Hyperpolarization-activated, cyclic-nucleotide gated ion channels (HCN channels) are activated by membrane hyperpolarization and modulated by cyclic nucleotides. HCN channels are important to maintain the resting membrane potential and input resistance in neurons and have important physiological functions in the brain and heart. Four mammalian HCN isoforms, HCN1-4, and the isoform cloned from sea urchin, spHCN, have been extensively studied. Among these, only spHCN channel shows a voltage dependent inactivation. Previous studies have shown that the ligand binding in mHCN2 channel is activity dependent: cAMP binding increases along with channel opening or channels in the open state have higher binding affinity for cAMP. But to date, information pertaining to the ligand binding to an inactivated ion channel or desensitized receptor is lacking. To address this gap, we used fluorescently labelled cAMP analogues in conjunction with patch clamp fluorometry (PCF) to study the ligand binding to the spHCN channel in various conformational states. We show that inactivated spHCN channel shows reduced binding affinity for cAMP, compared to that of the closed or open channel. Parallelly, we noticed significant changes to channel function when a combination of laser and photosensitizer was used to study ligand binding. A reactive oxygen species called singlet oxygen has been confirmed to be the major player in this process. Both photo-dynamically generated and chemically generated singlet oxygen modifies spHCN channel by removing the inactivation. The effect of singlet oxygen on channel can be abolished by the mutation of a key histidine (H462) residue in the ion conducting pore. Taken together, these two projects expanded our understanding about the physicochemical nature of fluorophores from two aspects: (i) the release of photon as a valuable tool to study the conformational dynamics in proteins; (ii) the generation of singlet oxygen as an effective modulator of protein function.

Bilayer embedded proteins are affected by stress. This general affirmation is, in this thesis, embodied by two types of proteins: membrane spandex and voltage-gated sodium channels. In this work, we essentially explore, using methods from physics, the theoretical consequences of ideas drawn from experimental biology. Membrane spandex was postulated to exist and we study the theoretical implications and possible benefits for a cell to have such proteins embedded in its bilayer. There are no specific membrane spandex proteins, rather any protein with a transition involving a large enough area change between two non-conducting states could act as spandex. Bacterial cells have osmovalve channels which open at near-lytic tensions to protect themselves against rupture. Spandex expanding at tensions just below the osmovalves’ opening tension could relieve tension enough as to avoid costly accidental osmovalve opening due to transient bilayer tension excursions. Another possible role for spandex is a tension-damper: spandex could be used to maintain bilayer tension at a fixed level. This would be useful as many bilayer embedded channels are known to be modulated by tension. The Stress/shear experienced in traumatic brain injury cause an immediate (< 2 min) and irreversible TTX-sensitive rise in axonal calcium. In situ, this underlies an untreatable condition, diffuse axonal injury. TTX sensitivity indicates that leaky voltage-gated sodium (Nav) channels mediate the calcium increase. Wang et al. showed that the mammalian adult CNS Nav isoform, Nav1.6, expressed in Xenopus oocytes becomes “leaky” when subjected to bleb-inducing pipette aspiration. This “leaky” condition is caused by a hyperpolarized-shift (left-shift or towards lower potentials, typically 20 mV) of the kinetically coupled processes of activation and inactivation thus effectively degrading a well-confined window conductance into a TTX-sensitive Na leak. We propose experimental protocols to determine whether this left-shift is the result of an all-or-none or graded process and whether persistent Na currents are also left-shifted by trauma. We also use modeling to assess whether left-shifted Nav channel kinetics could lead to Na+ (and hence Ca2+ ) loading of axons and to study saltatory propagation after traumatizing a single node of Ranvier.

Bilayer embedded proteins are affected by stress. This general affirmation is, in this thesis, embodied by two types of proteins: membrane spandex and voltage-gated sodium channels. In this work, we essentially explore, using methods from physics, the theoretical consequences of ideas drawn from experimental biology. Membrane spandex was postulated to exist and we study the theoretical implications and possible benefits for a cell to have such proteins embedded in its bilayer. There are no specific membrane spandex proteins, rather any protein with a transition involving a large enough area change between two non-conducting states could act as spandex. Bacterial cells have osmovalve channels which open at near-lytic tensions to protect themselves against rupture. Spandex expanding at tensions just below the osmovalves’ opening tension could relieve tension enough as to avoid costly accidental osmovalve opening due to transient bilayer tension excursions. Another possible role for spandex is a tension-damper: spandex could be used to maintain bilayer tension at a fixed level. This would be useful as many bilayer embedded channels are known to be modulated by tension. The Stress/shear experienced in traumatic brain injury cause an immediate (< 2 min) and irreversible TTX-sensitive rise in axonal calcium. In situ, this underlies an untreatable condition, diffuse axonal injury. TTX sensitivity indicates that leaky voltage-gated sodium (Nav) channels mediate the calcium increase. Wang et al. showed that the mammalian adult CNS Nav isoform, Nav1.6, expressed in Xenopus oocytes becomes “leaky” when subjected to bleb-inducing pipette aspiration. This “leaky” condition is caused by a hyperpolarized-shift (left-shift or towards lower potentials, typically 20 mV) of the kinetically coupled processes of activation and inactivation thus effectively degrading a well-confined window conductance into a TTX-sensitive Na leak. We propose experimental protocols to determine whether this left-shift is the result of an all-or-none or graded process and whether persistent Na currents are also left-shifted by trauma. We also use modeling to assess whether left-shifted Nav channel kinetics could lead to Na+ (and hence Ca2+ ) loading of axons and to study saltatory propagation after traumatizing a single node of Ranvier.

Due to two different mutations in the gene that encodes the a1A subunit of voltage-activated CaV 2.1 calcium ion channels, the compound heterozygous tottering/leaner (tg/tgla) mouse exhibits numerous neurological deficits. Human disorders that arise from mutations in this voltage dependent calcium channel are familial hemiplegic migraine, episodic ataxia-2, and spinocerebellar ataxia 6. The tg/tgla mouse exhibits ataxia, movement disorders and memory impairment, suggesting that both the cerebellum and hippocampus are affected. To gain greater understanding of the many neurological abnormalities that are exhibited by the 90-120 day old tg/tgla mouse the following aspects were investigated: 1) the morphology of the cerebellum and hippocampus, 2) proliferation and death in cells of the hippocampal dentate gyrus and 3) changes in basic biochemical parameters in granule cells of the cerebellum and hippocampus. This study revealed no volume abnormalities within the hippocampus of the mutant mice, but a decrease in cell density with the pyramidal layer of CA3 and the hilus of the dentate gyrus. Cell size in the CA3 region was unaffected, but cell size in the hilus of the dentate gyrus did not exhibit the gender difference seen in the wild type mouse. The cerebellum showed a decrease in volume without any decrease in cerebellar cellular density. Cell proliferation and differentiation in the subgranular zone of the hippocampal dentate gyrus remained normal. This region also revealed a decrease in cell death in the tg/tgla mice. Basal intracellular calcium levels in granule cells show no difference within the hippocampus, but an increase in the tg/tgla male cerebellum compared to the wild type male cerebellum. There was no significant difference in granule cell mitochondrial membrane potential within the wild type and mutant animals in either the hippocampus or cerebellum. The rate of reactive oxygen species (ROS) production in granule cells revealed no variation within the hippocampus or cerebellum. The amount of ROS was decreased in cerebellar granule cells, but not granule cells of the hippocampus. Inducing ROS showed no alteration in production or amount of ROS produced in the hippocampus, but did show a ceiling in the amount of ROS produced, but not rate of production, in the cerebellum.

The fundamental attribute of neurons is their cellular electrical excitability, which is based on the expression of a plethora of ligand- and voltage-gated membrane channels that give rise to prominent membrane currents and membrane potential variations that represent the biophysical substrate underlying the transfer and integration of information at the cellular level. Dendrites have both an electrical and a biochemical character, which are closely linked. In contrast, glial cells are non-electrically excitable but nevertheless display a form of excitability that is based on variations of the Ca2+ concentration in the cytosol rather than electrical changes in the membrane. Cytoplasmic Ca2+ serves as an intracellular signal that is responsible for controlling a multitude of cellular processes. The key to this pleiotropic role is the complex spatiotemporal organization of the [Ca2+]i rise evoked by extracellular agonists, which allows selected effectors to be recruited and specific actions to be initiated. Ca2+ handling in the cell is maintained by operation of multiple mechanisms of Ca2+ influx, internal release, diffusion, buffering and extrusion. Ca2+ tends to be a rather parochial operator with a small radius of action from its point of entry at the cytoplasm resulting in the concept of microdomains. Dendritic Ca2+ signaling have been shown to be highly compartmentalized and astrocytic processes have been reported to be constituted by hundreds of microdomains that represent the elementary units of the astrocyte Ca2+ signal, from where it can eventually propagate to other regions of the cell. The astrocyte Ca2+ elevation may thus act as intra and intercellular signal that can propagate within and between astrocytes, signaling to different regions of the cell and to different cells. The spatio-temporal features of neuron-to-astrocyte communication, results from diverse neurotransmitters and signaling pathways that converge and cooperate to shape the Ca2+ signal in astrocytes. Alterations in Ca2+ homeostasis have been shown to be associated with major pathological conditions of the brain such as epilepsy, ischemia and neurodegenerative diseases. Although there are evidences of Ca2+ rise in hippocampal neurons in in vitro models of epilepsy (Pal et al., 1999; Limbrick et al., 2001), there is no information on the Ca2+ regulatory mechanisms operating in discrete compartments of the epileptic neuron following Ca2+ influx through voltage gated calcium channels (VGCCs). In the first part of the work, the spatial and temporal profiles of depolarization induced changes in the intracellular Ca2+ concentration in the dendrites of cultured autaptic hippocampal pyramidal neurons rendered epileptic experimentally have been addressed. Our in vitro epilepsy model consisted of hippocampal neurons in autaptic culture that were grown in the presence of kynurenate and high Mg2+, and subsequently washing the preparation free of the blockers. To understand the differences in Ca2+ handling mechanisms in different compartments of a control neuron and the kynurenate treated neuron, a combination of whole-cell patch-clamp recording and fast Ca2+ imaging methods using the Ca2+ indicator Oregon Green 488 BAPTA-1 was applied. All our analysis was focused on localized regions in the dendrite that showed pronounced Ca2+ transients upon activation of high voltage activated (HVA) Ca2+ channels. The spatial extent of Ca2+ signals suggested the presence of distinct dendritic compartments that respond to the depolarizing stimulus. Further, the local Ca2+ transients were observed even in the presence of NMDA and AMPA receptor antagonists, suggesting that the opening of VGCCs primarily triggered the local Ca2+ changes. The prominent changes in intracellular Ca2+ observed in these dendritic regions appear to be sites where Ca2+ evoked dendritic exocytosis (CEDE) takes place. Since cellular Ca2+ buffers determine the amplitude and diffusional spread of neuronal Ca2+ signals, quantitative estimates of the time-dependent spread of intracellular Ca2+ in the dendritic compartments in the control and treated neurons were done using image processing techniques. Physiological changes in Ca2+ channel functioning were also induced by kynurenate treatment and one such noticeable difference was the observation of Ca2+ dependent inactivation in the treated neurons. We provide evidences of localized Ca2+ changes in the dendrites of hippocampal neurons that are rendered epileptic by kynurenate treatment, suggesting that these sites are more vulnerable (Padmashri et al., 2006). This might contribute to the epileptiform activity by local changes in cellular and membrane properties in complex ways that remains to be clearly understood. Status Epilepticus (SE), stroke and traumatic brain injury are all associated with large increases in extracellular glutamate concentrations. The concentration of glutamate in the extracellular fluid is around 3-4 µM and astrocytes are primarily responsible for the uptake of glutamate at the synapses. The extracellular levels of glutamate has been shown to increase dramatically (16 fold) in human SE suggesting an important role of glutamate in the mechanism of seizure activity and seizure related brain damage (Carlson et al., 1992). Several other studies have also shown a persistent increase in extracellular glutamate concentration to potentially neurotoxic concentrations in the epileptogenic hippocampus (During and Spencer, 1993; Sherwin, 1999; Cavus et al., 2005). We addressed the problem related to the effects of prolonged glutamate pretreatment on Ca2+ signaling in an individual astrocyte and its adjoining astrocyte (astrocyte pair), rather than on a syncytium of astrocytes in culture. Individual astrocytes may have functional domains that respond to an agonist through distinct receptor signaling systems. These are difficult to observe in studies that are done on glial syncytium because of spatial limits of image capture. This was examined with simultaneous somatic patch-pipette recording of a single astrocyte to evoke voltage-gated calcium currents, and Ca2+ imaging using the Ca2+ indicator Oregon Green 488 BAPTA-1 to identify the Ca2+ microdomains. Transient Ca2+ changes locked to the depolarization were observed in certain compartments in the astrocyte processes of the depolarized astrocyte and the responses were more pronounced in the adjoining astrocyte of the astrocyte pair. The Ca2+ transient amplitudes were enhanced on pretreatment of cells with glutamate (500 µM for 20 minutes). Estimation of local Ca2+ diffusion coefficients in the astrocytic processes indicated higher values in the adjoining astrocyte of the glutamate pretreated group. In order to understand the underlying mechanisms, we performed the experiments in the presence of different blockers for the metabotropic glutamate receptor, inositol 1,4,5 triphosphate (IP3) receptors and gap junctions. Ca2+ transients recorded on pretreatment of cells with glutamate showed attenuated responses in the presence of the metabotropic glutamate receptor (mGluR) antagonist α-Methyl(4-Carboxy-Phenyl) Glycine (MCPG). Intracellular heparin (an antagonist of IP3 receptor) introduced in the depolarized astrocyte did not affect the Ca2+ transients in the heparin loaded astrocyte, but attenuated the [Ca2+]i responses in the adjoining astrocyte suggesting that IP3 may be the transfer signal. The uncoupling agent 1-Octanol attenuated the [Ca2+]i responses in the adjoining cell of the astrocyte pair in both the control and glutamate pretreated astrocytes indicating the role of gap junctional communication. The findings of [Ca2+]i responses within discrete regions of astrocytic processes suggest that astrocytes may be comprised of microdomains whose properties are altered by glutamate pretreatment. The data also indicates that glutamate induced alterations in Ca2+ signaling in the astrocyte pair may be mediated through phospholipase C (PLC), IP3, internal Ca2+ stores, VGCCs and gap junction channels (Padmashri and Sikdar, 2006). Neuronal (EAAC-1) and glial (GLT-1 and GLAST) glutamate transporters facilitate glutamate reuptake after synaptic release. Transgenic mice with GLT-1 knockout display spontaneous epileptic activity (Tanaka et al., 1997) and loss of glial glutamate transporters using chronic antisense nucleotide administration was reported to result in elevated extracellular glutamate levels and neurodegeneration characteristic of excitotoxity (Rothstein et al., 1996). Dysfunction of glutamate transporters and the resulting increase of glutamate have been speculated to play an important role in infantile epilepsies (Demarque et al., 2004). We examined the effects of pretreatment with glutamate in the presence of the glutamate transport inhibitor threo-β-hydroxy-aspartate (TBHA) and in Na+-free extracellular medium to understand whether this resulted in any alteration in the astrocytic intracellular Ca2+ dynamics following activation of voltage gated calcium channels. The Ca2+ responses were found to be attenuated in both the cases indicating that the elevated levels of extracellular glutamate due to blockade of glutamate transporters may influence the responses mediated by the astrocytic glutamate receptors. Our studies indicate that the heightened extracellular glutamate concentration is not gliotoxic in our experimental system, although it may have a profound effect on altering the activity of surrounding neurons which was not addressed in the present work. Several studies have indicated that neurons control the level of gap junction mediated communication between astrocytes (Giaume and McCarthy, 1996; Rouach et al, 2000). All our earlier studies were done on process bearing astrocytes that were co-cultured with neurons. We have addressed the question as to whether the spatio-temporal changes in [Ca2+]i in astrocyte pairs differ if the astrocytes are cultured in the absence of neurons. The results indicate that there is indeed a significant reduction in the responses that are evoked in response to the depolarization pulse in the adjoining cell of the astrocyte pair. These experiments demonstrate that neurons in the cocultures may selectively enhance the Ca2+ responses possibly by increasing the coupling between the two cells.

No description available.

Les syndromes myotoniques congénitaux atypiques dus à des mutations du canal sodé voltage-dépendant Nav1.4 se distinguent des myotonies congénitales classiques (canal chlore ClC-1) par la présence de traits atypiques incluant des myotonies douloureuses aggravées au froid et à l’ingestion de potassium. La caractérisation clinique et moléculaire de plusieurs familles atteintes de ces conditions rares dans la région du Saguenay-Lac-St-Jean nous a permis de découvrir une nouvelle mutation SCN4A à effet fondateur causant un phénotype de myotonies douloureuses aggravées au froid, parfois accompagné de phénomènes dystrophiques ou paralytiques. L’ampleur de notre cohorte nous permet de commenter sur l’hétérogénité phénotypique observée, sur les traits caractéristiques des syndromes associés au gène SCN4A, sur les implications physiologiques probables d’une telle mutation ainsi que sur les facteurs modulant le phénotype observé. Enfin, notre étude nous permet de souligner l’importance du dépistage familial systématique afin de prévenir les complications anesthésiques potentielles associées à ces conditions. / Congenital myotonic syndromes due to mutations of the voltage-gated sodium channel Nav1.4 differ from those due to mutations of the chloride channel CLC-1 as they tend to present atypical traits including painful myotonias and aggravation of symptoms with cold and potassium ingestion. Indeed, after completing the clinical and molecular characterization of a large cohort of patients affected with these rare conditions in the Saguenay Lac-St-Jean area, we were able to describe a new founder SCN4A mutation presenting with painful cold-induced myotonias and occasional dystrophic and paralytic episodes. Our study illustrates the wide phenotypic variability and the typical traits of SCN4A mutations. In addition, we were able to speculate on the probable physiological consequences of such mutations. Finally, we conclude by stressing the importance of familial screening in order to reduce the incidence of anesthetic complications associated with these conditions.

<p>The capacity of marine organisms to adapt to natural and anthropogenic stressors is an integral component of ocean health. Harmful algal blooms (HABs), which are one of many growing threats in coastal marine ecosystems, represent a historically present natural stressor that has recently intensified and expanded in geographic distribution partially due to anthropogenic activities. In the Gulf of Mexico, HABs of <italic>Karenia brevis</italic> occur almost annually and produce neurotoxic brevetoxins that have been associated with large-scale mortality events of many marine species, including the common bottlenose dolphin (<italic>Tursiops truncatus</italic>). The factors resulting in large-scale dolphin mortality associated with HABs are not well understood, particularly in regards to the seemingly different impacts of HABs in geographically disjunct dolphin populations. My dissertation investigates a genetic basis for resistance to HABs in bottlenose dolphins in central-west Florida and the Florida Panhandle. I used both genome-wide and candidate gene approaches to analyze genetic variation in dolphins that died putatively due to brevetoxicosis and live dolphins from the same geographic areas that survived HAB events. Using restriction site-associated DNA sequencing, I identified genetic variation that suggested both a common genetic basis for resistance to HABs in bottlenose dolphins across the Gulf coast of Florida and regionally specific resistance. Many candidate genes involved in the immune, nervous, and detoxification systems were found in close genomic proximity to survival-associated polymorphisms throughout the bottlenose dolphin genome. I further investigated two groups of candidate genes, nine voltage-gated sodium channel genes selected because of their putative role in brevetoxin binding and four major histocompatibility complex (MHC) loci selected because of their genomic proximity to a polymorphism exhibiting a strong association with survival. I found little variation in the sodium channel genes and conclude that bottlenose dolphins have not evolved resistance to HABs via mutations in the toxin binding site. The immunologically relevant MHC loci were highly variable and exhibited patterns of genetic differentiation among geographic regions that differed from neutral loci; however, genetic variation at the MHC also could not fully explain variation in survival of bottlenose dolphins exposed to HABs. In my final chapter, I consider the advantages and drawbacks of the genome-wide approach in comparison to a candidate gene approach and, as laid out in my dissertation, I recommend using both complementary approaches in future investigations of adaptation in genome-enabled non-model organisms.</p> / Dissertation

Calmodulin (CaM) is a ubiquitous calcium-binding protein responsible for the binding and activation of a vast number of enzymes and signaling pathways. It contains two lobes that bind two calcium ions each, separated by a flexible central linker. This structural flexibility allows CaM to bind and regulate a large number of diverse protein targets within the cell in response to Ca2+ gradients. Voltage gated calcium channels (CaVs), as main sources of extracellular Ca2+, are crucial for a number of physiological processes, from muscle contraction to neurotransmission and endocrine function. These large transmembrane proteins open in response to membrane depolarization and allow gated entry of Ca2+ ions into the cytoplasm. Their regulation is currently the subject of intense investigation due to its pharmacological and scientific importance. CaM has been previously shown to pre-associate and act as a potent inhibitor of one class of high-voltage activated (HVA) channels called L-type channels via its interaction with their C-terminal cytoplasmic region. This interaction is primarily mediated by a conserved CaM-binding motif called the ‘IQ’ motif (for conserved isoleucine and glutamine residues), although the exact molecular details of its involvement in inactivation are currently unclear. Elucidation of these details was the primary objective of this dissertation. Recently, a novel sequence motif within this channel called ‘NSCaTE’ (N-terminal spatial calcium transforming element) has been described as an important contributor to calcium-dependent inactivation (CDI) of L-type channels. It was presumed to be unique to vertebrates, but we also show its conservation in a distantly related L-type channel homolog of Lymnaea stagnalis (pond snail). The interaction of CaM with a number of peptides representing the different regulatory motifs (IQ and NSCaTE) for both mammalian and snail isoforms was characterized in an attempt to better understand their role in CDI. Biophysical work with peptides as well as electrophysiology recordings with an N-terminal truncation mutant of Lymnaea CaV1 homolog were performed to expand our understanding of how the interplay between these channel elements might occur. In brief, the most striking feature of the interaction concerns the strong evidence for a CaM-mediated bridge between the N- and C-terminal elements of L-type channels. Further investigation of the CaM interaction with both IQ and NSCaTE peptides using Ca2+-deficient CaM mutants reveals a preference of both peptides for the Ca2+-C-lobe of CaM, and a much higher affinity of CaM for the IQ peptide, suggesting that the N-lobe of CaM is the main interaction responsible for the physiological effects of NSCaTE. These results are consistent with our electrophysiology findings that reveal a distinct buffer-sensitive CDI in wild type LCaV1 that can be abolished by the N-terminal truncation spanning the NSCaTE region. In addition to L-type channels, CaM has also been shown to have an indirect role in the regulation of low-voltage activated (LVA) or T-type channels (CaV3.x), via their phosphorylation by CaM-dependent protein kinase II (CaMKII). Using a primary sequence scanning algorithm, a CaM-binding site was predicted in a cytoplasmic region of these channels that was also previously shown to be important in channel gating. Biophysical experiments with synthetic peptides spanning this gating brake region from the three human and the single Lymnaea isoform strongly suggest that there is a novel, bona fide CaM interaction in this channel region, and also hint that this interaction may be a Ca2+-dependent switch of some sort. The results confirm a possible new role for CaM in the direct regulation of these channels, although the exact mechanism remains to be elucidated.

Bilayer embedded proteins are affected by stress. This general affirmation is, in this thesis, embodied by two types of proteins: membrane spandex and voltage-gated sodium channels. In this work, we essentially explore, using methods from physics, the theoretical consequences of ideas drawn from experimental biology. Membrane spandex was postulated to exist and we study the theoretical implications and possible benefits for a cell to have such proteins embedded in its bilayer. There are no specific membrane spandex proteins, rather any protein with a transition involving a large enough area change between two non-conducting states could act as spandex. Bacterial cells have osmovalve channels which open at near-lytic tensions to protect themselves against rupture. Spandex expanding at tensions just below the osmovalves’ opening tension could relieve tension enough as to avoid costly accidental osmovalve opening due to transient bilayer tension excursions. Another possible role for spandex is a tension-damper: spandex could be used to maintain bilayer tension at a fixed level. This would be useful as many bilayer embedded channels are known to be modulated by tension. The Stress/shear experienced in traumatic brain injury cause an immediate (< 2 min) and irreversible TTX-sensitive rise in axonal calcium. In situ, this underlies an untreatable condition, diffuse axonal injury. TTX sensitivity indicates that leaky voltage-gated sodium (Nav) channels mediate the calcium increase. Wang et al. showed that the mammalian adult CNS Nav isoform, Nav1.6, expressed in Xenopus oocytes becomes “leaky” when subjected to bleb-inducing pipette aspiration. This “leaky” condition is caused by a hyperpolarized-shift (left-shift or towards lower potentials, typically 20 mV) of the kinetically coupled processes of activation and inactivation thus effectively degrading a well-confined window conductance into a TTX-sensitive Na leak. We propose experimental protocols to determine whether this left-shift is the result of an all-or-none or graded process and whether persistent Na currents are also left-shifted by trauma. We also use modeling to assess whether left-shifted Nav channel kinetics could lead to Na+ (and hence Ca2+ ) loading of axons and to study saltatory propagation after traumatizing a single node of Ranvier.