosome hypotonic swelling. The results suggest the existence in synaptosomal plasma membrane of volume-dependent calcium-permeable channels with properties distinct from those of the voltage-dependent calcium channels. Activation of these channels may constitute an early event in volume regulation of nerve terminals in anisoosmotic conditions.     

Sodium channels in presynaptic nerve terminals. Regulation by neurotoxins

Regulation of Na+ channels by neurotoxins has been studied in pinched- off nerve endings (synaptosomes) from rat brain. Activation of Na+ channels by the steroid batrachotoxin and by the alkaloid veratridine resulted in an increase in the rate of influx of 22Na into the synaptosomes. In the presence of 145 mM Na+, these agents also depolarized the synaptosomes, as indicated by increased fluorescence in the presence of a voltage-sensitive oxacarbocyanine dye [diO-C5(3)]. Polypeptide neurotoxins from the scorpion Leiurus quinquestriatus and from the sea anemone Anthopleura xanthogrammica potentiated the stimulatory effects of batrachotoxin and veratridine on the influx of 22Na into synaptosomes. Saxitoxin and tetrodotoxin blocked the stimulatory effects of batrachotoxin and veratridine, both in the presence and absence of the polypeptide toxins, but did not affect control 22Na influx or resting membrane potential. A three-state model for Na+ channel operation can account for the effects of these neurotoxins on Na+ channels as determined both by Na+ flux measurements in vitro and by electrophysiological experiments in intact nerve and muscle.     

Two types of calcium channels coexist in peptide-releasing vertebrate nerve terminals

The properties of the Ca2+ channels mediating transmitter release in vertebrate neurons have not yet been described with voltage-clamp techniques. Several types of voltage-dependent Ca2+ channels are known to exist on neuronal somata, but the small size and inaccessibility of most vertebrate nerve endings have precluded direct characterization of the presynaptic channels. However, large nerve endings, which release the peptides oxytocin and vasopressin in a Ca2(+)-dependent manner, can be dissociated from the rat neurohypophysis. Using both single-channel and whole-cell patch-clamp techniques, we have characterized two types of Ca2+ channels that coexist in these terminals. One is a large-conductance, high-threshold, dihydropyridine-sensitive channel that contributes a slowly inactivating current. The second is a smaller conductance channel, which is also activated at high thresholds, but underlies a rapidly inactivating, dihydropyridine-insensitive current. Both types of Ca2+ channels may participate in the peptide release process.     

Aminoglycoside antibiotics block voltage-dependent calcium channels in intact vertebrate nerve terminals

Intrinsic and extrinsic optical signals recorded from the intact nerve terminals of vertebrate neurohypophyses were used to investigate the anatomical site and physiological mechanism of the antagonistic effects of aminoglycoside antibiotics on neurotransmission. Aminoglycoside antibiotics blocked the intrinsic light scattering signal closely associated with neurosecretion in the mouse neurohypophysis in a concentration-dependent manner with an IC50 of approximately 60 microM and the block was relieved by increasing [Ca2+]o. The rank order potency of different aminoglycoside antibiotics for blocking neurosecretion in this preparation was determined to be: neomycin greater than gentamicin = kanamycin greater than streptomycin. Optical recordings of rapid changes in membrane potential using voltage-sensitive dyes revealed that aminoglycoside antibiotics decreased the Ca(2+)-dependent after-hyperpolarization of the normal action potential and both the magnitude and after-hyperpolarization of the regenerative Ca2+ spike. The after-hyperpolarization results from a Ca-activated potassium conductance whose block by aminoglycoside antibiotics was also reversed by increased [Ca2+]o. These studies demonstrate that the capacity of aminoglycoside antibiotics to antagonize neurotransmission can be attributed to the block of Ca channels in the nerve terminal.     

Beta-adrenergic regulation of amiloride-sensitive lung sodium channels

We investigated the mechanism by which cAMP increases sodium transport in lung epithelial cells. Alveolar type II (ATII) cells have two types of amiloride-sensitive, cation channels: a nonselective cation channel (NSC) and a highly selective channel (HSC). Exposure of ATII cells to cAMP, beta-adrenergic agonists, or other agents that increase adenylyl cyclase activity increased activity of both channel types, albeit by different mechanisms. NSC open probability (P(o)) increased severalfold when exposed to terbutaline, isoproterenol, forskolin, or cAMP analogs without any change in NSC number. In contrast, terbutaline increased HSC number with no significant change in HSC P(o). For both channels, the effect of terbutaline was blocked by propranolol and H-89, suggesting a protein kinase A (PKA) requirement for beta-adrenergic-induced changes in channel activity. Terbutaline increased cAMP levels in ATII cells, but intracellular calcium also increased. Calcium sequestration with BAPTA blocked beta-adrenergic-induced increases in NSC P(o) but did not alter HSC activity. These observations suggest that beta-adrenergic stimulation increases intracellular cAMP and activates PKA. PKA increases HSC number and increases intracellular calcium. The increase in calcium increases NSC P(o). Thus increased cAMP levels are likely to increase lung sodium transport regardless of which channel type is present.     

Modulation of nerve membrane sodium channels by chemicals

1. Modulations of sodium channel kinetics by grayanotoxins and pyrethroids have been studied using voltage clamped, internally perfused giant axons from crayfish and squid. 2. Grayanotoxin I and alpha-dihydrograyanotoxin II greatly depolarize the nerve membrane through an increase in resting sodium channel permeability to sodium ions. 3. Grayanotoxins modify a fraction of sodium channel population to give rise to a slow conductance increase with little or no inactivation, and the slow conductance-membrane potential curve is shifted toward hyperpolarization. This accounts for the depolarization. 4. The tail current associated with step repolarization during the slow current in grayanotoxins decays with a dual exponential time course. 5. (+)-trans tetramethrin and (+)-trans allethrin also modify a fraction of sodium channel population in generating a slow current, which attains a maximum slowly and decays very slowly during a maintained depolarizing step. The membrane is depolarized only slightly. 6. The tail current associated with step repolarization during the slow current in the pyrethroids is very large in initial amplitude and decays very slowly. 7. The rate at which the sodium channel arrives at the modified open state in the presence of pyrethroids is expressed by a dual exponential function, and the slow phase disappears following removal of the sodium inactivation mechanism by internal perfusion of pronase. 8. A kinetic model is proposed to account for the actions of both grayanotoxins and pyrethroids on sodium channels. Both chemicals interact with the channel at both open and closed states to yield a modified open state which results in a slow sodium current.     

Dopamine regulation of amiloride-sensitive sodium channels in lung cells

Dopamine increases lung fluid clearance. This is partly due to activation of basolateral Na-K-ATPase. However, activation of Na-K-ATPase by itself is unlikely to produce large changes in transepithelial transport. Therefore, we examined apical and basolateral dopamine's effect on apical, highly selective sodium channels [epithelial sodium channels (ENaC)] in monolayers of an alveolar type 2 cell line (L2). Dopamine increased channel open probability (P(o)) without changing the unitary current. The D(1) receptor blocker SCH-23390 blocked the dopamine effect, but the D(2) receptor blocker sulpiride did not. The dopamine-mediated increase in ENaC activity was not a secondary effect of dopamine stimulation of Na-K-ATPase, since ouabain applied to the basolateral surface to block the activity of Na-K-ATPase did not alter dopamine-mediated ENaC activity. Protein kinase A (PKA) was not responsible for dopamine's effect since a PKA inhibitor, H89, did not reduce dopamine's effect. However, cpt-2-O-Me-cAMP, which selectively binds and activates EPAC (exchange protein activated by cAMP) but not PKA, increased ENaC P(o). An Src inhibitor, PP2, and the phosphatidylinositol-3-kinase inhibitor, LY-294002, blocked dopamine's effect on ENaC. In addition, an MEK blocker, U0126, an inhibitor of phospholipase A(2), and a protein phosphatase inhibitor also blocked the effect of dopamine on ENaC P(o). Finally, since the cAMP-EPAC-Rap1 pathway also activates DARPP32 (32-kDa dopamine response protein phosphatase), we confirmed that dopamine phosphorylates DARPP32, and okadaic acid, which blocks phosphatases (DARPP32), also blocks dopamine's effect. In summary, dopamine increases ENaC activity by a cAMP-mediated alternative signaling pathway involving EPAC and Rap1, signaling molecules usually associated with growth-factor-activated receptors.     

Pharmacological modifications of the sodium channels of frog nerve

Voltage clamp measurements on myelinated nerve fibers show that tetrodotoxin, saxitoxin, and DDT specifically affect the sodium channels of the membrane. Tetrodotoxin and saxitoxin render the sodium channels impermeable to Na ions and to Li ions and probably prevent the opening of individual sodium channels when one toxin molecule binds to a channel. The apparent dissociation constant of the inhibitory complex is about 1 nM for the cationic forms of both toxins. The zwitter ionic forms are much less potent. On the other hand, DDT causes a fraction of the sodium channels that open during a depolarization to remain open for a longer time than is normal. The effect cannot be described as a specific change in sodium inactivation or as a specific change in sodium activation, for both processes continue to govern the opening of the sodium channels and neither process is able to close the channels. The effects of DDT are very similar to those of veratrine.     

The regulation of cytosolic pH in isolated presynaptic nerve terminals from rat brain

Cytosolic pH (pHi) was measured in presynaptic nerve terminals isolated from rat brain (synaptosomes) using a fluorescent pH indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The synaptosomes were loaded with BCECF by incubation with the membrane-permanent acetoxy-methyl ester derivative of BCECF, which is hydrolyzed by intracellular esterases to the parent compound. pHi was estimated by calibrating the fluorescence signal after permeabilizing the synaptosomal membrane by two different methods. Synaptosomes loaded with 15-90 microM BCECF were estimated to have a pHi of 6.94 +/- 0.02 (mean +/- standard error; n = 54) if the fluorescence signal was calibrated after permeabilizing with digitonin; a similar value was obtained using synaptosomes loaded with 10 times less BCECF (6.9 +/- 0.1; n = 5). When the fluorescence signal was calibrated by permeabilizing the synaptosomal membrane to H+ with gramicidin and nigericin, pHi was estimated to be 7.19 +/- 0.03 (n = 12). With the latter method, pHi = 6.95 +/- 0.09 (n = 14) when the synaptosomes were loaded with 10 times less BCECF. Thus, pHi in synaptosomes was approximately 7.0 and could be more precisely monitored using the digitonin calibration method at higher BCECF concentrations. When synaptosomes were incubated in medium containing 20 mM NH4Cl and then diluted into NH4Cl-free medium, pHi immediately acidified to a level of approximately 6.6. After the acidification, pHi recovered over a period of a few minutes. The buffering capacity of the synaptosomes was estimated to be approximately 50 mM/pH unit. Recovery was substantially slowed by incubation in an Na-free medium, by the addition of amiloride (KI = 3 microM), and by abolition of the Nao/Nai gradient. pHi and its recovery after acidification were not affected by incubation in an HCO3-containing medium; disulfonic stilbene anion transport inhibitors (SITS and DIDS, 1 mM) and replacement of Cl with methylsulfonate did not affect the rate of recovery of pHi. It appears that an Na+/H+ antiporter is the primary regulator of pHi in mammalian brain nerve terminals.     

Quinidine blocking of sodium and potassium channels in myelinated nerve fibers

Experiments by the voltage clamp method showed that external application of quinidine (5 × 10^–5 M) to the Ranvier node membrane of the frog nerve fiber inhibitis both sodium and potassium currents. Blocking of the sodium current is considerably intensified by repetitive depolarization of the membrane (1–10 Hz); the rate of development of the block increases with an increase in stimulation frequency. After the end of stimulation the sodium current gradually returns to its initial level (with a time constant of the order of 30 sec at 12°C). Unlike repetitive depolarization with short (5 msec) stimuli, a prolonged shift (1 sec) of potential toward depolarization has no significant effect on quinidine blocking of the sodium current. Analysis of the current-voltage characteristic curves showed that quinidine blocks outward sodium current more strongly than inward. Batrachotoxin protects sodium channels against the blocking action of quinidine in a concentration of 10^–5 M. Inhibition of the outward potassium currents by quinidine is distinctly time-dependent in character: Initially the potassium current rises to a maximum, then falls steadily to a new stationary level. The results agree with the view that quinidine, applied externally, penetrates through the membrane in the basic form and blocks open sodium and potassium channels from within in the charged (protonated) form. The similarity in principle between the action of quinidine and local anesthetics on the sodium suggests that these compounds bind with the same receptor, located in the inner mouth of the sodium channel.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 14, No. 3, pp. 324–330, May–June, 1982.     

Modification of nerve membrane sodium channels by the insecticide pyrethroids

1. The synthetic pyrethroids exert potent and selective actions on nerve membrane sodium channels. (+)-trans tetramethrin and (+)-trans allethrin cause repetitive discharges to be produced in the isolated crayfish and squid giant axons in response to a single stimulus as a result of an increase in depolarizing after-potential. 2. The latter effect is due to slowing of the sodium channel kinetics which causes a prolonged sodium current following the normal peak sodium current. 3. A kinetic model is proposed to account for the action of the pyrethroids in which the pyrethroid molecule binds to the sodium channels at both closed and open states to produce a modified open state. 4. (-)-trans and (-)-cis isomers of tetramethrin are ineffective in causing the effects, but prevent the active (+)-trans and (+)-cis isomers from exerting the effects. This stereospecificity provides us with an excellent opportunity for the study of binding sites of pyrethroids and other sodium channel modulators.     

Current-dependent block of nerve membrane sodium channels by paragracine.

Paragracine, isolated from the coelenterate species Parazoanthus gracilis, selectively blocks sodium channels of squid axon membranes in a frequency-dependent manner. The blocking action depends on the direction and magnitude of the sodium current rather than on the absolute value of the membrane potential. Paragracine blocks the channels only from the axoplasmic side and does so only when the current is in the outward direction. This block may be reversed by generating inward sodium currents. In axons in which sodium inactivation has been removed by pronase, the frequency-dependent block persists, and a slow time-dependent block is observed. A slow interaction with its binding site in the channel may account for the frequency-dependent block.     

Hydrogen currents through aconitine-modified sodium channels in the nerve fiber membrane

Ionic currents through aconitine-modified sodium channels of the Ranvier node membrane were measured by a voltage clamp method in an external medium free from sodium ions. A shift of pH of the solution below 4.6 led to the appearance of inward ionic currents, whose kinetics and activation region were characteristic of aconitine-modified sodium channels at low pH. These currents were blocked by the local anesthetic benzocaine in a concentration of 2 mM. Experiments with variation of the concentration of Ca⁺⁺, Tris⁺, TEA⁺, and choline⁺ in acid sodium-free solutions showed that these cations make no appreciable contribution to the inward current. It is concluded that the inward currents observed under these conditions are carried by H⁺ (or H₃O⁺) through aconitine-modified sodium channels. From the shifts of reversal potentials of the ionic currents the relative permeability (P_H/P_Na) for H⁺ was determined: 1059 ± 88. The results agree with the view that the aconitine-modified sodium channel is a relatively wide water pore, and that movement of H⁺ through it is limited by its binding with an acid group.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 508–516, September–October, 1982.