Sulayman D. Dib-Hajj PhD

Senior Research Scientist in Neurology

Research Interests

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

Current Projects

• Identification and characterization of mutations in peripheral voltage-gated sodium channels in patients with heritable pain disorders.
• Investigate the contribution of individual sodium channel isoforms to firing properties of pain-sensing neurons
• Identification and characterization of sodium channel partners that modulate channel function, protein stability and trafficking.
• Characterization of sequence motifs that regulate channel targeting to different neuronal compartments.

Research Summary

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.

Extensive Research Description

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. Recently, 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 Nav1.8 channel is a substrate 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 to investigate 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.

Pain in IEM is triggered by warmth and is localized to the distal extremities (feet and hands). IEM has been reported as early as 1-year-old (early-onset), in the second decade (delayed-onset), and in adults (adult-onset). Although mutations in Nav1.7 have been identified in patients with early- and delayed-onset IEM, the molecular basis of adult-onset IEM remains elusive. Our studies have shown that all IEM mutations in Nav1.7 characterized thus far shift activation voltage-dependence in a hyperpolarized direction, allowing the mutant channels to open (activate) in response to a weaker stimulus, compared to wild-type channels. At the cellular level, mutant Nav1.7 channels studied thus far lower threshold for single action potentials and increase firing frequency in DRG neurons, with all but one causing a depolarizing shift in resting potential. While pharmacotherapy for IEM is largely ineffective, cooling is effective in relieving pain although cold-induced injury is a serious outcome of this practice. A recent study from our group, however, identified a new IEM case in which patients responded favorably to the anticonvulsant drug carbamazepine. Surprisingly, while carbamazepine blocked mutant and wild-type channels equally, it induced a depolarizing shift of activation of the mutant channel, suggesting an allosteric effect of the drug on the mutant channel.

A second set of mutations of Nav1.7 underlies many of the PEPD cases. Severe perirectal pain in PEPD along with skin flushing can start in infancy and possibly in utero, but with no reported involvement of feet and hands. While seizures and cardiac symptoms may accompany PEPD, a link to the mutant Nav1.7 channel in sympathetic neurons, which normally express this channel, has not yet been established. As patients age, pain extends to ocular and maxillary/mandibular areas and is triggered by cold, eating or emotional state. Our group and others have shown that the PEPD mutations shift the voltage-dependence of steady-state fast-inactivation of Nav1.7 in a depolarizing direction and, depending upon the specific mutation, may make inactivation incomplete resulting in a persistent current. PEPD symptoms, in contrast to IEM, are well controlled by carbamazepine; impaired inactivation of PEPD Nav1.7 mutant channels could explain the favorable response of the patients to carbamazepine. At the cellular level, we have shown that PEPD Nav1.7 mutant channels lower threshold for single action potential and increase frequency of firing in DRG neurons, but without altering resting potential.

Together with the published data that have shown that Nav1.7-related CIP is not accompanied by other major sensory deficits, except impaired olfaction, motor or cognitive deficits, our data provide a compelling rationale to target Nav1.7 for the development of new pain therapeutic agents which are predicted to have minimal side effects.

Selected Publications