Single channel properties of rat acid-sensitive ion channel-1alpha, -2a,and -3 expressed in Xenopus oocytes

The mammalian nervous system expresses proton-gated ion channels known as acid-sensing ion channels (ASICs). Depending on their location and specialization some neurons express more than one type of ASIC where they may form homo- or heteromeric channels. Macroscopic characteristics of the ASIC currents have been described, but little is known at the single channel level. Here, we have examined the properties of unitary currents of homomeric rat ASIC1alpha, ASIC2a, and ASIC3 expressed in Xenopus oocytes with the patch clamp technique. We describe and characterize properties unique to each of these channels that can be used to distinguish the various types of ASIC channels expressed in mammalian neurons. The amplitudes of the unitary currents in symmetrical Na(+) are similar for the three types of channels (23-18 pS) and are not voltage dependent. However, ASIC1alpha exhibits three subconductance states, ASIC2a exhibits only one, and ASIC3 none. The kinetics of the three types of channels are different: ASIC1alpha and ASIC2a shift between modes of activity, each mode has different open probability and kinetics. In contrast, the kinetics of ASIC3 are uniform throughout the burst of activity. ASIC1alpha, ASIC2a, and ASIC3 are activated by external protons with apparent pH(50) of 5.9, 5.0, and 5.4, respectively. Desensitization in the continual presence of protons is fast and complete in ASIC1alpha and ASIC3 (2.0 and 4.5 s(-1), respectively) but slow and only partial in ASIC2a (0.045 s(-1)). The response to external Ca(2+) also differs: micro M concentrations of extracellular Ca(2+) are necessary for proton gating of ASIC3 (EC(50) = 0.28 micro M), whereas ASIC1alpha and ASIC2a do not require Ca(2+). In addition, Ca(2+) inhibits ASIC1alpha (K(D) = 9.2 +/- 2 mM) by several mechanisms: decrease in the amplitude of unitary currents, shortening of the burst of activity, and decrease in the number of activated channels. Contrary to previous reports, our results indicate that the Ca(2+) permeability of ASIC1alpha is very small.     

背根神经节神经元阿片受体和离子通道的研究进展

阿片及阿片受体与外周神经系统镇痛机制的研究,随着分子生物学技术的发展,已在受体的分子结构、形态学、分子药理学、离子通道和细胞内信号转导系统等方面取得了显著进展。μ、δ、κ阿片受体分子结构上的部分差异决定了它们各自的功能特征。三种受体在初级感觉神经元分布的比例不同,但都能介导细胞Ｃａ＾２＋通道的抑制和Ｋ＾＋电流增加及减少。阿片受体和通道之间由多种第二信使系统偶联。分子药理学研究表明它们还存在亚型受体     

Characterization of na(+) and ca(2+) channels in zebrafish dorsal root ganglion neurons

Background

Dorsal root ganglia (DRG) somata from rodents have provided an excellent model system to study ion channel properties and modulation using electrophysiological investigation. As in other vertebrates, zebrafish (Danio rerio) DRG are organized segmentally and possess peripheral axons that bifurcate into each body segment. However, the electrical properties of zebrafish DRG sensory neurons, as compared with their mammalian counterparts, are relatively unexplored because a preparation suitable for electrophysiological studies has not been available.

Methodology/Principal Findings

We show enzymatically dissociated DRG neurons from juvenile zebrafish expressing Isl2b-promoter driven EGFP were easily identified with fluorescence microscopy and amenable to conventional whole-cell patch-clamp studies. Two kinetically distinct TTX-sensitive Na⁺ currents (rapidly- and slowly-inactivating) were discovered. Rapidly-inactivating I_Na were preferentially expressed in relatively large neurons, while slowly-inactivating I_Na was more prevalent in smaller DRG neurons. RT-PCR analysis suggests zscn1aa/ab, zscn8aa/ab, zscn4ab and zscn5Laa are possible candidates for these I_Na components. Voltage-gated Ca²⁺ currents (I_Ca) were primarily (87%) comprised of a high-voltage activated component arising from ω-conotoxin GVIA-sensitive Ca_V2.2 (N-type) Ca²⁺ channels. A few DRG neurons (8%) displayed a miniscule low-voltage-activated component. I_Ca in zebrafish DRG neurons were modulated by neurotransmitters via either voltage-dependent or -independent G-protein signaling pathway with large cell-to-cell response variability.

Conclusions/Significance

Our present results indicate that, as in higher vertebrates, zebrafish DRG neurons are heterogeneous being composed of functionally distinct subpopulations that may correlate with different sensory modalities. These findings provide the first comparison of zebrafish and rodent DRG neuron electrical properties and thus provide a basis for future studies.     

Kv3 channels contribute to the delayed rectifier current in small cultured mouse dorsal root ganglion neurons

Delayed rectifier voltage-gated K(+) (K(V)) channels are important determinants of neuronal excitability. However, the large number of K(V) subunits poses a major challenge to establish the molecular composition of the native neuronal K(+) currents. A large part (～60%) of the delayed rectifier current (I(K)) in small mouse dorsal root ganglion (DRG) neurons has been shown to be carried by both homotetrameric K(V)2.1 and heterotetrameric channels of K(V)2 subunits with silent K(V) subunits (K(V)S), while a contribution of K(V)1 channels has also been demonstrated. Because K(V)3 subunits also generate delayed rectifier currents, we investigated the contribution of K(V)3 subunits to I(K) in small mouse DRG neurons. After stromatoxin (ScTx) pretreatment to block the K(V)2-containing component, application of 1 mM TEA caused significant additional block, indicating that the ScTx-insensitive part of I(K) could include K(V)1, K(V)3, and/or M-current channels (KCNQ2/3). Combining ScTx and dendrotoxin confirmed a relevant contribution of K(V)2 and K(V)2/K(V)S, and K(V)1 subunits to I(K) in small mouse DRG neurons. After application of these toxins, a significant TEA-sensitive current (～19% of total I(K)) remained with biophysical properties that corresponded to those of K(V)3 currents obtained in expression systems. Using RT-PCR, we detected K(V)3.1-3 mRNA in DRG neurons. Furthermore, Western blot and immunocytochemistry using K(V)3.1-specific antibodies confirmed the presence of K(V)3.1 in cultured DRG neurons. These biophysical, pharmacological, and molecular results demonstrate a relevant contribution (～19%) of K(V)3-containing channels to I(K) in small mouse DRG neurons, supporting a substantial role for K(V)3 subunits in these neurons.     

Effect of pH on fast sodium channels in neurons of the rat dorsal root ganglion

Ionic currents through fast sodium channels in the neuronal somatic membrane were measured under voltage clamp conditions using external solutions of normal and low pH. Voltage-dependent inhibition of ionic currents through open channels was observed in acidic solutions. The voltage-dependent block of sodium channels may be explained by the presence of two acid groups at the channel. The parameters of the inner and outer acid groups calculated according to this model are similar to those reported for the nodal membrane.     

Agonistic effects of xanthines on tetrodotoxin-sensitive sodium channels in the rat dorsal root ganglion neurons

The effects of four xanthine derivatives, caffeine, caffeine benzoate, theophylline, and bromtheophylline, on sodium channels in internally perfused rat dorsal root ganglion neurons were studied under voltage-clamp and whole-cell patch-clamp conditions. Reversible acceleration, enhancement of the amplitude of sodium currents, and shifts of the current-voltage relation (plotted for their maxima), as well as of the steady-state inactivation curve toward more negative potentials, were observed at external applications of the above substances in the concentrations of 0.2–4.0 mM. Under long exposures, inactivation of sodium currents became slower in a part of the cells. Yet, when the exposure to 4 mM or higher concentrations was longer than 10 min, a rise in the passive conductance was obvious, and functional state of the cells became worse. Blocking effects of the xanthine derivatives on transient or delayed potassium currents were not observed. Thus, agonistic action of xanthines on sodium channels has been demonstrated, and it is supposed that a considerable component of their pharmacological effects is provided by the action on Na⁺/Ca²⁺ exchange.     

Potassium permeability of voltage-operated calcium channels of dorsal root ganglion neurons in a calcium-free medium

In freshly isolated neurons of the rat spinal ganglia, we studied the behavior of voltage-operated calcium channels of these cells under conditions of the absence of calcium ions in the extracellular solution; a patch-clamp technique in the whole-cell configuration was used. We found that such channels in a part of the studied neurons lose their selectivity in a calcium-free potassium-containing solution and become capable of passing an inward potassium current. This current was inhibited by blockers of voltage-operated calcium channels, nifedipine and nickel, and also was to some extent inhibited by caffeine. The latter effect is realized, perhaps, due to calcium-dependent inactivation of calcium channels induced by the action of calcium ions released from the endoplasmic reticulum upon caffeine-induced activation of ryanodine receptors. The peculiarities of current-voltage relationships and characteristics of activation/inactivation of calcium channels modified in calcium-free medium and the possible mechanisms of such modification are discussed. Neirofiziologiya/Neurophysiology, Vol. 40, No. 2, pp. 93–99, March–April, 2008.     

Gating properties of connexin32 cell-cell channels and their mutants expressed in Xenopus oocytes.

Carboxyl-terminal deletion mutants of the gap junction protein connexin32 were tested in the oocyte cell-cell channel assay. Oocytes expressing a mutant lacking 58 carboxyl terminal amino acids were found to exhibit junctional conductances of the same magnitude as oocytes expressing wild-type connexin32. The gating properties of the channels formed by this mutant of connexin32 with respect to transjunctional voltage and cytoplasmic acidification are indistinguishable from those found with wild-type connexin32 channels. This includes a novel pH-dependent voltage gate. In another mutant, two carboxyl terminal serine residues, Ser233 and Ser240, were replaced by Asn residues. This double mutant has properties indistinguishable from wild-type connexin32, suggesting that phosphorylation of either of these serines is not required for channel opening.     

Analysis of receptive fields revealed by in vivo patch-clamp recordings from dorsal horn neurons and in situ intracellular recordings from dorsal root ganglion neurons

It has been thought that spinal dorsal horn neurons receive convergent inputs from not only somatosensory but also visceral pathways. For instance, the referred pain is presumed to be due to the convergence of sensory inputs from cardiac and shoulder receptive fields. However, precise investigation has not been made from dorsal horn neurons yet, because of difficulty in studying the pathways from those regions by means of conventional electrophysiology. The purpose of this study is to clarify the convergent inputs to single dorsal horn neurons from wide receptive fields using an in vivo patch-clamp recording technique from the superficial spinal dorsal horn and an intracellular recording from dorsal root ganglion neurons that keep physiological connections with the peripheral sites. Identified dorsal root ganglion neurons received an input from a quite small area, about 1 x 1 mm in width of the skin. In contrast, substantia gelatinosa neurons in the spinal cord received inputs from an unexpectedly wide area of the skin. Previous extracellular recordings have, however, revealed that substantia gelatinosa neurons have small receptive field. This discrepancy is probably due mainly to an availability of the in vivo patch-clamp method to analyze sub-threshold synaptic responses. In contrast, the extracellular recording technique allows us to analyze predominantly the firing frequency of neurons. Thus, the in vivo patch-clamp recordings from dorsal horn neurons and the intracellular recordings from DRG neurons will be useful for well understanding the sensory processing in the spinal cord.     

Differential G protein-mediated coupling of neurotransmitter receptors to Ca2+ channels in rat dorsal root ganglion neurons in vitro

The peptides neuropeptide Y (NPY) and bradykinin (BK) both inhibited Ca2+ currents in rat dorsal root ganglion neurons (DRG) in vitro. The effects of both peptides were completely blocked by treatment of cells with pertussis toxin. Based on antigenic determinants, DRG cells contained at least two pertussis toxin substrates, alpha o (Mr, 39 kd) and alpha i2 (Mr, 40 kd). We examined the ability of three purified bovine alpha subunits (identified with antibodies as alpha o, alpha i1, and alpha i2) to reconstitute the inhibitory effects of NPY and BK. Reconstitution of NPY effects occurred according to the potency series alpha o greater than alpha i1 much greater than alpha i2. However, in the case of BK all three G proteins were approximately equally effective. Whereas complete reconstitution of NPY effects could be obtained with alpha o, no single alpha subunit produced complete reconstitution of BK. Combinations of alpha o and alpha i2, however, were able to completely reconstitute the effects of BK. Thus several G proteins can effect the regulation of Ca2+ channels in these cells. However, neurotransmitters may be selective in the G proteins or combinations of G proteins utilized to achieve this regulation.     

The effect of acute dissociation on the electrophysiological properties of rat dorsal root ganglion neurons

The acutely dissociated neurons from the dorsal root ganglia (DRGs) are extensively used. The effects of acute dissociation on the properties of these neurons are, however, not clear. In this study, the action potentials (APs) were recorded from both acutely dissociated and in vivo identified DRG neurons with patch clamp and sharp electrode recording techniques, respectively. We found that acute dissociation slowed both the depolarizing and repolarizing rate of APs, and elongated the AP duration (APD). The lower recording temperature presented in the acutely dissociated neurons contributed to about 10% of these differences. The major contributor of these differences was possibly modulation of the mRNA expression especially those of the ion channels, as suggested by our observation that acute dissociation significantly reduced the mRNA abundance of Nav1.6–1.9. In conclusion, acute dissociation altered the electrophysiological properties of the DRG neurons; the disrupted gene-expression pattern may contribute to this effect.     

Regulation of galanin and galanin receptor 2 expression by capsaicin in primary cultured dorsal root ganglion neurons

Galanin is a 29-amino-acid neuropeptide expressed in dorsal root ganglion (DRG) neurons which is thought to play a role in modulation of nociception in neuropathic states. Activation of galanin receptor 2 (GalR2) plays a pronociceptive role and enhances capsaicin-induced nociception in the periphery. GalR2 and vanilloid receptor 1 (VR1) are co-expressed in DRG neurons. Capsaicin evokes acute pain via activation of VR1 expressed in primary sensory neurons. It is not known to what extent galanin and its receptor GalR2 expression is regulated by capsaicin in DRG neurons. Effects of acute (4 h) or chronic (4 d) treatment with capsaicin at different concentrations (0.01, 0.1, 1 micromol/L) on galanin and GalR2 expression in primary cultured DRG neurons were investigated in the present study. Our results showed that acute exposure of high concentration capsaicin (1 micromol/L) increased galanin expression, whereas chronic exposure of low concentration capsaicin (0.01, 0.1 micromol/L) promoted galanin expression. Only chronic exposure of 0.1 micromol/L concentration capsaicin could elevate GalR2 expression, whereas capsaicin did not have this effect at any other conditions in this experiment. These results indicated that certain concentrations or exposure time of capsaicin stimulation may be relevant to upregulation of galanin and its receptor GalR2 expression in DRG cultures suggesting a response to peripheral neuronal stimulation. And also, capsaicin-induced GalR2 expression may be also modulated by capsaicin-induced galanin expression. The possible significance of the neurotransmission of nociceptive information involved in galanin or GalR2 expression caused by capsaicin is still to be clarified.     

Characteristics of store-operated calcium channels activated by depletion of the endoplasmic reticulum ryanodine-sensitive calcium stores in rat dorsal root ganglion neurons

In neurons of the rat dorsal root ganglia (DRG), using a patch-clamp technique in the whole-cell configuration, we studied the characteristics of calcium channels activated by depletion of the ryanodine-sensitive calcium stores of the endoplasmic reticulum. Current-voltage (I-V) relationships of these store-operated calcium channels were obtained by subtraction of the integral I-V characteristics after application of caffeine from the integral I-V characteristics of calcium channels in the control. Currents through store-operated calcium channels could be induced by application of a series of hyperpolarization current pulses to the cell under conditions of replacement of a calcium-free solution containing caffeine by a caffeine-free solution containing 2 mM Ca²⁺. In this case, the following two main conditions were abserved: Voltage-operated calcium channels were inactivated, while a gradient of the electrochemical potential for calcium ions was increased, which made easier passing of these currents through store-operated calcium channels. Therefore, we found that in DRG neurons, despite the presence of great numbers of both voltage-operated and receptor-dependent calcium channels, one more mechanism underlying the entry of calcium through store-operated channels does exist. Neirofiziologiya/Neurophysiology, Vol. 39, No. 3, pp. 195–200, May–June, 2007.     

藜芦碱和乌头碱在受损背根节神经元诱发不同的放电模式

为了研究钠通道失活门阻断后受损背根节神经元放电模式的变化特征,在大鼠背根节慢性压迫模型上采用单纤维技术记录A类神经元的自发放电。藜芦碱和乌头碱是钠通道失活门的抑制剂,但二者作用于不同的位点,前者结合于D2-S6,后者结合于D3-S6。我们比较了这两种试剂引发的放电模式。结果发现,在同一神经元,藜芦碱(1．5～5．0μmol／L)可以引起放电峰峰间期的慢波振荡,即峰峰间期由大逐渐减小,然后又逐渐增大,形成重复的振荡波形,每个振荡持续约数十秒至数分钟：而乌头碱(10～200μmol／L)则引起强直性放电,即峰峰间期逐渐减小,然后维持在一个稳定的水平。这两种不同的放电模式不因背景放电或试剂浓度的不同而发生明显的改变。实验结果表明,藜芦碱和乌头碱在受损的背根节神经元可以引发不同的放电模式,这可能与它们结合于钠通道上不同位点的抑制作用有关。     

Versatile regulation of cytosolic Ca2+ by vanilloid receptor I in rat dorsal root ganglion neurons

Analysis of small dorsal root ganglion (DRG) neurons revealed novel functions for vanilloid receptor 1 (VR1) in the regulation of cytosolic Ca(2+). The VR1 agonist capsaicin induced Ca(2+) mobilization from intracellular stores in the absence of extracellular Ca(2+), and this release was inhibited by the VR1 antagonist capsazepine but was unaffected by the phospholipase C inhibitor xestospongins, indicating that Ca(2+) mobilization was dependent on capsaicin receptor binding and was not due to intracellular inositol-1,4,5-trisphosphate generation. Confocal microscopy revealed extensive expression of VR1 on endoplasmic reticulum, consistent with VR1 operating as a Ca(2+) release receptor. The main part of the capsaicin-releasable Ca(2+) store was insensitive to thapsigargin, a selective endoplasmic reticulum Ca(2+)-ATPase inhibitor, suggesting that VR1 might be predominantly localized to a thapsigargin-insensitive endoplasmic reticulum Ca(2+) store. In addition, VR1 was observed to behave as a store-operated Ca(2+) influx channel. In DRG neurons, capsazepine attenuated Ca(2+) influx following thapsigargin-induced Ca(2+) store depletion and inhibited thapsigargin-induced inward currents. Conversely, transfected HEK-293 cells expressing VR1 showed enhanced Ca(2+) influx and inward currents following Ca(2+) store depletion. Combined data support topographical and functional diversity for VR1 in the regulation of cytosolic Ca(2+) with the plasma membrane-associated form behaving as a store-operated Ca(2+) influx channel and endoplasmic reticulum-associated VR1 possibly functioning as a Ca(2+) release receptor in sensory neurons.     

Analgesic effect of electroacupuncture on chronic neuropathic pain mediated by P2X3 receptors in rat dorsal root ganglion neurons

Adenosine 5'-triphosphate disodium (ATP) gated P2X receptors, especially the subtype P2X(3), play a key role in transmission of pain signals in neuropathic pain, ATP has been documented to play a significant role in the progression of pain signals, suggesting that control of these pathways through electroacupuncture (EA) is potentially an effective treatment for chronic neuropathic pain. EA has been accepted to effectively manage chronic pain by applying the stimulating current to acupoints through acupuncture needles. To determine the significance of EA on neuropathic pain mediated by P2X(3) receptors in the dorsal root ganglion (DRG) neurons, mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were recorded, and the expression of P2X(3) receptors in the DRG neurons was assessed by immunohistochemistry (IHC) and in situ hybridization (ISH). In addition, the currents which were evoked in DRG neurons isolated from rats following chronic constriction injury (CCI) by the P2X(3) receptors agonists i.e. ATP and α,β-methylen-ATP (α,β-meATP) were examined through the experimental use of whole cell patch clamp recording. The present study demonstrates that EA treatment can increase the MWT and TWL values and decrease the expression of P2X(3) receptors in DRG neurons in CCI rats. Simultaneously, EA treatment attenuates the ATP and α,β-meATP evoked currents. EA may be expected to induce an apparent induce analgesic effect by decreasing expression and inhibiting P2X(3) receptors in DRG neurons of CCI rats. There is a similar effect on analgesic effect between rats with contralateral EA and those with ipsilateral EA.     

Role of acidosis,NMDA receptors,and acid-sensitive ion channel 1a (ASIC1a) in neuronal death induced by ischemia

This review collects data on the influence of intracellular and extracellular acidosis on neuronal viability and the effect of acidosis on neuronal damage progressing under brain ischemia/hypoxia. Particular attention is devoted to the involvement of ionotropic glutamic receptors and acid-sensitive ion channel 1a in these processes. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 11, pp. 1461–1466.