My research focuses on understanding the role of individual sodium channels, a class of proteins that conduct electrical currents, in the transmission of nerve impulses. A few sodium channels have been implicated in the hyperexcitability of pain-sensing neurons in acquired and inherited pain disorders. We are currently investigating the contribution of individual channels to the excitability of neurons under normal and pathological conditions. These studies aim to identify new targets for treatment of neurological disorders including neuropathic pain.
Specialized Terms: Molecular biology of voltage-gated sodium channels; Quantitative analysis of gene expression in normal and injured neurons; Structure-function relationship of sodium channel alpha subunits; Identification of proteins that modulate channel properties
My research has focused on studying voltage-gated sodium channels regulation by accessory proteins and phosphorylation, and the contribution of specific channels to electrogenesis in dorsal root ganglion (DRG) neurons under normal conditions and in inherited channelopathies. Sodium channels are heterotrimers consisting of a large pore-forming alpha-subunit (referred to as channel), and smaller auxiliary beta-subunits. Sodium channels are large polypeptides (1700-2000 amino acids) which fold into four domains (DI-DIV), each domain including six transmembrane segments, linked by three loops (L1-L3). Nine alpha-subunits (Nav1.1-Nav1.9) encoded by the SCN1A-SCN5A and SCN8A-SCN11A genes, have been identified in mammals, and their expression is spatially-, and temporally-regulated. Different channels activate and inactivate with different kinetics and voltage-dependent properties, with six channels (Nav1.1-1.4, Nav1.6 and Nav1.7) sensitive to block by nanomolar concentrations of tetrodotoxin (TTX-S), and three channels (Nav1.5, Nav1.8 and Nav1.9) resistant to this blocker (TTX-R). Because channel properties are cell-type dependent and sodium channel properties can be modulated in a cell-type-specific manner, we have developed methods to study these channels within native neurons.
Neuronal sodium channels, Nav1.3, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 have been intensively investigated because of their potential role in nervous system disorders. Specifically, Nav1.6-Nav1.9 are the main channels in DRG neurons, and their altered expression and modulation following injury or inflammation have been linked to acquired neuropathic pain in animal models. Electrophysiological studies over the past decade from our group and several other research groups have attributed specific roles for individual channels to specific aspects of action potential firing. Thus, in small-diameter nociceptive neurons Nav1.7 and Nav1.9 are considered threshold channels that boost weak stimuli and Nav1.8 is the channel that carries the main sodium current of action potentials with Nav1.6 contributing to the first few spikes. In large-diameter DRG neurons Nav1.6 is the main sodium channel with Nav1.7 and Nav1.8 present in a smaller number of these cells. Mutations in Nav1.7 have been shown to underlie two distinct pain disorders, while its complete loss results in congenital insensitivity to pain. Ongoing studies aim to better understand the contribution of these channels to the pathophysiology of pain and other neurological disorders, including multiple sclerosis and spinal cord injury.
Using genetic, biochemical, and electrophysiological approaches we have identified channel partners that may be important for channel trafficking and /or modulation. We have shown that members of the intracellular fibroblast growth factors (FGF11-14) can regulate biophysical properties of sodium channels Nav1.2, Nav1.3, Nav1.6 and Nav1.7. We have also identified and characterized CAP-1A, a cytosolic protein which binds selectively to Nav1.8, among sodium channels, and induce a reduction in the current density in DRG neurons. CAP-1A also binds to clathrin, and may represent a new class of adaptor proteins which link sodium channels and clathrin and regulate sodium channel density by clathrin-mediated endocytosis. We have also reported that contactin, a GPI-anchored cell adhesion moleculae, regulates the sodium channel density of Nav1.3, Nav1.8 and Nav1.9, but not Nav1.6 and Nav1.7. We are continuing this line of investigation to investigate isoform-specific regulation of sodium channels by these, and other newly-discovered channel partners.
Phosphorylation of ion channels is a rapid and reversible that may significantly alter neuronal physiology, and phosphorylation of sodium channels is predicted to acutely regulate DRG neuron firing under pathological conditions. It is well-established that tissue and nerve injury cause the release of pro-nociceptive cytokines and growth factors, and alter ion conductances, leading to sensitization and hyperexcitability of nociceptive neurons. For example, TNF-a, a major pro-nociceptive cytokine and other pro-nociceptive factors including neurotrophic growth factor (NGF) activate downstream signaling pathways including the mitogen-activated protein kinase (MAPK) p38 (stress activated MAPK) and ERK1/2 (extracellular regulated kinase), a process which has been implicated in inducing hyperexcitability of injured DRG neurons. In fact, published work has shown that acute application of TNF-a to DRG neurons in culture increases the TTX-R current density in a p38-dependent manner. These results suggest that Nav1.8 current density may be regulated by activated p38. We have designed experiments to answer the question of whether the Nav1 channels are substrates for direct phosphorylation by activated p38 MAPK, or whether phosphorylation of the channel is necessary for the increase in current density. Additional studies aim to investigate the effect of p38 and ERK1/2 on different sodium channel isoforms that are co-expressed within the same neuron.
MAP kinases are proline-directed serine/threonine kinases which phosphorylate SP or TP sites in their substrates. We have identified several potential MAPK phosphoacceptor sites within cytoplasmic regions of sodium channels, suggesting that they may be MAPK substrates during pain signal transduction. Using in vitro kinase assays on individual channel fragments, we have now shown that loop 1 (L1), which joins domains I and II, carries a single p38 phosphorylation site in Nav1.6 and two sites in Nav1.8. Interestingly these phosphoacceptor sites are part of a PXSP motif, the minimal PXXP motif that binds proteins with SH3 domains. Also PXpSP motif is a potential binding motif of the type 4 WW domain of some ubiquitin ligases. Thus phosphorylation of these sites within Nav1.6 and Nav1.8 may act as a switch that permits binding or un-binding of channel partners, leading to regulation of these sodium channels. Indeed, we have shown that activation of p38 increases Nav1.8 current density while it reduces Nav1.6 current density. Our findings suggest that p38 directly modulates Nav1.6 and Nav1.8 in vivo, providing a rapid mechanism that can regulate nociceptive neuron excitability following injury. Ongoing studies aim to elucidate mechanisms that underlie the p38-mediated, isoform-specific regulation of sodium channels, and toinvestigate the effect of p38 and ERK1/2 on other sodium channels within DRG neurons.
While the role of sodium channels in acquired channelopathies leading to neuropathic pain is well-established, their role in inherited painful neuropathies has been less clear. However, the recent discovery of a monogenic link of Nav1.7 to pain disorders in humans provided a compelling case for establishing Nav1.7 as central to pain-signaling. Dominant gain-of-function mutations in SCN9A, the gene that encodes sodium channel Nav1.7, have been linked to two severe pain syndromes, inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD), while recessive loss-of-function mutations have been linked to complete insensitivity to pain (CIP). Electrophysiological characterization of these mutations has elucidated molecular basis for altered excitability of DRG neurons that express these mutant channels, thus establishing a mechanistic link to human pain. Additional genetic studies have validated to role of Nav1.8 and Nav1.9 in human pain disorders including small fiber neuropathy.
More recently we developed tools and methods to study the trafficking of Nav channels in live sensory neurons. These studies are beginning to describe dynamic regulation of Nav and other ion channels in sensory axons under conditions that mimic disease states.
Together, these data provide a compelling rationale to target peripheral Nav channels (Nav1.7, Nav1.8 and Nav1.9) for the development of new pain therapeutic agents which are predicted to have minimal side effects.
Molecular Biology; Nervous System Diseases; Neurology; Neurons; Sodium Channels; Voltage-Gated Sodium Channels