Our previous studies implicated glycosylation of the Ca_V3.2 isoform of T-type Ca²⁺ channels (T-channels) in the development of Type 2 painful peripheral diabetic neuropathy (PDN). Here we investigated biophysical mechanisms underlying the modulation of recombinant Ca_V3.2 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 in vitro study we used whole-cell recordings of current-voltage relationships to confirm that Ca_V3.2 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 in vivo by injections of streptozotocin (STZ) in adult female C57Bl/6j wild type (WT) mice, adult female Sprague Dawley rats and Ca_V3.2 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 Ca_V3.2 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 Ca_V3.2 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 Ca_V3.2 channels in vivo 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 Ca ²⁺通道（T 通道）的 Ca _{V 3.2 同种型在 2 型疼痛性周围糖尿病神经病变 (PDN) 的发展中的糖基化。在这里，我们研究了神经氨酸酶 (NEU) 和 PNGase-F (PNG) 等去糖基化酶调节重组 Ca V} 3.2 通道的生物物理机制，以及它们在疼痛性 PDN 1 型中的行为和生化效应。体外研究我们使用电流-电压关系的全细胞记录来确认去糖基化后 Ca _V 3.2 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着的去极化转变，而对电流失活和失活恢复的动力学几乎没有影响。PDN被诱导体内_{通过在成年雌性 C57Bl/6j 野生型 (WT) 小鼠、成年雌性 Sprague Dawley 大鼠和 Ca V} 3.2 敲除 (KO 小鼠)中注射链脲佐菌素 (STZ )。将 NEU 或载体（盐水）局部注射到右后爪中或鞘内注射。我们发现注射 NEU 而不是载体，可以完全逆转糖尿病 WT 大鼠和小鼠的热和机械痛觉过敏。相比之下，NEU 没有改变 Ca _V 3.2 KO 小鼠的基线热敏感性和机械敏感性，这些小鼠也未能发展出疼痛的 PDN。最后，我们使用生化方法和凝胶位移分析来直接证明天然 Ca _{V的 N 端片段}3.2 背根神经节 (DRG) 中的通道在健康和糖尿病动物中都被糖基化。我们的研究结果表明，在感觉神经元中，糖基化诱导的 Ca _V 3.2 通道改变体内直接增强糖尿病痛觉过敏，糖基化抑制剂可用于改善 1 型糖尿病的疼痛症状。我们希望我们的研究可以更好地理解疼痛 PDN 背后的分子机制，以促进发现这种难治性疾病的新疗法。