Neuropathic pain is a complex clinical syndrome characterized by increased sensitivity to thermal and/or mechanical stimuli that may or may not be accompanied by the phenomenon of spontaneous or aberrant pain sensations. <p>Over the past decade, the mechanisms underlying the behavioral manifestations of inflammatory neuropathic pain have become more clearly elucidated. These include the involvement of: 1) transient receptor potential vanilloid receptor 1 (TRPV1) in the generation of thermal hyperalgesia; 2) acid sensing ion channel 3 (ASIC3) in some aspects of the development/maintenance of mechanical hypersensitivity; 3) the tetrodotoxin resistant sodium channels Nav1.8 and Nav1.9 in both hyperalgesia and spontaneous pain; and 4) activation of the MAP Kinases p38 and ERK1/2 in the regulation of expression of the aforementioned molecules.<p>Interestingly, it is the pro-inflammatory neurotrophin nerve growth factor (NGF) that is the common link between all of these mediators of neuropathic pain. Increased availability of NGF under conditions of inflammation has been shown to drive increased expression/upregulation of TRPV1, ASIC3, Nav1.8 and Nav1.9, as well as phospho-p38 and phospho-ERK1/2.<p>Evidence presented here continues to support a role for neurotrophin-3 (NT-3) in antagonizing the effects of increased NGF on trkA signaling, neuropathic pain behaviors and some of the molecules associated with the generation of such behaviors.<p>More specifically, the work culminating in this thesis demonstrates a novel role for NT-3 in negative modulation of TRPV1, ASIC3, Nav1.8 and Nav1.9, as well as phospho-p38 expression in response to the chronic constriction injury model of neuropathic pain. Finally, initial insights into how this negative regulation of these nociceptive markers might occur is elucidated in studies demonstrating that NT-3 differentially affects levels of the key signaling molecule phospho-ERK in trkA-positive versus trkC-positive neurons in naïve dorsal root ganglia (DRG).

Neuropathic pain is a complex clinical syndrome characterized by increased sensitivity to thermal and/or mechanical stimuli that may or may not be accompanied by the phenomenon of spontaneous or aberrant pain sensations. <p>Over the past decade, the mechanisms underlying the behavioral manifestations of inflammatory neuropathic pain have become more clearly elucidated. These include the involvement of: 1) transient receptor potential vanilloid receptor 1 (TRPV1) in the generation of thermal hyperalgesia; 2) acid sensing ion channel 3 (ASIC3) in some aspects of the development/maintenance of mechanical hypersensitivity; 3) the tetrodotoxin resistant sodium channels Nav1.8 and Nav1.9 in both hyperalgesia and spontaneous pain; and 4) activation of the MAP Kinases p38 and ERK1/2 in the regulation of expression of the aforementioned molecules.<p>Interestingly, it is the pro-inflammatory neurotrophin nerve growth factor (NGF) that is the common link between all of these mediators of neuropathic pain. Increased availability of NGF under conditions of inflammation has been shown to drive increased expression/upregulation of TRPV1, ASIC3, Nav1.8 and Nav1.9, as well as phospho-p38 and phospho-ERK1/2.<p>Evidence presented here continues to support a role for neurotrophin-3 (NT-3) in antagonizing the effects of increased NGF on trkA signaling, neuropathic pain behaviors and some of the molecules associated with the generation of such behaviors.<p>More specifically, the work culminating in this thesis demonstrates a novel role for NT-3 in negative modulation of TRPV1, ASIC3, Nav1.8 and Nav1.9, as well as phospho-p38 expression in response to the chronic constriction injury model of neuropathic pain. Finally, initial insights into how this negative regulation of these nociceptive markers might occur is elucidated in studies demonstrating that NT-3 differentially affects levels of the key signaling molecule phospho-ERK in trkA-positive versus trkC-positive neurons in naïve dorsal root ganglia (DRG).

Thesis (Ph. D.)--University of Hawaii at Manoa, 1990. / Includes bibliographical references (leaves 135-153) / Microfiche. / xi, 153 leaves, bound ill. 29 cm

Thesis (Ph. D. in Pharmacology)--Vanderbilt University, Dec. 2008. / Title from title screen. Includes bibliographical references.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Genetic mutations perturbing inactivation of human cardiac voltage-gated sodium channels (VGSCs), specifically Nav1.5, can cause long QT syndrome type 3 (LQT3). LQT3 is a cardiac disorder in which patients experience syncope and ventricular tachyarrhythmia, and are thus predisposed to sudden cardiac death. Deeper understanding of the structural dynamics of VGSC inactivation is needed to inform treatment of and drug design for potentially life-threatening arrhythmias. A well supported hypothesis is that the VGSC inactivated state is stabilized by hydrophobic interactions between the inactivation gate and an unknown binding site potentially involving the underside of the channel pore, C-terminus (C-T), and auxiliary proteins. Despite advances in biophysical and structural characterization of VGSCs, the specific molecular components and timing of their interactions within the inactivation complex remain unclear. Fluorescence imaging approaches that connect conformational change with channel function in mammalian cells could provide much needed mechanistic insight on the structural dynamics of the VGSC inactivation complex. This thesis describes the development of a site-specific fluorescent unnatural amino acid (UAA) labeling and spectral imaging methodology to probe the cardiac VGSC, Nav1.5, inactivation complex in live mammalian cells. First, UAA mutagenesis experiments were performed to validate orthogonal synthetase-tRNA (aaRS-tRNA) technology for fluorescent labeling of intracellular and membrane proteins in mammalian cells. Next, towards investigating conformational dynamics and intramolecular interactions related to inactivation, the Nav1.5 inactivation gate was labeled with a single environmentally sensitive fluorescent UAA L-anap. While the function of L-anap labeled channels was altered, their function remained within pathophysiological range. Then, imaging of L-anap labeled Nav1.5 in mammalian cells afforded characterization of unique L-anap spectra at different sites in the inactivation gate. Finally, using potassium-depolarization (K-depolarization) as rough means of voltage control, L-anap spectral shifts demonstrated conformational changes between the closed and open-inactivated states, which depended on the presence of the distal C-T (DCT). Site-specific L-anap labeling of the inactivation gate combined with spectral imaging and K-depolarization affords a general imaging assay to directly monitor conformational rearrangements of the Nav1.5 inactivation gate in channels expressed in live mammalian cells. While interactions with the DCT are specifically probed, this general assay provides an opportunity to bring necessary unification of ideas about VGSC inactivation, as well as insight on outstanding questions of VGSC regulation.

Proper functioning of the voltage gated sodium channel, NaV1.5, is essential for maintenance of normal cardiac electrophysiological properties. Changes to the biophysical properties of sodium channels can take many forms and can affect the peak component of current carried during phase zero of the action potential; the “persistent” or “late” current component conducted during the repolarizing phases of the action potential; the availability of the channel as seen by changes in window current; and the kinetics of channel transitions between closed, opened and inactivated states. Mutations in NaV1.5 that alter these parameters of channel function are linked to a number of cardiac diseases including arrhythmias such as atrial fibrillation. In addition, mutations in many of the auxiliary proteins that form part of the sodium channel macromolecular complex have likewise been associated with diseases of the heart. Mutations in regions of the sodium channel responsible for interactions with these auxiliary proteins have also been linked to various dysfunctional cardiac states. Indeed, a large number of disease causing mutations are localized to the C-terminal domain of NaV1.5, a hotspot for interacting proteins. Using a transgenic mouse model, we show that expression of a mutant sodium channel with gain-of-function properties conferring increased persistent current, is sufficient to cause both structural and electrophysiological abnormalities in the heart driving the development of spontaneous and prolonged episodes of atrial fibrillation. The sustained and spontaneous atrial arrhythmias, an unusual if not unique phenotype in mice, enabled explorations of mechanisms of atrial fibrillation using in vivo (telemetry), ex vivo (optical voltage mapping), and in vitro (cellular electrophysiology) techniques. Since persistent sodium current was the driver of the structural and electrophysiological abnormalities leading to atrial fibrillation, we subsequently pursued studies exploring the mechanisms of persistent sodium current. Prior work of heterologously expressed sodium channels identified calmodulin as a regulator of persistent current. Mutation of the calmodulin binding site in the C-terminus of the cardiac sodium channel caused increased persistent current when the channel was expressed heterologously. The role of calmodulin in the regulation of the sodium channel in cardiomyocytes has not been definitively determined. We created transgenic mice expressing human sodium channels harboring a mutation of the calmodulin binding site. Using whole cell patch clamping, we found, in contrast to previously reported findings, that ablation of the calmodulin binding site did not induce increased persistent sodium current. Instead, loss of calmodulin binding stabilized the inactivated state by shifting the V50 for steady-state inactivation in the hyperpolarizing direction. Furthermore, loss of calmodulin binding sped up the transition to the inactivated state demonstrated by a significantly shortened tau of inactivation. In contrast to studies performed in heterologous expression systems, our findings thus suggest that in heart cells, calmodulin binding increases availability, similar to its role in regulating NaV1.4 channels. The studies were then expanded to explore the role of other interacting proteins, fibroblast growth factor (FGF) homologous factors (FHF), in the presence and absence of calmodulin binding. Using whole cell patch clamping, we found that a mutation (H1849R) of the sodium channel causing decreased FHF binding affinity leads to a rightward shift in steady-state inactivation and a slowed rate of inactivation of INa. A third mutant channel, with concurrent decreased FHF and calmodulin binding affinity similarly results in a rightward shift in steady-state inactivation suggesting a dominant effect of the H1849R mutation. Persistent current was not elevated in either of these mutant channels. Importantly, the methodology that we report enables us and other groups to carry out studies of human sodium channels in the native environment of NaV1.5. Our investigation into calmodulin’s role, which yielded conclusions distinct from prior findings in heterologous expression systems, demonstrates the value of this approach.

The epithelial sodium channel (ENaC) is the central component of the sodium absorption pathway in epithelia. It is critical for sodium homeostasis and blood pressure control, which is demonstrated by rare genetic disorders such as Liddle�s syndrome and pseudohypoaldosteronism type I, that are associated with hyper- and hypotension, respectively. ENaC is mainly regulated by mechanisms that control the expression of active channels at the cell surface. Ubiquitin ligases of the Nedd4-like family, such as Nedd4 and Nedd4-2 decrease epithelial sodium absorption by binding to and targeting ENaC for endocytosis and degradation. This is most likely achieved by catalyzing the ubiquitination of ENaC. Conversely the serum- and glucocorticoid regulated kinase (SGK) increases ENaC activity. This effect is partly mediated by the interaction of SGK with the ubiquitin ligases Nedd4 and Nedd4-2. SGK is able to bind to both Nedd4 and Nedd4-2, however only Nedd4-2 is phosphorylated by SGK. The phosphorylation of Nedd4-2 inhibits its interaction with ENaC, thus reducing ENaC ubiquitination, thereby increasing surface expression and sodium absorption. Nedd4-like proteins interact with ENaC via their WW-domains. These domains bind PY-motifs (PPXY) present in ENaC subunits. Nedd4 and Nedd4-2 both have four highly similar WW-domains. Previous studies have shown that interaction between Nedd4 and ENaC is mainly mediated by WW-domain 3. SGK also has a PY-motif; therefore it was analyzed whether the WW-domains of Nedd4 and Nedd4-2 mediate binding to SGK. Here, it is shown that single or tandem WW-domains of Nedd4 and Nedd4-2 mediate binding to SGK and that, despite their high similarity, different WW-domains of Nedd4 and Nedd4-2 are involved. These data also suggest that WW-domains 2 and 3 of Nedd4-2 mediate the interaction with SGK in a concerted manner, and that in vitro the phosphorylation of SGK at serine residue 422 increases its affinity for the WW-domains of Nedd4-2. The stimulatory effect of SGK on ENaC activity is partly mediated via Nedd4-2 and will decrease if competition between Nedd4 and Nedd4-2 for binding to SGK occurs. Here it is shown that Nedd4 and Nedd4-2 are located in the same subcellular compartment and that they compete for binding to SGK. Besides its function in the proteasomal degradation pathway ubiquitination is involved in the regulation of membrane protein trafficking, including their endocytosis. ENaC was shown previously to be ubiquitinated. Here, we provide evidence that ENaC can be ubiquitinated differentially depending on its cellular location. Channels residing in the plasma membrane are multiubiquitinated and we suggest that this serves as an internalization signal for ENaC and a control for further trafficking. Cytosolic ENaC is mainly polyubiquitinated, and therefore probably targeted for proteasomal degradation. However, mono- and multiubiquitination of ENaC located within the cytosol is very likely to occur as well. In addition, it is shown that both proteasomal and lysosomal pathways are involved in the regulation of ENaC.

Hyperkalemic periodic paralysis (HyperKPP) is an autosomal dominant human skeletal muscle channelopathy that causes periods of myotonic discharge and periodic paralysis due to defective Nav1.4 sodium channels. Patients are asymptomatic at birth, attacks become short and frequent during childhood, and more severe during adolescence. Since the Nav1.4 content in the cell membrane is relatively constant during childhood, it was hypothesized that some symptoms start with the defective Nav1.4 channels, while other symptoms start after some changes occur in gene expression affecting other membrane channel content and/or activity. To test the hypothesis, the contractile characteristics of EDL and soleus muscles from HyperKPP mice from the age of 0.5 to 12 months were tested in vitro. For both EDL and soleus, contractile defects, including low force generation, instability and large unstimulated force were observed by two weeks of age. With aging, the defects did not worsen, but muscles actually showed some improvement. Considering that Nav1.4 protein content reaches maximum at three weeks of age, the data suggests that HyperKPP symptoms are solely due to the defective Nav1.4 channels.

Hyperkalemic periodic paralysis (HyperKPP) is an autosomal dominant human skeletal muscle channelopathy that causes periods of myotonic discharge and periodic paralysis due to defective Nav1.4 sodium channels. Patients are asymptomatic at birth, attacks become short and frequent during childhood, and more severe during adolescence. Since the Nav1.4 content in the cell membrane is relatively constant during childhood, it was hypothesized that some symptoms start with the defective Nav1.4 channels, while other symptoms start after some changes occur in gene expression affecting other membrane channel content and/or activity. To test the hypothesis, the contractile characteristics of EDL and soleus muscles from HyperKPP mice from the age of 0.5 to 12 months were tested in vitro. For both EDL and soleus, contractile defects, including low force generation, instability and large unstimulated force were observed by two weeks of age. With aging, the defects did not worsen, but muscles actually showed some improvement. Considering that Nav1.4 protein content reaches maximum at three weeks of age, the data suggests that HyperKPP symptoms are solely due to the defective Nav1.4 channels.