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![Functional Regulation of Ion Channels in Dorsal Root Ganglion Neurons by Magi-1: Implications in Pain Signaling için kapak resmi Functional Regulation of Ion Channels in Dorsal Root Ganglion Neurons by Magi-1: Implications in Pain Signaling için kapak resmi](/client/assets/d79c3e4af2b6d196/ctx/images/no_image.png)
Functional Regulation of Ion Channels in Dorsal Root Ganglion Neurons by Magi-1: Implications in Pain Signaling
Başlık:
Functional Regulation of Ion Channels in Dorsal Root Ganglion Neurons by Magi-1: Implications in Pain Signaling
Yazar:
Pryce, Kerri D., author.
ISBN:
9780438050013
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (134 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-10(E), Section: B.
Advisors: Arindam Bhattacharjee Committee members: David Dietz; Jun-xu Li; Malcolm Slaughter.
Özet:
Persistent pain leads to alterations in ion channel expression in neurons along the pain-axis. Ion channels provide neurons with a mechanism to detect and respond to changes in their environment. Adaptive changes in ion channel expression are major driving factors for the increased neuronal excitability seen in pain states. Understanding the molecular mechanisms underlying alterations to ion channel expression in pain-sensing neurons (nociceptors) during pain signaling is essential for the development of novel analgesics. Two critical subsets of ion channels necessary for the detection and transmission of painful stimuli are the voltage-gated sodium channels (Navs) and sodium-activated potassium channels (KNa) family. This study will focus on Nav1.8, which is preferentially expressed in pain-sensing Dorsal Root Ganglion (DRG) neurons along with the KNa channel subunits called Slack and Slick. The membrane expression of Nav1.8 channels (promotes nociceptor excitability) and KNa channels (decrease nociceptor excitability) are altered during pain signaling, the expression of Nav1.8 channels increase whereas KNa channels decrease. Studies have suggested that Navs are functionally coupled to the KNa channel Slack; however, the molecular machinery responsible for this coupling is relatively unknown. In this study, I have identified the Membrane-associated Guanylate Kinase with PDZ and WW containing domain protein 1 (Magi-1), an understudied scaffolding protein, as a novel interacting partner for the KNa channels Slack/Slick, and Nav1.8. To determine whether Magi-1 is essential for DRG neuronal excitability, siRNA silencing strategies were used to knockdown Magi-1 expression in cultured embryonic DRG neurons. A significant decrease in neuronal excitability was observed after Magi-1 knockdown as measured by the ability of the neurons to fire action potentials (AP). In addition, Nav1.8, and Slack KNa channel membrane expression was significantly decreased after Magi-1 knockdown. To determine the physiological role of Magi-1 in vivo, I first characterized the expression of Magi-1 in primary sensory neurons of the DRG. Abundant expression of Magi-1 was observed, especially in small- and medium-sized DRG neurons. The highest expression of Magi-1 immunoreactivity in the spinal cord was detected in the superficial lamina of the spinal dorsal horn were nociceptive DRG neurons project their afferents. Next, to identify a specific role of Magi-1 in pain, I performed a novel spinal nerve injection technique of shRNAs targeted against Magi-1, a method I modified for mice, achieving long-lasting Magi-1 knockdown in DRG neurons. To this extent, profound alterations in thermal nociception and inflammatory pain behaviors were observed. Moreover, after Magi-1 knockdown, an almost complete loss of Nav1.8 expression was detected, indicating that Magi-1 is an obligate protein interacting partner for this channel. In a neuropathic pain model, significant upregulation of Magi-1 protein expression was seen in the sciatic nerve and DRGs. These results suggest that Magi-1 is critical to the development and maintenance of both inflammatory and neuropathic pain.
Nav1.8 channels are targeted for proteasome degradation through binding and subsequent ubiquitination by the Neural Precursor Cell Expressed, Developmentally Down-Regulated 4 (Nedd4) family of E-3 ubiquitin ligases, specifically Nedd4-2. Nedd4-2 interacts with Nav1.8 through WW (Tryptophan- Tryptophan ) domain-binding to an evolutionarily conserved PY motif (PPXY ) in the C-terminus of Nav1.8 channels. Similarly, Magi-1 proteins contained two WW domains that have approximately 60% homology to Nedd-2 WW domains. Furthermore, the Magi family of proteins have been previously shown to protect proteins from Nedd4-2 mediated ubiquitination and subsequent degradation. Therefore, to further investigate the interaction between Magi-1/Nav1.8 we designed a competing, cell-penetrating peptide mimetic derived from the Nav1.8 PY motif. Incubation of competing peptide mimetic designated PY peptide, decreased sodium currents, reduced Nav1.8 protein expression, and produced hypoexcitability in cultured DRG neurons. Remarkably, a phosphorylated variant (phospho-PY peptide) of the very same peptide caused an opposing increase in Nav1.8 surface expression and repetitive action potential firing. To next assess the role of our competitor peptides in a model of acute inflammatory pain the formalin assay was performed. To this extent, both peptides were shown to alter pain responses in mice, the PY peptide decreased pain responses, whereas the phosphorylated increased pain responses when compared to controls.
In summary, this study provides evidence that Magi-1 is widely expressed in sensory neurons regulating ion channel trafficking, stability and plasma membrane expression. Furthermore, Magi-1 deficiency decreased pain responses through decreases in membrane scaffolding of Nav1.8 and Slack KNa channels. Here we demonstrate the ability to modulate Nav1.8 plasma membrane expression and protein stability, and Slack channel membrane expression using peptide mimetics to compete with PY motif binding and PDZ binding respectively. These effects may have therapeutic relevance given the established role of Nav1.8 and the Slack KNa channels in clinical pathologies such as chronic pain and epilepsy. Overall, this thesis provides evidence suggesting that Magi-1 is an essential scaffold for ion transport in DRG neurons and is a central player in pain.
Notlar:
School code: 0656
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(682045.1) | 682045-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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