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

描述（由申请人提供）：背根神经节（DRG）的痛觉感觉神经元可响应于病理状况（如糖尿病）而变得敏感（过度兴奋）。由于对这种致敏机制的认识不足，目前对疼痛性糖尿病神经病变的治疗仅限于一些非特异性全身药物，如阿片类药物或加巴喷丁，这些药物可引起显著的副作用，并有很高的滥用可能性。最近的研究已经证实，T通道通过增强伤害感受器的兴奋性对疼痛反应的敏化做出了以前未被认识的贡献。我们最近发现，在链脲佐菌素（STZ）诱导的糖尿病神经病变和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，我们表明，糖基化抑制剂神经氨酸酶抑制T-电流和逆转这些动物模型中的热和机械痛觉过敏。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，从而导致疼痛性糖尿病神经病变的症状。我们的具体目标是：目标1：采用膜片钳技术和生物物理学方法研究糖基化对离体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2：研究重组细胞、天然和培养的DRG神经元中CaV3.2 T通道糖基化的位点。我们建议，糖基化的特定细胞外天冬酰胺残基的CaV3.2通道增加电流密度和膜表达的通道。目的3：验证外周感觉神经元轴突CaV 3.2 T通道糖基化参与痛性PDN的假说。我们推测，逆转糖尿病动物中CaV3.2通道的糖基化将逆转这些通道在伤害性DRG细胞的胞体和外周轴突中的异常膜表达，降低细胞的过度兴奋性，并逆转体内神经病理性疼痛的进展。本文的创新之处在于提出了一种新的渠道监管机制。它具有医学意义，因为了解这一调控途径的细节将有助于开发针对这一途径中治疗疼痛性神经病的新药物。我们希望这种方法可以减少药物的副作用，并减少疼痛性糖尿病神经病变患者滥用药物的可能性。

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.