Modulating Action of Hyperforin on the P-Type Calcium Channels in the Membranes of Rat Cerebellar Purkinje Neurons

A modulating action of hyperforin (an active compound of the extract from Hypericum perforatum) on a high-threshold component of the calcium current, sensitive to application of 100 nM -Aga-IVA toxin and identified as P current, was studied on freshly isolated Purkinje neurons with the use of a patch-clamp technique in the whole-cell configuration. It was shown that extracellular application of 0.8 M hyperforin caused a shift of the current-voltage (I-V) relationship of P current by -(8 ± 2) mV, slowdown of the activation kinetics, and a decrease in the amplitude of this current. The shift of the I-V relationship and slowdown of activation kinetics developed for less than 10 sec, while the P-current amplitude decreased for a much longer time (several minutes) and depended on the intracellular concentration of Ca²⁺ ions. -Aga-IVA toxin at the concentration of 100 nM completely blocked the recorded inward current in the presence of 0.8 M hyperforin. In experiments with intracellular perfusion of Purkinje neurons, we found that interaction of hyperforin with its binding site occurs at the external side of the cell membrane. The study of the mechanisms involved in the hyperforin-induced P-current modulation revealed that 1 mM GTPS (activating G_Ts proteins, as well as activating or blocking G_Ms proteins) or 1-2 mM GDPS (blocking G_Ts and G_Ms proteins) in the intracellular solution did not affect the hyperforin-induced modulation of P current. Hyperforin-induced Ca²⁺-independent shift of the I-V relationship and slowdown of the activation kinetics of P current were abolished in the presence of 0.5 M calmidazolium in the extracellular medium.     

Characterization of Opioid Receptors in Cultured Neurons

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.     

Modulation of Voltage-Gated Ca2+ Channels by G Protein-Coupled Receptors in Celiac-Mesenteric Ganglion Neurons of Septic Rats

Septic shock, the most severe complication associated with sepsis, is manifested by tissue hypoperfusion due, in part, to cardiovascular and autonomic dysfunction. In many cases, the splanchnic circulation becomes vasoplegic. The celiac-superior mesenteric ganglion (CSMG) sympathetic neurons provide the main autonomic input to these vessels. We used the cecal ligation puncture (CLP) model, which closely mimics the hemodynamic and metabolic disturbances observed in septic patients, to examine the properties and modulation of Ca²⁺ channels by G protein-coupled receptors in acutely dissociated rat CSMG neurons. Voltage-clamp studies 48 hr post-sepsis revealed that the Ca²⁺ current density in CMSG neurons from septic rats was significantly lower than those isolated from sham control rats. This reduction coincided with a significant increase in membrane surface area and a negligible increase in Ca²⁺ current amplitude. Possible explanations for these findings include either cell swelling or neurite outgrowth enhancement of CSMG neurons from septic rats. Additionally, a significant rightward shift of the concentration-response relationship for the norepinephrine (NE)-mediated Ca²⁺ current inhibition was observed in CSMG neurons from septic rats. Testing for the presence of opioid receptor subtypes in CSMG neurons, showed that mu opioid receptors were present in ~70% of CSMG, while NOP opioid receptors were found in all CSMG neurons tested. The pharmacological profile for both opioid receptor subtypes was not significantly affected by sepsis. Further, the Ca²⁺ current modulation by propionate, an agonist for the free fatty acid receptors GPR41 and GPR43, was not altered by sepsis. Overall, our findings suggest that CSMG function is affected by sepsis via changes in cell size and α2-adrenergic receptor-mediated Ca²⁺ channel modulation.     

The Adenosine Receptor Agonist,APNEA, Increases Calcium Influx into Rat Cortical Synaptosomes through N-Type Channels Associated with A2a Receptors

N⁶-2-(4-aminophenyl)ethyladenosine (APNEA) is a nonselective adenosine receptor agonist known to have a high affinity for the adenosine A₁ and A₃ receptors. It was found to be able to dose-dependently increase the sustained (4 min) Ca²⁺ influx into rat cortical synaptosomes while 2-chloro-N⁶-(3-iodobenzyl)-adenosine-5-N-methyluronamide (Cl-IB-MECA), a selective A₃ agonist has no effect. However, this effect of APNEA was not affected by the presence of 8-cyclopentyl-l,3-dimethylxanthine (CPT), a selective A₁ antagonist; but instead completely abolished by 8-(3-chlorostyryl)caffeine (CSC), a selective A_2a antagonist, or -conotoxin GVIA. These results show that in the rat cortex, presynaptic A_2a receptors can mediate neurotransmitter release by increasing Ca²⁺ influx through the N-type calcium channels. A₁ and A₃ receptors appear not to be involved.     

Ion-dependent Inactivation of Barium Current through L-type Calcium Channels

It is widely believed that Ba²⁺ currents carried through L-type Ca²⁺ channels inactivate by a voltage- dependent mechanism similar to that described for other voltage-dependent channels. Studying ionic and gating currents of rabbit cardiac Ca²⁺ channels expressed in different subunit combinations in tsA201 cells, we found a phase of Ba²⁺ current decay with characteristics of ion-dependent inactivation. Upon a long duration (20 s) depolarizing pulse, I_Ba decayed as the sum of two exponentials. The slow phase (τ ≈ 6 s, 21°C) was parallel to a reduction of gating charge mobile at positive voltages, which was determined in the same cells. The fast phase of current decay (τ ≈ 600 ms), involving about 50% of total decay, was not accompanied by decrease of gating currents. Its amplitude depended on voltage with a characteristic U-shape, reflecting reduction of inactivation at positive voltages. When Na⁺ was used as the charge carrier, decay of ionic current followed a single exponential, of rate similar to that of the slow decay of Ba²⁺ current. The reduction of Ba²⁺ current during a depolarizing pulse was not due to changes in the concentration gradients driving ion movement, because Ba²⁺ entry during the pulse did not change the reversal potential for Ba²⁺. A simple model of Ca²⁺-dependent inactivation (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1993. J. Gen. Physiol. 102:1005–1030) robustly accounts for fast Ba²⁺ current decay assuming the affinity of the inactivation site on the α₁ subunit to be 100 times lower for Ba²⁺ than Ca²⁺.     

Mechanisms of Gain Control by Voltage-Gated Channels in Intrinsically-Firing Neurons

Gain modulation is a key feature of neural information processing, but underlying mechanisms remain unclear. In single neurons, gain can be measured as the slope of the current-frequency (input-output) relationship over any given range of inputs. While much work has focused on the control of basal firing rates and spike rate adaptation, gain control has been relatively unstudied. Of the limited studies on gain control, some have examined the roles of synaptic noise and passive somatic currents, but the roles of voltage-gated channels present ubiquitously in neurons have been less explored. Here, we systematically examined the relationship between gain and voltage-gated ion channels in a conductance-based, tonically-active, model neuron. Changes in expression (conductance density) of voltage-gated channels increased (Ca²⁺ channel), reduced (K⁺ channels), or produced little effect (h-type channel) on gain. We found that the gain-controlling ability of channels increased exponentially with the steepness of their activation within the dynamic voltage window (voltage range associated with firing). For depolarization-activated channels, this produced a greater channel current per action potential at higher firing rates. This allowed these channels to modulate gain by contributing to firing preferentially at states of higher excitation. A finer analysis of the current-voltage relationship during tonic firing identified narrow voltage windows at which the gain-modulating channels exerted their effects. As a proof of concept, we show that h-type channels can be tuned to modulate gain by changing the steepness of their activation within the dynamic voltage window. These results show how the impact of an ion channel on gain can be predicted from the relationship between channel kinetics and the membrane potential during firing. This is potentially relevant to understanding input-output scaling in a wide class of neurons found throughout the brain and other nervous systems.     

Activity of TRPV1 Channels in Primary Nociceptive Neurons of Rats: Modulation by Changes in Intracellular Calcium Level

In rat neurons of the dorsal root ganglia (DRG) with mid- (35 to 25 μm) and small-sized (less than 25 μm) somata, we studied calcium transients induced by application of capsaicin (selective agonist of TRPV1 channels) under conditions of the development of other calcium transients caused by preliminary depolarization of the plasma membrane of these neurons. The above transients in rat DRG neurons were measured using the calcium-sensitive fluorescent dye Fura 2/AM. At delays of 3, 7, and 10 sec with respect to the beginning of preliminary potassium depolarization, the amplitudes of capsaicin-induced responses were smaller, as compared with the control, on average, by 26.8, 22.1, and 4.5%, respectively, in the population of mid-sized neurons and by 35.3, 21.1, and 22.4% in small neurons. Under such conditions, we observed noticeable delays of reactions to applications of capsaicin and a certain decrease in the level of intracellular calcium at the moment of beginning of development of these reactions with respect to the corresponding values in isolated depolarization-induced transients. We conclude that excitation of primary nociceptive neurons and activation of voltage-operated calcium channels result in noticeable modulation of the activity of TRPV1 channels and change their role during pain reception.     

Coriaria Lactone Increased the Intracellular Level of Calcium through the Voltage-gated Calcium Channels in Rat Hippocampal Neurons

To investigate the effects of Coriaria Lactone (CL), an epileptogenic substance, on intracellular levels of calcium ([Ca²⁺]_i) and physiological properties of voltage-gated calcium channels (VGCCs). Ratiometric calcium imaging using Fura Red and whole-cell voltage patch-clamp technique were explored on freshly isolated rat hippocampal neurons exposed to CL. Coriaria Lactone increased [Ca²⁺]_i from 118 ± 21 to 440 ± 35 nM; VGCCs and calcium influx through NMDA receptor served as the main routes of entry. Coriaria Lactone could enhance both Low voltage activated (LVA) and High voltage activated calcium currents in a concentration-dependent way, and its effect on LVA current was more potent (about 60%). The increased calcium currents were accompanied by the shift of voltage-dependent steady-state inactivation to more positive potentials. These effects of CL, especially its impact on LVA current, could activate different calcium-dependent signaling pathways, and influence cellular excitable properties as well, which might play an important role in CL’s epileptogenic process. Q. Zhang and X. Lai contributed equally to this article.     

Subcellular Compartmentation of Opioid Receptors: Modulation by Enkephalin and Alkaloids

Abstract: A subclone of NG108–15 neuroblastoma-glioma hybrid cells was used to study the intracellular distribution of opioid receptors. Subcellular organelles were separated on self-generating Percoll-sucrose gradients and the enzymes β-glucuronidase, galactosyltransferase, 5′-nucleotidase, and glucose-6-phosphatase were used as markers to localize the various structures. Analysis of the receptor distribution from untreated cells shows that the plasma membranes contained the highest receptor density, but a significant portion of the opioid binding sites was unevenly distributed between the lysosomes, microsomes, and Golgi elements. The enzyme markers indicated that appearance of opioid receptors in these intracellular structures does not result merely from contamination with plasma membranes. About 11% of the receptors appeared in a fraction lighter than plasma membranes. The antilysosomal agent chloroquine altered the intracellular compartmentation of the receptors, possibly by blocking their translocation in the cells. Leu-enkephalin induced time-dependent loss of receptors from all four intracellular compartments examined, but a kinetic analysis showed that the rate of receptor loss in these fractions was not identical. Thus, the percent of receptors appearing in the lysosomal fraction that could still bind [³H]-D-Ala²D-Leu⁵-enkephalin in vitro was increased on treatment with Leu-enkephalin. As an additional approach to follow the intracellular fate of the receptors, cells were labeled with [³H]diprenorphine, chased with various unlabeled opiates, and the distribution of ³H-ligand-receptors in the cells was monitored. Leu-enkephalin and etorphine altered the distribution of receptor-bound [³H]diprenorphine between the plasma membranes, lysosomes, and Golgi elements, whereas morphine had no such effect. The study sheds light on the role of intracellular structures in the metabolism of opioid receptors in untreated and opioid-treated cells.     

Modulation of Calcium Channels by Taurine Acting Via a Metabotropic-like Glycine Receptor

Taurine is one of the most abundant free amino acids in the central nervous system, where it displays several functions. However, its molecular targets remain unknown. It is well known that taurine can activate GABA-A and strychnine-sensitive glycine receptors, which increases a chloride conductance. In this study, we describe that acute application of taurine induces a dose-dependent inhibition of voltage-dependent calcium channels in chromaffin cells from bovine adrenal medullae. This taurine effect was not explained by the activation of either GABA-A, GABA-B or strychnine-sensitive glycine receptors. Interestingly, glycine mimicked the modulatory action exerted by taurine on calcium channels, although the acute application of glycine did not elicit any ionic current in these cells. Additionally, the modulation of calcium channels exerted by both taurine and glycine was prevented by the intracellular dialysis of GDP-β-S. Thus, the modulation of voltage-dependent calcium channels by taurine seems to be mediated by a metabotropic-like glycinergic receptor coupled to G-protein activation in a membrane delimited pathway.     

Block of N-type Calcium Channels in Chick Sensory Neurons by External Sodium

L-type Ca²⁺ channels select for Ca²⁺ over sodium Na⁺ by an affinity-based mechanism. The prevailing model of Ca²⁺ channel permeation describes a multi-ion pore that requires pore occupancy by at least two Ca²⁺ ions to generate a Ca²⁺ current. At [Ca²⁺] < 1 μM, Ca²⁺ channels conduct Na⁺. Due to the high affinity of the intrapore binding sites for Ca²⁺ relative to Na⁺, addition of μM concentrations of Ca²⁺ block Na⁺ conductance through the channel. There is little information, however, about the potential for interaction between Na⁺ and Ca²⁺ for the second binding site in a Ca²⁺ channel already occupied by one Ca²⁺. The two simplest possibilities, (a) that Na⁺ and Ca²⁺ compete for the second binding site or (b) that full time occupancy by one Ca²⁺ excludes Na⁺ from the pore altogether, would imply considerably different mechanisms of channel permeation. We are studying permeation mechanisms in N-type Ca²⁺ channels. Similar to L-type Ca²⁺ channels, N-type channels conduct Na⁺ well in the absence of external Ca²⁺. Addition of 10 μM Ca²⁺ inhibited Na⁺ conductance by 95%, and addition of 1 mM Mg²⁺ inhibited Na⁺ conductance by 80%. At divalent ion concentrations of 2 mM, 120 mM Na⁺ blocked both Ca²⁺ and Ba²⁺ currents. With 2 mM Ba²⁺, the IC₅₀ for block of Ba²⁺ currents by Na⁺ was 119 mM. External Li⁺ also blocked Ba²⁺ currents in a concentration-dependent manner, with an IC₅₀ of 97 mM. Na⁺ block of Ba²⁺ currents was dependent on [Ba²⁺]; increasing [Ba²⁺] progressively reduced block with an IC₅₀ of 2 mM. External Na⁺ had no effect on voltage-dependent activation or inactivation of the channel. These data suggest that at physiological concentrations, Na⁺ and Ca²⁺ compete for occupancy in a pore already occupied by a single Ca²⁺. Occupancy of the pore by Na⁺ reduced Ca²⁺ channel conductance, such that in physiological solutions, Ca²⁺ channel currents are between 50 and 70% of maximal.     

FK-506结合蛋白对钙释放通道的调控

细胞内自由钙作为一种重要的细胞信使广泛地参与细胞生理功能调控.胞内钙库(内质网系和肌浆网系)对调节细胞内自由钙水平起着重要的作用.钙库膜上的钙释放通道(ryanodine受体和三磷酸肌醇受体)受许多因素调控,其中之一就是新近研究得相当多的FK506结合蛋白.免疫抑制剂FK506能特异地结合钙库上一种分子质量为12 ku左右的蛋白,这种FK506结合蛋白与钙释放通道形成一种紧密连接的复合体,在正常生理情况下对钙释放通道起着十分重要的调控作用.     

Modulation of Binding at Opioid Receptors by Monoand Divalent Cations and by Cholinergic Compounds

Abstract

In membrane suspensions from guinea-pig brain, NaCl, LiCl, NH₄Cl and KCl, inhibit the equilibrium binding (25°C) of the selective μ-agonist [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin, the selective δ-agonist [³H]-[D-Pen²,D-Pen⁵]enkephalin and the selective δ-agonist [³H]-dynorphin A (1-9). Choline chloride inhibits the binding of the μ- and δ-agonists but not of the δ-agonist; the choline derivative, methacholine, inhibits also the binding of the δ-agonist. Binding of the δ-agonist is potentiated by CaCl₂, MgCl₂ and MnCl₂; these salts inhibit binding of the δ-agonist. As far as binding of the μ-agonist is concerned, MgCl₂ and MnCl₂ may potentiate or inhibit whereas CaCl₂ is only inhibitory. The binding of the μ-antagonist [³H]-naloxone is potentiated by NaCl; while the threshold of inhibition by LiCl is increased there is no potentiation. In membrane suspensions of the rabbit cerebellum about 80% of the opioid binding sites are of the μ-type; the binding of the μ-agonist [³H]-[D-Ala², MePhe⁴, Gly-ol⁵]enkephalin is inhibited by NaCl, LiCl, KCl and choline chloride whereas that of the μ-antagonists [³H]-naloxone and [³H]-(-)-bremazocine is potentiated at low concentrations but inhibited at higher concentrations of NaCl. In membranes of the guinea-pig cerebellum about 80% of the opioid binding sites are of the δ-type; they are particularly effective for assays of K-receptors when the selective K-agonist [³H]-dynorphin A (1-9) is used as ligand.     

Ca2+-Dependent Inactivation of P-Type Calcium Channels in Nerve Terminals

Abstract: Rapid Ca²⁺ signals evoked by K⁺ depolarization of rat cerebral cortical synaptosomes were measured by dual-channel Ca²⁺ spectrofluorometry coupled to a stopped-flow device. Kinetic analysis of the signal rise phase at various extracellular Ca²⁺ concentrations revealed that the responsible voltage-dependent Ca²⁺ channels, previously identified as P-type Ca²⁺ channels, inactivate owing to the rise in intracellular Ca²⁺ levels. At millimolar extracellular Ca²⁺ concentrations the channels were inactivated very rapidly and the rate was dependent on the high influx rate of Ca²⁺, thus limiting the Ca²⁺ signal amplitudes to 500–600 n M. A slower, probably voltage-dependent regulation appears to be effective at lower Ca²⁺ influx rates, leading to submaximal Ca²⁺ signal amplitudes. The functional feedback regulation of calcium channels via a sensor for intracellular Ca²⁺ levels appears to be responsible for the different inhibition characteristics of Cd²⁺ versus ω-agatoxin IVa.     

Modulation of Neuronal Voltage-Activated Calcium and Sodium Channels by Polyamines and pH

The endogenous polyamines spermine, spermidine and putrescine are present at high concentrations inside neurons and can be released into the extracellular space where they have been shown to modulate ion channels. Here, we have examined polyamine modulation of voltage-activated Ca2+ channels (VACCs) and voltage-activated Na+ channels (VANCs) in rat superior cervical ganglion neurons using whole-cell voltage-clamp at physiological divalent concentrations. Polyamines inhibited VACCs in a concentration-dependent manner with IC50s for spermine, spermidine, and putrescine of 4.7 ± 0.7, 11.2 ± 1.4, and 90 ± 36 mM, respectively. Polyamines caused inhibition by shifting the VACC half-activation voltage (V0.5) to depolarized potentials and by reducing total VACC permeability. The shift was described by Gouy-Chapman-Stern theory with a surface charge density of 0.120 ± 0.005 e- nm-2 and a surface potential of -19 mV. Attenuation of spermidine and spermine inhibition of VACC at decreased pH was explained by H+ titration of surface charge. Polyamine-mediated effects also decreased at elevated pH due to the inhibitors having lower valence and being less effective at screening surface charge. Polyamines affected VANC currents indirectly by reducing TTX inhibition of VANCs at high pH. This may reflect surface charge induced decreases in the local TTX concentration or polyamine-TTX interactions. In conclusion, polyamines inhibit neuronal VACCs via complex interactions with extracellular H+ and Ca. Many of the observed effects can be explained by a model incorporating polyamine binding, H+ binding and surface charge screening.     

Modulation of the Acetylcholine-Induced Input Current by Noopept in Helix Lucorum Neurons

Biophysics - Abstract—Possible causes of the positive modulating effect of noopept (in a concentration range of 0.1 to 10&nbsp;nM) on the amplitude of the acetylcholine-induced input...