Sodium channels and mammalian sensory mechanotransduction

BACKGROUND: Members of the degenerin/epithelial (DEG/ENaC) sodium channel family are mechanosensors in C elegans, and Nav1.7 and Nav1.8 voltage-gated sodium channel knockout mice have major deficits in mechanosensation. β and γENaC sodium channel subunits are present with acid sensing ion channels (ASICs) in mammalian sensory neurons of the dorsal root ganglia (DRG). The extent to which epithelial or voltage-gated sodium channels are involved in transduction of mechanical stimuli is unclear. RESULTS: Here we show that deleting β and γENaC sodium channels in sensory neurons does not result in mechanosensory behavioural deficits. We had shown previously that Nav1.7/Nav1.8 double knockout mice have major deficits in behavioural responses to noxious mechanical pressure. However, all classes of mechanically activated currents in DRG neurons are unaffected by deletion of the two sodium channels. In contrast, the ability of Nav1.7/Nav1.8 knockout DRG neurons to generate action potentials is compromised with 50% of the small diameter sensory neurons unable to respond to electrical stimulation in vitro. CONCLUSION: Behavioural deficits in Nav1.7/Nav1.8 knockout mice reflects a failure of action potential propagation in a mechanosensitive set of sensory neurons rather than a loss of primary transduction currents. DEG/ENaC sodium channels are not mechanosensors in mouse sensory neurons

TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells

Transient receptor potential (TRP) channels TRPC3 and TRPC6 are expressed in both sensory neurons and cochlear hair cells. Deletion of TRPC3 or TRPC6 in mice caused no behavioural phenotype, although loss of TRPC3 caused a shift of rapidly adapting (RA) mechanosensitive currents to intermediate-adapting currents in dorsal root ganglion sensory neurons. Deletion of both TRPC3 and TRPC6 caused deficits in light touch and silenced half of small-diameter sensory neurons expressing mechanically activated RA currents. Double TRPC3/TRPC6 knock-out mice also showed hearing impairment, vestibular deficits and defective auditory brain stem responses to high-frequency sounds. Basal, but not apical, cochlear outer hair cells lost more than 75 per cent of their responses to mechanical stimulation. FM1-43-sensitive mechanically gated currents were induced when TRPC3 and TRPC6 were co-expressed in sensory neuron cell lines. TRPC3 and TRPC6 are thus required for the normal function of cells involved in touch and hearing, and are potential components of mechanotransducing complexes

Functional regulation of N-methyl-D-aspartate receptors by serine/threonine protein kinases

grantor: University of TorontoThe N-methyl-D-aspartate (NMDA) subtype of glutamate receptor plays a crucial role in a variety of neuronal processes such as long-term potentiation (LTP). A major consequence of NMDA receptor activation is a large influx of calcium into the cell which in turn leads to the activation of protein kinases. Chen and Huang (1992) reported that PKC enhances NMDA currents by reducing the Mg\sp{2+} sensitivity of these channels in trigeminal neurons. In the hippocampal neurons activation of PKC has been reported to enhance or reduce the NMDA activated currents (Wang et al., 1994, Markram and Segal 1992). In these experiments, we examined the effects of intracellular perfusion of PKCM (a constituitively active form of PKC) on NMDA-evoked currents in cultured and acutely isolated hippocampal neurons. Also the effects of Ca\sp{2+}/calmodulin dependent protein kinase II (CaMK II) on NMDA-evoked currents were examined. Results indicate that PKC acts via a phosphorylation-dependent mechanism to enhance NMDA-evoked currents in hippocampal neurons without altering the Mg\sp{2+} sensitivity of the channels. (Abstract shortened by UMI.)M.Sc

Insights into the molecular physiology of the P2X receptor family

Adenosine triphosphate (ATP), the source of metabolic energy for a majority of chemical reactions in the cell, is now considered as an ancient messenger molecule used in intercellular communications. The ionotropic effects of ATP are mediated by P2X receptors which constitute a novel family of ligand-gated ion channels (LGICs). The seven vertebrate subunits, which form homomeric as well as heteromeric channel complexes, show little structural resemblance to other families of LGICs such as the Cys-loop family exemplified by the nicotinic acetylcholine receptors. P2X receptors have been shown to play important physiological roles in processes such as transmission of pain, vasoconstriction, and inflammatory processes involving release of cytokines. The potential importance of these receptors as therapeutic targets in a number of pathophysiological conditions has focused many efforts on the understanding of their molecular physiology and modulation in vivo.This thesis reports the rationale and the results of experiments carried out to advance our knowledge of the molecular physiology of P2X receptors in several fronts: First, development of more selective tools, based on structure-function data, for the knockdown of the receptor function in vivo (manuscripts #1 and #2); second, contributions made to our understanding of the phylogeny and structure-function relationship in this protein family through functional analysis of an invertebrate P2X subunit (manuscripts #3 and #4); and third, advancing our understanding of the modulation of the P2X function through second messenger signalling pathways which would aid the efforts in drug development aimed at this receptor family (manuscript #5)

High zinc sensitivity and pore formation in an invertebrate P2X receptor

AbstractTo investigate fast purinergic signaling in invertebrates, we examined the functional properties of a P2X receptor subunit cloned from the parasitic platyhelminth Schistosoma mansoni. This purinoceptor (SmP2X) displays unambiguous homology of primary sequence with vertebrate P2X subunits. SmP2X subunits assemble into homomeric ATP-gated channels that exhibit slow activation kinetics and are blocked by suramin and PPADS but not TNP-ATP. SmP2X mediates the uptake of the dye YO-PRO-1 through the formation of large pores and can be blocked by submicromolar concentrations of extracellular Zn2+ ions (IC50=0.4 μM). The unique receptor phenotype defined by SmP2X suggests that slow kinetics, modulation by zinc and the ability to form large pores are ancestral properties of P2X receptors. The high sensitivity of SmP2X to zinc further reveals a zinc regulation requirement for the parasite's physiology that could potentially be exploited for therapeutic purposes

Probing functional properties of nociceptive axons using a microfluidic culture system

Pathological changes in axonal function are integral features of many neurological disorders, yet our knowledge of the molecular basis of axonal dysfunction remains limited. Microfluidic chambers (MFCs) can provide unique insight into the axonal compartment independent of the soma. Here we demonstrate how an MFC based cell culture system can be readily adapted for the study of axonal function in vitro. We illustrate the ease and versatility to assay electrogenesis and conduction of action potentials (APs) in naïve, damaged or sensitized DRG axons using calcium imaging at the soma for pharmacological screening or patch-clamp electrophysiology for detailed biophysical characterisation. To demonstrate the adaptability of the system, we report by way of example functional changes in nociceptor axons following sensitization by neurotrophins and axotomy in vitro. We show that NGF can locally sensitize axonal responses to capsaicin, independent of the soma. Axotomizing neurons in MFC results in a significant increase in the proportion of neurons that respond to axonal stimulation, and interestingly leads to accumulation of Nav1.8 channels in regenerating axons. Axotomy also augmented AP amplitude following axotomy and altered activation thresholds in a subpopulation of regenerating axons. We further show how the system can readily be used to study modulation of axonal function by non-neuronal cells such as keratinocytes. Hence we describe a novel in vitro platform for the study of axonal function and a surrogate model for nerve injury and sensitization