Joksimovic Sonja Lj, Evans J Grayson, McIntire William E, Orestes Peihan, Barrett Paula Q, Jevtovic-Todorovic Vesna, Todorovic Slobodan M
Department of Anesthesiology, University of Colorado Denver, Aurora, CO, United States.
Undergraduate School of Arts and Sciences, University of Virginia, Charlottesville, VA, United States.
Front Cell Neurosci. 2020 Dec 15;14:605312. doi: 10.3389/fncel.2020.605312. eCollection 2020.
Our previous studies implicated glycosylation of the Ca3.2 isoform of T-type Ca channels (T-channels) in the development of Type 2 painful peripheral diabetic neuropathy (PDN). Here we investigated biophysical mechanisms underlying the modulation of recombinant Ca3.2 channel by de-glycosylation enzymes such as neuraminidase (NEU) and PNGase-F (PNG), as well as their behavioral and biochemical effects in painful PDN Type 1. In our study we used whole-cell recordings of current-voltage relationships to confirm that Ca3.2 current densities were decreased ~2-fold after de-glycosylation. Furthermore, de-glycosylation induced a significant depolarizing shift in the steady-state relationships for activation and inactivation while producing little effects on the kinetics of current deactivation and recovery from inactivation. PDN was induced by injections of streptozotocin (STZ) in adult female C57Bl/6j wild type (WT) mice, adult female Sprague Dawley rats and Ca3.2 knock-out (KO mice). Either NEU or vehicle (saline) were locally injected into the right hind paws or intrathecally. We found that injections of NEU, but not vehicle, completely reversed thermal and mechanical hyperalgesia in diabetic WT rats and mice. In contrast, NEU did not alter baseline thermal and mechanical sensitivity in the Ca3.2 KO mice which also failed to develop painful PDN. Finally, we used biochemical methods with gel-shift analysis to directly demonstrate that N-terminal fragments of native Ca3.2 channels in the dorsal root ganglia (DRG) are glycosylated in both healthy and diabetic animals. Our results demonstrate that in sensory neurons glycosylation-induced alterations in Ca3.2 channels directly enhance diabetic hyperalgesia, and that glycosylation inhibitors can be used to ameliorate painful symptoms in Type 1 diabetes. We expect that our studies may lead to a better understanding of the molecular mechanisms underlying painful PDN in an effort to facilitate the discovery of novel treatments for this intractable disease.
我们之前的研究表明，T型钙通道（T通道）的Ca3.2亚型糖基化与2型疼痛性外周糖尿病神经病变（PDN）的发展有关。在此，我们研究了神经氨酸酶（NEU）和PNGase - F（PNG）等去糖基化酶对重组Ca3.2通道调节的生物物理机制，以及它们在1型疼痛性PDN中的行为和生化效应。在我们的研究中，我们使用全细胞电流 - 电压关系记录来证实去糖基化后Ca3.2电流密度降低了约2倍。此外，去糖基化在激活和失活的稳态关系中引起了显著的去极化偏移，而对电流失活和从失活恢复的动力学影响很小。通过向成年雌性C57Bl/6j野生型（WT）小鼠、成年雌性Sprague Dawley大鼠和Ca3.2基因敲除（KO）小鼠注射链脲佐菌素（STZ）诱导PDN。将NEU或溶剂（生理盐水）局部注射到右后爪或鞘内。我们发现，注射NEU而非溶剂，可完全逆转糖尿病WT大鼠和小鼠的热痛觉过敏和机械性痛觉过敏。相比之下，NEU并未改变Ca3.2 KO小鼠的基线热敏感性和机械敏感性，这些小鼠也未发展出疼痛性PDN。最后，我们使用凝胶迁移分析的生化方法直接证明，背根神经节（DRG）中天然Ca3.2通道的N端片段在健康动物和糖尿病动物中均被糖基化。我们的结果表明，在感觉神经元中，糖基化诱导的Ca3.2通道改变直接增强了糖尿病性痛觉过敏，并且糖基化抑制剂可用于改善1型糖尿病的疼痛症状。我们期望我们的研究可能有助于更好地理解疼痛性PDN的分子机制，以便促进针对这种难治性疾病的新疗法的发现。
