Lakshmi Sangameswaran

Distribution of the Tetrodotoxin-Resistant Sodium Channel PN3 in Rat Sensory Neurons in Normal and Neuropathic Conditions

The novel sodium channel PN3/α-SNS, which was cloned from a rat dorsal root ganglion (DRG) cDNA library, is expressed predominantly in small sensory neurons and may contribute to the tetrodotoxin-resistant (TTXR) sodium current that is believed to be associated with central sensitization in chronic neuropathic pain states. To assess further the role of PN3, we have used electrophysiological, in situ hybridization and immunohistochemical methods to monitor changes in TTXRsodium current and the distribution of PN3 in normal and peripheral nerve-injured rats. (1) Whole-cell patch-clamp recordings showed that there were no significant changes in the TTXR and TTX-sensitive sodium current densities of small DRG neurons after chronic constriction injury (CCI) of the sciatic nerve. (2) Additionally, in situ hybridization showed that there was no change in the expression of PN3 mRNA in the DRG up to 14 d after CCI. PN3 mRNA was not detected in sections of brain and spinal cord taken from either normal or nerve-injured rats. (3) In contrast, immunohistochemical studies showed that major changes in the subcellular distribution of PN3 protein were caused by either CCI or complete transection of the sciatic nerve. The intensity of PN3 immunolabeling decreased in small DRG neurons and increased in sciatic nerve axons at the site of injury. The alteration in immunolabeling was attributed to translocation of presynthesized, intracellularly located PN3 protein from neuronal somata to peripheral axons, with subsequent accumulation at the site of injury. The specific subcellular redistribution of PN3 after peripheral nerve injury may be an important factor in establishing peripheral nerve hyperexcitability and resultant neuropathic pain.

Functional Analysis of a Voltage‐Gated Sodium Channel and Its Splice Variant from Rat Dorsal Root Ganglia

Abstract: Neurons of the dorsal root ganglia (DRG) express a diversity of voltage‐gated sodium channels. From rat DRG we have cloned and functionally expressed a tetrodotoxin‐sensitive sodium channel α subunit, NaCh6/Scn8a/rPN4, and a splice variant, rPN4a. Primary structure analysis shows NaCh6/Scn8a/rPN4 to be highly homologous (99%) to NaCh6 and most likely represents the same transcript. The splice variation in rPN4a is homologous in sequence and location to that of rat brain I. Tissue distribution analyzed by RT‐PCR showed NaCh6/Scn8a/rPN4 to be expressed at its highest levels in rat brain, at moderate levels in spinal cord, and at lower levels in DRG, nodose ganglia, and superior cervical ganglia and to be absent from sciatic nerve, heart, and skeletal muscle. In contrast, rPN4a shows no expression in brain and low‐level expression in spinal cord, whereas in DRG its expression is comparable to that of NaCh6/Scn8a/rPN4. Functional analysis of these channels expressed in Xenopus oocytes showed that NaCh6/Scn8a/rPN4 and rPN4a exhibited similar properties, with V1/2≅−100 mV for steady‐state inactivation and V1/2≅−40 mV for activation. rPN4a recovered from inactivation significantly faster than NaCh6/Scn8a/rPN4. NaCh6/Scn8a/rPN4 was inhibited by tetrodotoxin with an IC50≅ 1 nM. Coexpression of the β1 subunit accelerated inactivation kinetics, but the β2 subunit was without effect.

Increased acid-sensing ion channel ASIC-3 in inflamed human intestine.

Objectives Acid-sensing ion channels (ASICs) are expressed by rat sensory neurons and may mediate pain associated with tissue acidosis after inflammation or injury. Our aim was to examine the molecular forms and localization of ASICs in human intestine and dorsal root ganglia using immunochemical techniques, and to measure the effects of inflammation and injury. Design and methods Inflamed Crohns disease intestine and injured human dorsal root ganglia, with appropriate controls, were studied by Western blotting and immunohistochemistry, using specific affinity-purified ASIC antibodies. Results In the Western blot, there was a significant three-fold increase in the mean relative optical density of the ASIC-3 55-kDa band (but not ASIC-1 or ASIC-2) in full-thickness inflamed intestine, as well as in separated muscle and mucosal layers. There was a corresponding trend for an increased immunoreactive density and increased number of ASIC-3-positive neurons in the myenteric and sub-mucous plexus of inflamed intestine. In dorsal root ganglia, immunoreactivity for all ASICs was restricted to a sub-population (about 50%) of small-diameter (nociceptor) sensory neurons, and was generally less intense after injury. Conclusions Increased ASIC-3 in inflamed intestine suggests a role in pain or dysmotility, for which ASICs represent new therapeutic targets.

A tetrodotoxin-resistant voltage-gated sodium channel from human dorsal root ganglia, hPN3/SCN10A

Abstract Neuropathic pain may be produced, at least in part, by the increased activity of primary afferent neurons. Studies have suggested that an accumulation of voltage‐gated sodium channels at the site of peripheral nerve injury is a primary precursory event for subsequent afferent hyperexcitability. In this study, a human sodium channel (hPN3, SCN10A) has been cloned from the lumbar 4/5 dorsal root ganglia (DRG). Expression of hPN3 in Xenopus oocytes showed that this clone is a functional voltage‐gated sodium channel. The amino acid sequence of hPN3 is most closely related to the rat PN3/SNS sodium channels which are expressed primarily in the small neurons of rat DRGs. The homologous relationship between rPN3 and hPN3 is defined by (i) a high level of sequence identity (ii) sodium currents that are highly resistant to tetrodotoxin (TTX) (iii) similar tissue distribution profiles and (iv) orthologous chromosomal map positions. Since rPN3/SNS has been implicated in nociceptive transmission, hPN3 may prove to be a valuable target for therapeutic agents against neuropathic pain.

Differential distribution of the tetrodotoxin‐sensitive rPN4/NaCh6/Scn8a sodium channel in the nervous system

Voltage‐gated sodium channels underlie the generation of action potentials in excitable cells. Various sodium channel isoforms have been cloned, functionally expressed and distinguished on the basis of their biophysical properties or differential sensitivity to tetrodotoxin (TTX). In the present study, we have investigated the immunolocalization of the TTX‐sensitive sodium channel, rPN4/NaCh6/Scn8a, in discrete areas of the rat nervous system. Thus, in naïve animals, PN4 was abundantly expressed in brain, spinal cord, dorsal root ganglia (DRG) and peripheral nerve. The presence of PN4 at the nodes of Ranvier in the sciatic nerve suggests the importance of this sodium channel in peripheral nerve conduction. In addition, the pattern of PN4 immunolabeling was determined in DRG, spinal cord and sciatic nerve in rats subjected to chronic constriction nerve injury (CCI). J. Neurosci. Res. 60:37–44, 2000

SNS/PN3 and SNS2/NaN sodium channel-like immunoreactivity in human adult and neonate injured sensory nerves

Two tetrodotoxin‐resistant voltage‐gated sodium channels, SNS/PN3 and SNS2/NaN, have been described recently in small‐diameter sensory neurones of the rat, and play a key role in neuropathic pain. Using region‐specific antibodies raised against different peptide sequences of their α subunits, we show by Western blot evidence for the presence of these channels in human nerves and sensory ganglia. The expected fully mature 260 kDa component of SNS/PN3 was noted in all injured nerve tissues obtained from adults; however, for SNS2/NaN, smaller bands were found, most likely arising from protein degradation. There was increased intensity of the SNS/PN3 260 kDa band in nerves proximal to the site of injury, whereas it was decreased distally, suggesting accumulation at sites of injury; all adult patients had a positive Tinels sign at the site of nerve injury, indicating mechanical hypersensitivity. Injured nerves from human neonates showed similar results for both channels, but neonate neuromas lacked the SNS2/NaN 180 kDa molecular form, which was strongly present in adult neuromas. The distribution of SNS/PN3 and SNS2/NaN sodium channels in injured human nerves indicates that they represent targets for novel analgesics, and could account for some differences in the development of neuropathic pain in infants.

Assessment of differential gene expression in human peripheral nerve injury

BackgroundMicroarray technology is a powerful methodology for identifying differentially expressed genes. However, when thousands of genes in a microarray data set are evaluated simultaneously by fold changes and significance tests, the probability of detecting false positives rises sharply. In this first microarray study of brachial plexus injury, we applied and compared the performance of two recently proposed algorithms for tackling this multiple testing problem, Significance Analysis of Microarrays (SAM) and Westfall and Young step down adjusted p values, as well as t-statistics and Welch statistics, in specifying differential gene expression under different biological states.ResultsUsing SAM based on t statistics, we identified 73 significant genes, which fall into different functional categories, such as cytokines / neurotrophin, myelin function and signal transduction. Interestingly, all but one gene were down-regulated in the patients. Using Welch statistics in conjunction with SAM, we identified an additional set of up-regulated genes, several of which are engaged in transcription and translation regulation. In contrast, the Westfall and Young algorithm identified only one gene using a conventional significance level of 0.05.ConclusionIn coping with multiple testing problems, Family-wise type I error rate (FWER) and false discovery rate (FDR) are different expressions of Type I error rates. The Westfall and Young algorithm controls FWER. In the context of this microarray study, it is, seemingly, too conservative. In contrast, SAM, by controlling FDR, provides a promising alternative. In this instance, genes selected by SAM were shown to be biologically meaningful.