Molecular mechanisms of glycosylation of Cav3.2 channels in pain pathway

疼痛通路中Cav3.2通道糖基化的分子机制

• 批准号: 9471872
• 负责人: Vesna Jevtovic-Todorovic
• 金额: $ 36.97万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9471872
• 关键词: Acute; Adverse effects; Afferent Neurons; Animal Model; Animals; Asparagine; Ataxia; Axon; Calcium Channel; Canis familiaris; Capsaicin; Cells; Characteristics; Chronic Disease; Clinical; Complication; Constipation; Cream; Data; Development; Diabetes Mellitus; Diabetic Neuralgia; Diabetic Neuropathies; Diabetic mouse; Disease; Dizziness; Drug abuse; Electrophysiology (science); Euphoria; Functional disorder; Genetic; Human; Hyperalgesia; Hyperglycemia; Impaired cognition; In Vitro; Insulin-Dependent Diabetes Mellitus; Intractable Pain; Kinetics; Knowledge; Lead; Leptin; Lidocaine; Measures; Mechanics; Medical; Membrane; Modality; Modeling; Molecular; Morbid Obesity; Mus; Nerve; Neuraminidase inhibitor; Neurons; Nociception; Nociceptors; Non-Insulin-Dependent Diabetes Mellitus; Numbness; Obese Mice; Opioid; Optical Methods; Oral; Outcome; Pain; Pain management; Pathogenesis; Pathologic; Pathway interactions; Patients; Peripheral; Peripheral Nerves; Pharmaceutical Preparations; Pharmacology Study; Phenotype; Play; Post-Translational Protein Processing; Posterior Horn Cells; Preparation; Protein Conformation; Protein Isoforms; Protein Secretion; Proteins; Public Health; Quality of life; Recombinants; Regulation; Regulatory Pathway; Reporting; Research; Role; Sedation procedure; Site; Skin; Spinal Ganglia; Stimulus; Streptozocin; Symptoms; T-Type Calcium Channels; Testing; Therapeutic; Tissues; Topical application; Urinary Retention; Ventilatory Depression; Weight Gain; Wild Type Mouse; Work; addiction; allodynia; biophysical techniques; chronic pain; cognitive function; density; diabetic; diabetic patient; diabetic rat; disabling symptom; effective therapy; experience; extracellular; gabapentin; ganglion cell; glycosylation; human disease; in vivo; innovation; mouse model; neuronal cell body; new therapeutic target; nociceptive response; novel; novel therapeutics; pain sensation; pain symptom; painful neuropathy; patch clamp; pregabalin; protein transport; public health relevance; response; spontaneous pain; sugar; tool; transmission process; voltage

DESCRIPTION (provided by applicant): Pain-sensing sensory neurons of the dorsal root ganglion (DRG) can become sensitized (hyperexcitable) in response to pathological conditions such as diabetes. Due to insufficient knowledge concerning the mechanisms underlying this sensitization, current treatments for painful diabetic neuropathy are limited to somewhat non-specific systemic drugs, such as opioids or gabapentin, which can cause significant side effects and have high potential for abuse. Recent studies have established that T-channels make a previously unrecognized contribution to sensitization of pain responses by enhancing excitability of nociceptors. We recently showed that DRG T-currents are up-regulated in streptozotocin (STZ)-induced and ob/ob mouse models of diabetic neuropathy and contribute to enhanced pain transmission. In preliminary data, we show that the glycosylation inhibitor neuraminidase inhibits T-currents and reverses thermal and mechanical hyperalgesia in these animal models. This finding has led us to hypothesize that post-translational glycosylation of the CaV3.2 channel increases activity, enhances excitability of nociceptive DRG neurons, and consequently contributes to the symptoms of painful diabetic neuropathy. Our specific aims are to: Aim 1: To use patch-clamp recordings and biophysical methods to study glycosylation-induced alterations of CaV3.2 T-channel activity in acutely dissociated DRG neurons in vitro. We propose that alterations in T-current kinetics and density can directly influence excitability of nociceptive DR cells. Aim 2: To investigate sites at which glycosylation of CaV3.2 T-channels occur in recombinant cells, native, and cultured DRG neurons. We propose that glycosylation of specific extracellular asparagine residues of CaV3.2 channels increases current density and membrane expression of the channel. Aim 3: To test the hypothesis that glycosylation of CaV3.2 T-channels in the peripheral axons of sensory neurons participates in painful PDN. We postulate that reversing glycosylation of CaV3.2 channels in diabetic animals will reverse abnormal membrane expression of these channels in somas and peripheral axons of nociceptive DRG cells, diminish cellular hyper-excitability, and reverse neuropathic pain progression in vivo. The proposed work is innovative in that a new mechanism for channel regulation will be characterized. It is medically significant because understanding the details of this regulatory pathway will facilitate development of novel drugs targeting steps in this pathway for treatment of painful neuropathies. We expect that this approach may decrease side effects from medication and reduce the potential for drug abuse in patients with painful diabetic neuropathy.

描述(由申请人提供)：背根神经节(DRG)的痛觉感觉神经元可因糖尿病等病理情况而变得敏感(高度兴奋)。由于对这种致敏作用的机制了解不足，目前对痛性糖尿病神经病变的治疗仅限于一些非特异性的全身药物，如阿片类药物或加巴喷丁，这些药物可能会导致显著的副作用，并有很高的滥用潜力。最近的研究证实，T通道通过增强伤害性感受器的兴奋性，在痛觉反应的敏化过程中起到了以前未知的作用。我们最近发现，在链脲佐菌素(STZ)诱导的糖尿病神经病变模型和ob/ob小鼠模型中，DRG T电流上调，并有助于增强疼痛传递。在初步数据中，我们发现糖基化抑制剂神经氨酸酶抑制T-电流，逆转这些动物模型中的热和机械性痛敏。这一发现使我们假设CaV3.2通道的翻译后糖基化增加了活性，增强了伤害性DRG神经元的兴奋性，从而导致了疼痛的糖尿病神经病变的症状。我们的具体目标是：目的1：利用膜片钳记录和生物物理方法研究糖基化对急性分离的DRG神经元CaV3.2 T通道活性的影响。我们认为T电流动力学和密度的改变可以直接影响伤害性DR细胞的兴奋性。目的：研究重组细胞、天然和培养的背根节神经元中CaV3.2 T通道的糖基化位点。我们认为，CaV3.2通道的特定胞外天冬酰胺残基的糖基化增加了通道的电流密度和膜表达。目的：验证感觉神经元外周轴突CaV3.2T通道糖基化参与痛性PDN的假说。我们推测，逆转糖尿病动物CaV3.2通道的糖基化将逆转伤害性DRG细胞胞体和外周轴突中这些通道的异常膜表达，降低细胞的超兴奋性，并逆转体内神经病理性疼痛的进展。拟议的工作是创新的，因为将表征一种新的渠道监管机制。这具有重要的医学意义，因为了解这一调控途径的细节将促进针对这一途径中的步骤治疗痛性神经疾病的新药的开发。我们预计，这种方法可能会减少药物副作用，并减少患有痛苦的糖尿病神经病变患者滥用药物的可能性。