Mitochondrial potassium channels and reactive oxygen species

Pretreatment of tissues with potassium channel openers (KCO’s) has been observed to be cytoprotective in a broad variety of insults. This phenomenon has been proposed to be intimately linked to activation of mitochondrial potassium channels which apparently modulate the mitochondrial production of reactive oxygen species (ROS). This critical review summarizes literature findings about the mitochondrial production of ROS, the action of KCO’s on mitochondrial ROS production and the putative link to the cytoprotective action of these drugs.     

Two tetrodotoxin-resistant sodium channels in human dorsal root ganglion neurons

Two tetrodotoxin-resistant (TTX-R) voltage-gated sodium channels, SNS and NaN, are preferentially expressed in small dorsal root ganglia (DRG) and trigeminal ganglia neurons, most of which are nociceptive, of rat and mouse. We report here the sequence of NaN from human DRG, and demonstrate the presence of two TTX-R currents in human DRG neurons. One current has physiological properties similar to those reported for SNS, while the other displays hyperpolarized voltage-dependence and persistent kinetics; a similar TTX-R current was recently identified in DRG neurons of sns-null mouse. Thus SNS and NaN channels appear to produce different currents in human DRG neurons.     

Experimental colitis modulates the functional properties of NMDA receptors in dorsal root ganglia neurons

N-methyl-D-aspartate (NMDA) receptors (NMDARs) on spinal afferent neurons regulate the peripheral and central release of neuropeptides involved in the development of hyperalgesia. We examined the effect of experimental colitis on the molecular and functional properties of NMDARs on these neurons. Lumbosacral dorsal root ganglia (DRG) were collected from adult rats 5 days after the induction of colitis for whole cell patch-clamp recording, Western blot analysis, and quantitative RT-PCR. Compared with neurons from control rats, those taken from animals with colitis had a threefold higher density of NMDA currents in both retrograde-labeled, colon-specific, and unlabeled DRG neurons. Increased current densities were not observed in DRG neurons taken from thoracic spinal levels. There was no significant change in NMDA or glycine affinity or in voltage-dependent Mg2+ inhibition; however, there was a 10-fold decrease in sensitivity to the NR2B subunit-selective antagonist ifenprodil. Quantitative RT-PCR and Western blot analysis indicated a 28% increase in the expression of NR2B with little or no change in the other three NR2 subunits. The addition of the Src family tyrosine kinase inhibitor PP2 (10 microM) decreased NMDAR currents in neurons from colitis but not control rats. Conversely, pretreatment of DRG neurons from control animals with 100 microM sodium orthovanadate increased NMDAR currents and decreased ifenprodil sensitivity to levels similar to those observed in neurons from animals with colitis. In conclusion, colonic inflammation upregulates the activity of NMDARs in all DRG neurons within ganglia innervating this tissue through mechanisms involving increased expression and persistent tyrosine phosphorylation.     

Slow inactivation in voltage-gated sodium channels

Slow inactivation in voltage-gated sodium channels is a biophysical process that governs the availability of sodium channels over extended periods of time. Slow inactivation, therefore, plays an important role in controlling membrane excitability, firing properties, and spike frequency adaptation. Defective slow inactivation is associated with several diseases of cell excitability, such as hyperkalemic periodic paralysis, myotonia, idiopathic ventricular fibrillation and long-QT syndrome. These associations underscore the physiological importance of this phenomenon. Nevertheless, our understanding of the molecular substrates for slow inactivation is still fragmentary. This review covers the current state of knowledge concerning the molecular underpinnings of slow inactivation, and its relationship with other biophysical processes of voltage-gated sodium channels.     

Slow inactivation of tetrodotoxin-insensitive Na+ channels in neurons of rat dorsal root ganglia

Summary Whole-cell patch-clamp experiments were performed with neurons cultured from rat dorsal root ganglia (DRG). Two types of Na⁺ currents were identified on the basis of sensitivity to tetrodotoxin. One type was blocked by 0.1 nm tetrodotoxin, while the other type was insensitive to 10 m tetrodotoxin. The peak amplitude of the tetrodotoxin-insensitive Na⁺ current gradually decreased after depolarization of the membrane. The steady-state value of the peak amplitude was attained several minutes after the change of holding potential. Such a slow inactivation was not observed in tetrodotoxin-sensitive Na⁺ current. The slow inactivation of the tetrodotoxin-insensitive Na⁺ current was kinetically distinct from the ordinary short-time steady-state inactivation. The voltage dependence of the slow inactivation could be described by a sigmoidal function, and its time course had a double-exponential process. A decrease of external pH partially antagonized the slow inactivation, probably through an increased diffusion potential across the membrane. However, the slow inactivation was not due to change in surface negative charges, since a shift of the kinetic parameters along the voltage axis was not observed during the slow inactivation. Due to the slow inactivation, the inactivation curves for the tetrodotoxininsensitive Na⁺ current were shifted in the negative direction as the prepulse duration was increased. Consequently, the window current activated at potentials close to the resting membrane potential was markedly reduced. Thus, the slow inactivation may be involved in the long-term regulation of the excitability of sensory neurons.We thank Prof. Hirosi Kuriyama for his support and advice and Dr. M. Yoshii for helpful discussions. This study was supported by the Japanese Ministry of Education (Scientific Research 02670090).     

Conotoxin modulation of voltage-gated sodium channels

The rising phase of the action potential in excitable cells is mediated by voltage-gated sodium channels (VGSCs), of which there are nine mammalian subtypes with distinct tissue distribution and biophysical properties. The involvement of certain VGSC subtypes in disease states such as pain and epilepsy highlights the need for agents that modulate VGSCs in a subtype-specific manner. Conotoxins from marine snails of the Conus genus constitute a promising source of such modulators, since these peptide toxins have evolved to become selective for various membrane receptors, ion channels and transporters in excitable cells. This review covers the structure and function of three classes of conopeptides that modulate VGSCs: the pore-blocking mu-conotoxins, the delta-conotoxins which delay or inhibit VGSC inactivation, and the muO-conotoxins which inhibit VGSC Na(+) conductance independent of the tetrodotoxin binding site. Some of these toxins have potential therapeutic and research applications, in particular the muO-conotoxins, which may develop into potential drug leads for the treatment of pain states.     

Altered ATP release and metabolism in dorsal root ganglia of neuropathic rats

Recent advances in pain research provide a clear picture for the molecular mechanisms of acute pain; substantial information concerning plasticity that occurs during neuropathic pain has also become available. The peripheral mechanisms responsible for neuropathic pain are found in the altered gene/protein expression of primary sensory neurons. With damage to peripheral sensory fibers, a variety of changes in pain-related gene expression take place in dorsal root ganglion neurons. These changes, or plasticity, might underlie unique neuropathic pain-specific phenotype modifications – decreased unmyelinated-fiber functions, but increased myelinated A-fiber functions. Another characteristic change is observed in allodynia, the functional change of tactile to nociceptive perception. Throughout a series of studies, using novel nociceptive tests to characterize sensory-fiber or pain modality-specific nociceptive behaviors, it was demonstrated that communication between innocuous and noxious sensory fibers might play a role in allodynia mechanisms. Because neuropathic pain in peripheral and central demyelinating diseases develops as a result of aberrant myelination in experimental animals, demyelination seems to be a key mechanism of plasticity in neuropathic pain. More recently, we discovered that lysophosphatidic acid receptor activation initiates neuropathic pain, as well as possible peripheral mechanims of demyelination after nerve injury. These results lead to further hypotheses of physical communication between innocuous Aβ- and noxious C- or Aδ-fibers to influence the molecular mechanisms of allodynia.     

Serotonin modulates Arabidopsis root growth via changes in reactive oxygen species and jasmonic acid–ethylene signaling

Serotonin (5‐hydroxytryptamine) is a bioactive indoleamine with neurotransmitter function in vertebrates, which represents an emerging signaling molecule in plants, playing key roles in the development and defense. In this study, the role of reactive oxygen species (ROS) and jasmonic acid (JA)–ethylene (Et) signaling in root developmental alterations induced by serotonin was investigated. An Arabidopsis thaliana mutant defective at the RADICAL‐INDUCED CELL DEATH1 (RCD1) locus was resistant to paraquat‐induced ROS accumulation in primary roots and showed decreased inhibition or root growth in response to serotonin. A suite of JA‐ and Et‐related mutants including coronatine insensitive1, jasmonic acid resistant1 (jar1), etr1, ein2 and ein3 showed tolerance to serotonin in the inhibition of primary root growth and ROS redistribution within the root tip when compared with wild‐type (WT) seedlings. Competence assays between serotonin and AgNO₃, a well‐known blocker of Et action, showed that primary root growth in medium supplemented with serotonin was normalized by AgNO_3, whereas roots of eto3, an Et overproducer mutant, were oversensitive to serotonin. Comparison of ROS levels in WT, etr1, jar1 and rcd1 primary root tips using the ROS‐specific probe 2′,7′‐dichlorofluorescein diacetate and confocal imaging showed that serotonin inhibition of primary root growth likely occurs independently of its conversion into melatonin. Our results provide compelling evidence that serotonin affects ROS distribution in roots, involving RCD1 and components of the JA–Et signaling pathways.     

Functional and molecular characterization of voltage-gated sodium channels in uteri from nonpregnant rats

We investigated the function and expression of voltage-gated Na(+) channels (VGSC) in the uteri of nonpregnant rats using organ bath techniques, intracellular [Ca(2+)] fluorescence measurements, and RT-PCR. In longitudinally arranged whole-tissue uterine strips, veratridine, a VGSC activator, caused the rapid appearance of phasic contractions of irregular frequency and amplitude. After 50-60 min in the continuous presence of veratridine, rhythmic contractions of very regular frequency and slightly increasing amplitude occurred and were sustained for up to 12 h. Both the early and late components of the contractile response to veratridine were inhibited in a concentration-dependent manner by tetrodotoxin (TTX). In small strips dissected from the uterine longitudinal smooth muscle layer and loaded with Fura-2, veratridine also caused rhythmic contractions, accompanied by transient increases in [Ca(2+)](i), which were abolished by treatment with 0.1 microM TTX. Using end-point and real-time quantitative RT-PCR, we detected the presence of the VGSC alpha subunits Scn2a1, Scn3a, Scn5a, and Scn8a in the cDNA from longitudinal muscle. The mRNAs of the auxiliary beta subunits Scbn1b, Scbn2b, Scbn4b, and traces of Scn3b were also present. These data show for the first time that Scn2a1, Scn3a, Scn5a, and Scn8a, as well as all VGSC beta subunits are expressed in the longitudinal smooth muscle layer of the rat myometrium. In addition, our data show that TTX-sensitive VGSC are able to mediate phasic contractions maintained over long periods of time in the uteri of nonpregnant rats.     

A superfamily of voltage-gated sodium channels in bacteria

NaChBac, a six-alpha-helical transmembrane-spanning protein cloned from Bacillus halodurans, is the first functionally characterized bacterial voltage-gated Na(+)-selective channel. As a highly expressing ion channel protein, NaChBac is an ideal candidate for high resolution structural determination and structure-function studies. The biological role of NaChBac, however, is still unknown. In this report, another 11 structurally related bacterial proteins are described. Two of these functionally expressed as voltage-dependent Na(+) channels (Na(V)PZ from Paracoccus zeaxanthinifaciens and Na(V)SP from Silicibacter pomeroyi). Na(V)PZ and Na(V)SP share approximately 40% amino acid sequence identity with NaChBac. When expressed in mammalian cell lines, both Na(V)PZ and Na(V)SP were Na(+)-selective and voltage-dependent. However, their kinetics and voltage dependence differ significantly. These single six-alpha-helical transmembrane-spanning subunits constitute a widely distributed superfamily (Na(V)Bac) of channels in bacteria, implying a fundamental prokaryotic function. The degree of sequence homology (22-54%) is optimal for future comparisons of Na(V)Bac structure and function of similarity and dissimilarity among Na(V)Bac proteins. Thus, the Na(V)Bac superfamily is fertile ground for crystallographic, electrophysiological, and microbiological studies.     

Ribosomes in myelinated axons of dorsal root ganglia

Summary In the dorsal root ganglia of the rat, ribosomes were found not only in the initial segment, but they were also observed in the axoplasm of intraganglionar myelinated fibres and in the sensory portion of spinal nerves. Axons of seven-days-old rats contained more ribosomes than those of adult animals. The amount of particles decreased gradually from the initial segment trough intraganglionar internodes to the axons of spinal nerves. No ribosomes were found in axons of dorsal roots. In intraganglionar fibres, ribosomal particles were usually observed near the nodes of Ranvier, in the vicinity of Schmidt-Lantermann clefts and in axons near the Schwann cell nuclei. They were arranged in tetrads, pentads or in larger polysomes, and they were often observed adjacent to a group of mitochondria.The particles had invariably a stable size, their average diameters measuring 234 ± 2 × 197 ± 3 Å, which is practically equal to the diameters of 232 ± 2 × 203 ± 3 Å of ribosomes in the Schwann cell cytoplasm. These values fall within the range of diameters of ribosomes isolated from various cells of eukaryotic organisms as given in the literature. Since no other granular component of the cytoplasm has similarly stable dimensions, the measurements are considered to prove that the axonal particles described here are ribosomes.The author wishes to thank Dr. K. Smetana for his valuable suggestions and Mrs. M. Sobotková, Ing. M. Doubek and Mr. H. Kunz for their skillful technical assistance. The investigation was in part supported by a grant-in-aid from the Muscular Dystrophy Associations of America, Inc.     

Effects of hydroxyurea during final neuronal DNA synthesis in dorsal root ganglia of rats

The sequence of final neuronal DNA synthesis was investigated in developing lumbar dorsal root ganglia of rats. Patterns of final division were compared to permanent neuronal deficiencies produced by single doses of hydroxyurea (HU), a specific cytotoxic inhibitor of DNA synthesis. The purpose was to discern increased susceptibility of terminal cell cycles in order to evaluate possible phenotypic or mitotic commitments responsible for cessation of DNA synthesis. The normal period of final DNA synthesis was found to occur primarily on gestation Days 12, 13, and early 14. Precursors of larger (A cells) and smaller (B cells) neurons are generated in sequence, suggesting the presence of phenotypic commitments during terminal division. When HU was administered during the period of final DNA synthesis, severe neuronal depletions and altered phenotypic proportions were observed postnatally. With HU on Day 13, total neuronal numbers were reduced by an average of 62% and deficiencies were confined primarily to neurons originating at or near the time of treatment. Given 12 hr later (Day 13.5), HU produced a 48% depletion involving neurons of smaller diameters. With treatment on Day 14, some ganglia appeared normal histologically but quantitation revealed an average 21% numerical deficiency involving the smallest neuronal phenotypes. Later treatments did not appear to affect ganglion morphology even though other defects (primarily growth retardation and gait abnormalities) continued to occur. Earlier treatments, given during terminal division of large neurons on Day 12, produced resorption or early postnatal death. The results suggest the emergence of phenotypic commitments in final cell cycles which restrict the probability of continued DNA synthesis and, thus, the probability of regeneration.