Purinergic Modulation of Hippocampal Acetylcholine Release Involves α-Dendrotoxin-Sensitive Potassium Channels

Modulation of acetylcholine (ACh) release from superfused hippocampal slices was examined when the release of ACh was stimulated by exposure of slices to elevated K+ concentration. Evoked release was not sensitive to inhibition by 0.1 microM tetrodotoxin, but it could be inhibited in a dose-dependent manner by a muscarinic agonist (10-100 nM oxotremorine) and a purinergic agonist (10-100 nM 2-chloroadenosine). The alpha-dendrotoxin (100 nM), which selectively blocks voltage-gated inactivating K+ channels in nerve endings, did not affect the release of ACh under resting or depolarized conditions. However, alpha-dendrotoxin reduced the 2-chloroadenosine-induced inhibition of release, but did not alter the oxotremorine-induced inhibition. These results suggest that an alpha-dendrotoxin-sensitive K+ channel may be activated as an obligatory step in the modulation of ACh release by presynaptic purinergic receptor activation, but not in the modulation by presynaptic muscarinic receptors.     

Modeling study of the effects of overlapping Ca2+ microdomains on neurotransmitter release.

Although single-channel Ca2+ microdomains are capable of gating neurotransmitter release in some instances, it is likely that in many cases the microdomains from several open channels overlap to activate vesicle fusion. We describe a mathematical model in which transmitter release is gated by single or overlapping Ca2+ microdomains produced by the opening of nearby Ca2+ channels. This model accounts for the presence of a mobile Ca2+ buffer, provided either that the buffer is unsaturable or that it is saturated near an open channel with Ca2+ binding kinetics that are rapid relative to Ca2+ diffusion. We show that the release time course is unaffected by the location of the channels (at least for distances up to 50 nm), but paired-pulse facilitation is greater when the channels are farther from the release sites. We then develop formulas relating the fractional release following selective or random channel blockage to the cooperative relationship between release and the presynaptic Ca2+ current. These formulas are used with the transmitter release model to study the dependence of this form of cooperativity, which we call Ca2+ current cooperativity, on mobile buffers and on the local geometry of Ca2+ channels. We find that Ca2+ current cooperativity increases with the number of channels per release site, but is considerably less than the number of channels, the theoretical upper bound. In the presence of a saturating mobile buffer the Ca2+ current cooperativity is greater, and it increases more rapidly with the number of channels. Finally, Ca2+ current cooperativity is an increasing function of channel distance, particularly in the presence of saturating mobile buffer.     

Two components of high-threshold Ca2+ current inactivate by different mechanisms

High-threshold Ca2+ current triggers neurotransmitter release, but the existence, significance, and correct identification of different types of high-threshold Ca2+ channels remain controversial. We show selective inhibition of a rapidly inactivating component of high-threshold Ca2+ current in rat sensory neurons by bursts of brief pulses that mimic trains of action potentials and by prolonged depolarization just above the normal rest potential. In contrast, a slowly inactivating component decreases only when sufficient Ca2+ accumulates within the cell. Thus, there are physiologically important differences: whereas availability of the transient component depends on the value of the rest potential and the pattern of a prior stimulus, the sustained component seems to provide a baseline level of voltage-dependent Ca2+ entry that is lost only when intracellular Ca2+ rises.     

Both T- and L-type Ca2+ channels can contribute to excitation-contraction coupling in cardiac Purkinje cells.

Although L-type Ca2+ channels have been shown to play a central role in cardiac excitation-contraction (E-C) coupling, little is known about the role of T-type Ca2+ channels in this process. We used the amphotericin B perforated patch method to study the possible role of T-type Ca2+ current in E-C coupling in isolated canine Purkinje myocytes where both Ca2+ currents are large. T-type Ca2+ current was separated from L-type Ca2+ current using protocols employing the different voltage dependencies of the channel types and their different sensitivities to pharmacological blockade. We showed that Ca2+ admitted through either T- or L-type Ca2+ channels is capable of initiating contraction and that the contractions depended on Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR). The contractions, however, had different properties. Those initiated by Ca2+ entry through T-type Ca2+ channels had a longer delay to the onset of shortening, slower rates of shortening and relaxation, lower peak shortening, and longer time to peak shortening. These differences were present even when L-type Ca2+ current amplitude, or charge entry, was less than that of T-type Ca2+ current, suggesting that Ca2+ entry through the T-type Ca2+ channel is a less effective signal transduction mechanism to the SR than is Ca2+ entry through the L-type Ca2+ channel. We conclude that under our experimental conditions in cardiac Purkinje cells Ca2+ entry through the T-type Ca2+ channel can activate cell contraction. However, Ca2+ entry through the L-type Ca2+ channel is a more effective signal transduction mechanism. Our findings support the concept that different structural relationships exist between these channel types and the SR Ca2+ release mechanism.     

Receptor-mediated regulation of calcium channels and neurotransmitter release

Ca2+ influx into the nerve terminal is normally the trigger for the release of neurotransmitters. Many neurons possess presynaptic receptors whose activation results in changes in the quantity of neurotransmitter released by an action potential. This paper reviews studies that show that presynaptic receptors can regulate the activity of Ca2+ channels in the nerve terminal, resulting in changes in the influx of Ca2+ and in neurotransmitter release. Neurons possess several different types of voltage-sensitive Ca2+ channels. Ca2+ influx through N-type channels appears to trigger transmitter release in many instances. In other cases Ca2+ influx through L channels can influence transmitter release. Neurotransmitters can inhibit N channels through a G protein-mediated transduction mechanism. The G proteins are frequently pertussis toxin substrates. Inhibition of N channels appears to involve changes in their voltage dependence. Neurotransmitters can also regulate neuronal K+ channels. Activation of these K+ channels can lead to a reduction in Ca2+ influx and neurotransmitter release; these effects are also mediated by G proteins. Thus neurotransmitters may often regulate both presynaptic Ca2+ and K+ channels. These two effects may be synergistic mechanisms for the regulation of Ca2+ influx and neurotransmitter release.     

Calcium channel activity in rat brain synaptosomes: Effects of neuroleptics and other factors regulating phosphorylation and transmitter release

Neuroleptic drugs inhibit depolarization-induced Ca uptake in nerve endings, having IC₅₀ values in the micromolar range. Dopamine and a variety of other substances including opiates and PGE₁ are inactive. The effect is probably not mediated by the interaction of the neuroleptics with calmodulin, which itself is a potent inhibitor of stimulated Ca uptake. Dibutyryl cyclic AMP, but not fluoride, increases K⁺-stimulated Ca uptake. Phosphatidic acid, which is an intermediate in transmitter-stimulated phosphatidylinositol turnover, acts as a Ca ionophore in nerve endings and enhances K⁺-stimulated Ca uptake at a relatively low concentration. Carbamyl choline, a known stimulator of phosphatidylinositol turnover, did not, however, cause a significant increase in K⁺-stimulated Ca uptake. Treatment of the nerve ending fraction with relatively small amounts of phospholipase A₂ greatly inhibited depolarization-induced Ca uptake, demonstrating the importance of phospholipids for the functioning of the potential-dependent Ca channel in nerve endings. These studies suggest that the regulation of voltagesensitive Ca channels in nerve endings may be one mechanism controlling transmitter release.     

Calcium channel types involved in intrinsic amino acid neurotransmitters release evoked by depolarizing agents in cortical neurons

Although numerous biochemical and electrophysiological studies have already established many of the properties of the putative Ca2+ receptor for exocytosis at the synapse, the molecular mechanism that involves the influx of Ca2+ and the release of neurotransmitters has remained elusive. Several relationships have been established between neurotransmitter release and Ca2+ channel involved, but no work attempting to connect a particular neurotransmitter release, the effector which produces the release and the opening of a Ca2+ channel type has been performed. This work shows, data dealing with this subject. Based on our results, we have reached the following conclusions: (1) Ca2+ channel types P/Q, N and L mediate Ca2+ entry evoked by high KCl and veratridine, and P/Q and N but not L-type Ca2+ channels are involved when the effector is 4-aminopyridine (4-AP); (2) When we compare the relationship between the amino acid release and the Ca2+ channels which are opened by different depolarizing agents, we find that the release of a particular amino acid neurotransmitter not only depends on the opening of the voltage-dependent Ca2+ channel but also on the effector which produces the opening; and (3) the amount of amino acid release evoked by the different depolarizing agents is not correlated with the elevation of intracellular Ca2+ produced by them. From all of these results, we may conclude that calcium concentration in the active zone is not the only important factor in mediating amino acid release.     

Two types of store-operated channels in A431 cells

Activation of phospholipase C-coupled receptors leads to the release of Ca2+ from Ca2+ stores, and subsequent activation of store-operated cation (SOC) channels, promoting sustained Ca2+ influx. The most studied SOC channels are CRAC ("calcium-release activated calcium") channels exhibiting a very high selectivity for Ca2+. However, there are many SOC channels permeable for Ca2+ but having a lower selectivity. And while Ca2+ influx is important for many biological processes, little is known about the types of SOC channels and mechanisms of SOC channel activation. Previously, we described store-operated Imin channels in A431 cells. Here, by whole-cell recordings, we demonstrated that the store depletion activates two types of current in A431 cells--highly selective for divalent cations (presumably, ICRAC), and moderately selective (ISOC supported by Imin channels). These currents can be registered separately and have different developing time and amplitude. Coexisting of two different types of SOC channels in A431 cells seems to facilitate the control of intracellular Ca(2+)-dependent processes.     

KCa channel insensitivity to Ca2+ sparks underlies fractional uncoupling in newborn cerebral artery smooth muscle cells

In smooth muscle cells, localized intracellular Ca2+ transients, termed "Ca2+ sparks," activate several large-conductance Ca2+-activated K+ (KCa) channels, resulting in a transient KCa current. In some smooth muscle cell types, a significant proportion of Ca2+ sparks do not activate KCa channels. The goal of this study was to explore mechanisms that underlie fractional Ca2+ spark-KCa channel coupling. We investigated whether membrane depolarization or ryanodine-sensitive Ca2+ release (RyR) channel activation modulates coupling in newborn (1- to 3-day-old) porcine cerebral artery myocytes. At steady membrane potentials of -40, 0, and +40 mV, mean transient KCa current frequency was approximately 0.18, 0.43, and 0.26 Hz and KCa channel activity [number of KCa channels activated by Ca2+ sparksxopen probability of KCa channels at peak of Ca2+ sparks (NPo)] at the transient KCa current peak was approximately 4, 12, and 24, respectively. Depolarization between -40 and +40 mV increased KCa channel sensitivity to Ca2+ sparks and elevated the percentage of Ca2+ sparks that activated a transient KCa current from 59 to 86%. In a Ca2+-free bath solution or in diltiazem, a voltage-dependent Ca2+ channel blocker, steady membrane depolarization between -40 and +40 mV increased transient KCa current frequency up to approximately 1.6-fold. In contrast, caffeine (10 microM), an RyR channel activator, increased mean transient KCa current frequency but did not alter Ca2+ spark-KCa channel coupling. These data indicate that coupling is increased by mechanisms that elevate KCa channel sensitivity to Ca2+ sparks, but not by RyR channel activation. Overall, KCa channel insensitivity to Ca2+ sparks is a prominent factor underlying fractional Ca2+ spark uncoupling in newborn cerebral artery myocytes.     

ω-Conotoxin Inhibits the Acute Activation of Tyrosine Hydroxylase and the Stimulation of Norepinephrine Release by Potassium Depolarization of Sympathetic Nerve Endings

Increased Ca2+ influx serves as a signal that initiates multiple biochemical and physiological events in neurons following depolarization. The most widely studied of these phenomena is the release of neurotransmitters. In sympathetic neurons, depolarization also increases the rate of synthesis of the transmitter norepinephrine (NE), via an activation of the enzyme tyrosine hydroxylase (TH), and this effect also seems to involve Ca2+ entry. We have examined whether the mechanism of Ca2+ entry relevant to TH activation is via voltage-sensitive Ca2+ channels and, if so, whether the type of Ca2+ channel involved is the same as that involved in the stimulation of NE release. We have investigated the isolated rat iris, allowing us to examine transmitter biosynthesis and release in sympathetic nerve terminals in the absence of sympathetic cell bodies and dendrites. Potassium depolarization produced a three- to fivefold increase in TH activity and an approximately 100-fold increase in NE release. Both effects were dependent on Ca2+ being present in the extracellular medium, and both were inhibited by omega-conotoxin (1 microM), which inhibits N-type voltage-sensitive Ca2+ channels. In contrast, the dihydropyridine nimodipine (1-3 microM), which blocks L-type Ca2+ channels, had no effect on either measure. These data support the hypothesis that increases in NE biosynthesis and release in sympathetic nerve terminals during periods of depolarization are both initiated by an influx of Ca2+ through voltage-sensitive Ca2+ channels and that a similar type of Ca2+ channel is involved in both processes.     

Effects of Ca2+ channel antagonists on nerve stimulation-induced and ischemia-induced myocardial interstitial acetylcholine release in cats

Although an axoplasmic Ca(2+) increase is associated with an exocytotic acetylcholine (ACh) release from the parasympathetic postganglionic nerve endings, the role of voltage-dependent Ca(2+) channels in ACh release in the mammalian cardiac parasympathetic nerve is not clearly understood. Using a cardiac microdialysis technique, we examined the effects of Ca(2+) channel antagonists on vagal nerve stimulation- and ischemia-induced myocardial interstitial ACh releases in anesthetized cats. The vagal stimulation-induced ACh release [22.4 nM (SD 10.6), n = 7] was significantly attenuated by local administration of an N-type Ca(2+) channel antagonist omega-conotoxin GVIA [11.7 nM (SD 5.8), n = 7, P = 0.0054], or a P/Q-type Ca(2+) channel antagonist omega-conotoxin MVIIC [3.8 nM (SD 2.3), n = 6, P = 0.0002] but not by local administration of an L-type Ca(2+) channel antagonist verapamil [23.5 nM (SD 6.0), n = 5, P = 0.758]. The ischemia-induced myocardial interstitial ACh release [15.0 nM (SD 8.3), n = 8] was not attenuated by local administration of the L-, N-, or P/Q-type Ca(2+) channel antagonists, by inhibition of Na(+)/Ca(2+) exchange, or by blockade of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptor but was significantly suppressed by local administration of gadolinium [2.8 nM (SD 2.6), n = 6, P = 0.0283]. In conclusion, stimulation-induced ACh release from the cardiac postganglionic nerves depends on the N- and P/Q-type Ca(2+) channels (with a dominance of P/Q-type) but probably not on the L-type Ca(2+) channels in cats. In contrast, ischemia-induced ACh release depends on nonselective cation channels or cation-selective stretch activated channels but not on L-, N-, or P/Q type Ca(2+) channels, Na(+)/Ca(2+) exchange, or Ins(1,4,5)P(3) receptor-mediated pathway.     

N- and P/Q-type Ca2+ channels regulate synaptic efficacy between spinal dorsolateral funiculus terminals and motoneurons

Ca2+ influx through voltage-gated Ca2+ channels mediates synaptic transmission at numerous central synapses. However, electrophysiological and pharmacological evidence linking Ca+ channel activity with neurotransmitter release in the vertebrate mature spinal cord is scarce. In the current report, we investigated in a slice preparation from the adult turtle spinal cord, the effects of various Ca+ channel antagonists on neurotransmission at terminals from the dorsolateral funiculus synapsing motoneurons. Bath application of tetrodotoxin or NiCl2 prevented the monosynaptic excitatory postsynaptic potentials (EPSPs), and this effect was mimicked by exposure to a zero-Ca2+ solution. Application of polypeptide toxins that block N- and P/Q-type channels (omega-CTx-GVIA and omega-Aga-IVA) reduced the EPSP amplitude in a dose-dependent manner. By analyzing the input resistance and the EPSP time course, and using a paired pulse protocol we determined that both toxins act at presynaptic level to modulate neurotransmitter release. RT-PCR studies showed the expression of N- and P/Q-type channel mRNAs in the turtle spinal cord. Together, these results indicate that N- and P/Q-type Ca2+ channels may play a central role in the regulation of neurotransmitter release in the adult turtle spinal cord.     

Muscarinic modulation of cardiac activity

The goal of the present review is to report information concerning cardiac innervation or more precisely to approach the modulation of cardiac electrical and mechanical activity by parasympathetic innervation. Acetylcholine (ACh) release by nerve endings from the vagus nerve hyperpolarizes the membrane, shortens action potential (AP) duration and has a negative inotropic effect on cardiac muscle. Toxins are usefull tools in the study of membrane signals. The Caribbean ciguatoxin (C-CTX-1) has a muscarinic effect on frog atrial fibres. The toxin evokes the release of ACh from motoneuron nerve terminals innervating this tissue which allows us to propose a model, similar to the one of the neuromuscular junction (nmj), to describe the events occurring during the triggering and release of ACh. Trachynilysin (TLY) is a proteic toxin which causes an influx of Ca2+ into the cells and releases ACh from nmj synaptic vesicles. TLY has a muscarinic effect on atrial fibres which is explicated in the release of neurotransmitter from the nerve endings generated by the TLY-induced Ca2+ influx. It is known that ACh release from nmj is known to be due to exocytosis of synaptic vesicles via the activation of a proteic complex blocked by botulinum toxins. One of these proteins SNAP-25 is the target of type A botulinum toxin (BoNT/A). The study of hearts isolated from BoNT/A poisoned frogs show that atrial AP is lengthened and reveals the presence of SNAP-25 in nerve endings of this tissue. Moreover, the electrical activity of ventricular muscle is markedly altered; in BoNT/A treated frog, an important outward current activated by internal Ca2+ develops. ACh released from nerve terminals binds to a G protein coupled membrane receptor and activates a K+ channel and other effectors. Five subtypes of muscarinic receptors have been cloned from different tissue (M1, M2, M3, M4) subtypes have been identified in cardiac tissues throughout many species. These receptors coupled with different G-proteins activate different effectors. M1 receptors modulate the cardiac plateau and therefore the magnitude of the peak contraction. M2 receptors are mainly involved in the repolarization phase of the AP and modulate the duration of the peak contraction. The roles of M3 and M4 are not yet clearly defined; however, they may activate K+ currents. In conclusion, ACh releases from parasympathetic nerve endings which innervate cardiac cells follows to similar events (Ca2+ influx; presence of a SNAP-25 protein) to those which produce ACh release from nmj, stimulates different G proteins coupled muscarinic receptors, and activates different effectors involved in the modulation of cardiac electrical and mechanical activity.     

Calcium channels involved in neurotransmitter release at adult,neonatal and P/Q-type deficient neuromuscular junctions (Review)

Different types of voltage-dependent calcium channels (VDCCs) have been recognized based on their molecular structure as well as their pharmacological and biophysical properties. One of these, the P/Q type, is the main channel involved in nerve evoked neurotransmitter release at neuromuscular junctions (NMJs) and many central nervous system synapses. However, under particular experimental or biological conditions, other channels can be involved. L-type VDCC presence at the NMJ has been demonstrated by the contribution to the perineural calcium currents (I Ca ) at adult mice Bapta-loaded NMJs. This is probably a result of a reduction in Ca 2+ inactivation. The L-type current was not coupled to neurotransmitter release, but became coupled, as demonstrated by the release of acetylcholine, after the inhibition of serine/threonine protein phosphatases with okadaic acid (OA). Thus, under these conditions, L-type channels were unmasked at Bapta- but not at Egta-loaded NMJs. This suggests that the speed, not the capacity, of the calcium chelator was decisive in preventing Ca 2+ -inactivation and facilitating the contribution to neurotransmitter release. At neonatal rat NMJs, N-type VDCCs were involved early during development whereas P/Q-type VDCCs play a main role at all stages of development. Furthermore, P/Q-type VDCCs were more efficiently coupled to neurotransmitter release than N-type VDCCs. This difference could be accounted for by a differential location of these channels at the release site. Neuromuscular transmission in P/Q-type calcium channel knock out ataxic mice jointly depends on both N-type and R-type channels and shows several altered properties including low quantal content. Thus, calcium channels may be recruited to mediate neurotransmitter release with a functional hierarchy where the P/Q channel seems to be the channel most suited to mediate exocytosis at NMJs.     

Elementary properties and pharmacological sensitivities of calcium channels in mammalian peripheral neurons

The major component of whole-cell Ca2+ current in differentiated, neuron-like rat pheochromocytoma (PC12) cells and sympathetic neurons is carried by dihydropyridine-insensitive, high-threshold-activated N-type Ca2+ channels. We show that these channels have unitary properties distinct from those of previously described Ca2+ channels and contribute both slowly inactivating and large sustained components of whole-cell current. The N-type Ca2+ currents are modulated by GTP binding proteins. The snail toxin omega-conotoxin reveals two pharmacological components of N-type currents, one blocked irreversibly and one inhibited reversibly. Contrary to previous reports, neuronal L-type channels are insensitive to omega-conotoxin. N-type Ca2+ channels appear to be specific for neuronal cells, since their functional expression is greatly enhanced by nerve growth factor.     

Ca2+ Channels as Targets of Neurological Disease: Lambert–Eaton Syndrome and Other Ca2+ Channelopathies

In the nervous system, voltage-gated Ca2+ channels regulate numerous processes critical to neuronal function including secretion of neurotransmitters, initiation of action potentials in dendritic regions of some neurons, growth cone elongation, and gene expression. Because of the critical role which Ca2+ channels play in signaling processes within the nervous system, disruption of their function will lead to profound disturbances in neuronal function. Voltage-gated Ca2+ channels are the targets of several relatively rare neurological or neuromuscular diseases resulting from spontaneously-occurring mutations in genes encoding for parts of the channel proteins, or from autoimmune attack on the channel protein responses. Mutations in CACNAIA, which encodes for the alpha1A subunit of P/Q-type Ca2+ channels, lead to symptoms seen in familial hemiplegic migraine, episodic ataxia type 2, and spinocerebellar ataxia type 6. Conversely, autoimmune attack on Ca2+ channels at motor axon terminals causes peripheral cholinergic nerve dysfunction observed in Lambert-Eaton Myasthenic Syndrome (LEMS), the best studied of the disorders targeting voltage-gated Ca2+ channels. LEMS is characterized by decreased evoked quantal release of acetylcholine (ACh) and disruption of the presynaptic active zones, the sites at which ACh is thought to be released. LEMS is generally believed to be due to circulating antibodies directed specifically at the Ca2+ channels located at or near the active zone of motor nerve terminals (P/Q-type) and hence involved in the release of ACh. However, other presynaptic proteins have also been postulated to be targets of the autoantibodies. LEMS has a high degree of coincidence (approximately 60%) with small cell lung cancer; the remaining 40% of patients with LEMS have no detectable tumor. Diagnosis of LEMS relies on characteristic patterns of electromyographic changes; these changes are observable at neuromuscular junctions of muscle biopsies from patients with LEMS. In the majority of LEMS patients, those having detectable tumor, the disease is thought to occur as a result of immune response directed initially against voltage-gated Ca2+ channels found on the lung tumor cells. In these patients, effective treatment of the underlying tumor generally causes marked improvement of the symptoms of LEMS as well. Animal models of LEMS can be generated by chronic administration of plasma, serum or immunoglobulin G to mice. These models have helped dramatically in our understanding of the pathogenesis of LEMS. This "passive transfer" model mimics the electrophysiological and ultrastructural findings seen in muscle biopsies of patients with LEMS. In this model, we have shown that the reduction in amplitude of Ca2+ currents through P/Q-type channels is followed by "unmasking" of an L-type Ca2+ current not normally found at the motor nerve terminal which participates in release of ACh from terminals of mice treated with plasma from patients with LEMS. It is unclear what mechanisms underlie the development of this novel L-type Ca2+ current involved in release of ACh at motor nerve terminals during passive transfer of LEMS.     

Single calcium channels on a cholinergic presynaptic nerve terminal.

The calyx-type synapse of the chick ciliary ganglion was used to examine single calcium channels in a vertebrate cholinergic presynaptic nerve terminal by means of the cell-attached, patch-clamp technique. Calcium channels were recorded on the internal, transmitter-release face of the nerve terminal, but were not detected on the external face. These channels were recruited at -30 mV, with maximum activation at about +30 mV, and were sometimes clustered at high densities. Single-channel conductance estimates with voltage-pulse or -ramp techniques gave values of 11-14 pS with 110 mM barium, which is in the intermediate, N-type range for calcium channels on a control neuron. This nerve terminal calcium channel, termed the NPT-type, may link action potentials to transmitter release at many vertebrate fast-transmitting synapses.     

Omega-agatoxin-TK is a useful tool to study P-type Ca2+ channel-mediated changes in internal Ca2+ and glutamate release in depolarised brain nerve terminals

The present study shows that omega-agatoxin-TK, a toxin of the venom of Agelenopsis aperta, which is 10 times more concentrated than the P/Q type Ca(2+) channel blocker, omega-agatoxin-IVA in the venom, inhibits the high K(+) depolarisation-induced rise in internal Ca(2+) (Ca(i), as determined with fura-2) dose dependently in cerebral (striatal and hippocampal) isolated nerve endings, with calculated IC(50)'s of about 60nM. The maximal inhibition exerted by omega-agatoxin-TK in striatal synaptosomes (61 +/- 11%) is 10% larger than in hippocampal synaptosomes, suggesting a larger population of omega-agatoxin-TK-sensitive Ca(2+) channels in striatal than in hippocampal nerve endings. The N-type Ca(2+) channel blocker, omega-conotoxin-GVIA (1muM), inhibits part of the omega-agatoxin-TK-insensitive rise in Ca(i) induced by high K(+). In contrast to the inhibition exerted by omega-agatoxin-TK on the Ca(i) response to high K(+), omega-agatoxin-TK failed to inhibit the tetrodotoxin-sensitive elevations in Ca(i) and in internal Na(+) (Na(i), as determined with SBFI) induced by veratridine, indicating that the Ca(2+) influx activated by veratridine does not involve omega-agatoxin-TK-sensitive channels. High K(+) does not increase Na(i). In [(3)H]Glu preloaded hippocampal synaptosomes super-fused with low Na(+) Krebs Ringer HEPES (a condition that guarantees the elimination of neurotransmitter transporters-mediated release), the release of [(3)H]Glu induced by high K(+) is absolutely dependent on the entrance of external Ca(2+). This exocytotic release of [(3)H]Glu attained in the absence of a chemical Na(+) gradient is inhibited with the same potency and efficacy by omega-agatoxin-TK and by omega-agatoxin-IVA, which is known to differ from omega-agatoxin-TK in its amino terminal moiety. These results indicate that omega-agatoxin-TK represents a good pharmacological tool to study P/Q type Ca(2+) channel-mediated responses in cerebral nerve endings.     

Reconstitution and characterization of a nicotinic acid adenine dinucleotide phosphate (NAADP)-sensitive Ca2+ release channel from liver lysosomes of rats

Nicotinic acid adenine dinucleotide phosphate (NAADP) is capable of inducing global Ca2+ increases via a lysosome-associated mechanism, but the mechanism mediating NAADP-induced intracellular Ca2+ release remains unclear. The present study reconstituted and characterized a lysosomal NAADP-sensitive Ca2+ release channel using purified lysosomes from rat liver. Furthermore, the identity of lysosomal NAADP-sensitive Ca2+ release channels was also investigated. It was found that NAADP activates lysosomal Ca2+ release channels at concentrations of 1 nM to 1 microM, but this activating effect of NAADP was significantly reduced when the concentrations used increased to 10 or 100 microM. Either activators or blockers of Ca2+ release channels on the sarcoplasmic reticulum (SR) had no effect on the activity of these NAADP-activated Ca2+ release channels. Interestingly, the activity of this lysosomal NAADP-sensitive Ca2+ release channel increased when the pH in cis solution decreased, but it could not be inhibited by a lysosomal H+-ATPase antagonist, bafilomycin A1. However, the activity of this channel was significantly inhibited by plasma membrane L-type Ca2+ channel blockers such as verapamil, diltiazem, and nifedipine, or the nonselective Ca2+,Na+ channel blocker, amiloride. In addition, blockade of TRP-ML1 (transient receptor potential-mucolipin 1) protein by anti-TRP-ML1 antibody markedly attenuated NAADP-induced activation of these lysosomal Ca2+ channels. These results for the first time provide direct evidence that a NAADP-sensitive Ca2+ release channel is present in the lysosome of native liver cells and that this channel is associated with TRP-ML1, which is different from ER/SR Ca2+ release channels.     

Calcium-activated endonexin II forms calcium channels across acidic phospholipid bilayer membranes

Human endonexin II (annexin V) and recombinant human endonexin II can be activated by Ca2+ to interact with acidic phospholipid bilayers formed at the tip of a patch pipette. Once associated with the bilayer, endonexin II forms voltage-gated channels which are selective for divalent cations according to the following series Ca2+ greater than Ba2+ greater than Sr2+ much greater than Mg2+. However, endonexin II also expresses a selective affinity for Ca2+ which is manifest by an observed reduced current through the open channel when Ca2+ is the charge carrier. La3+ blocks endonexin II channels, as it does synexin (annexin VII) and other types of Ca2+ channels. However, as with synexin, the dihydropyridine Ca2+ channel antagonist nifedipine does not affect endonexin II channel activity. Endonexin II channels are also permeant to Li+, Cs+, Na+, and to a lesser extent, K+, resembling in this manner Ca2+ release channels from sarcoplasmic reticulum. Indeed, the low affinity of endonexin II channels for such ions as Cs+ or Li+ have allowed us to use these cations for measurement of the kinetic properties of the channel, with minimal concerns for the ion/channel interactions observed with the physiological substrate, Ca+. Finally, we observed that endonexin II channel activity always occurred in bursts, making necessary the use of two exponential functions to fit open- and closed-time histograms. We conclude from these data that the domain responsible for endonexin II channel activity, first observed by ourselves in the homologue synexin, is probably the C-terminal tetrad repeat common to both molecules.