Glycosylation of CaV3.2 Channels Contributes to the Hyperalgesia in Peripheral Neuropathy of Type 1 Diabetes

Abstract

Our previous studies implicated glycosylation of the CaV3.2 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 CaV3.2 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 CaV3.2 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 CaV3.2 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 CaV3.2 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 CaV3.2 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 CaV3.2 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.

Alpha lipoic acid attenuates evoked and spontaneous pain following surgical skin incision in rats

“…Here, we specifically focused on punctate mechanical hyperalgesia and spontaneous pain measures, given that they are the most relevant to clinical setting [ 36–38 ]. We have previously established that Ca V 3.2 channels play an important role in the development of both thermal and mechanical hyperalgesia in the incisional pain model in rats and mice [ 19 , 20 , 26 ]. We also demonstrated that hyperalgesia develops to a lesser extent in Ca V 3.2 knock-out (KO) mice as compared to WT mice [ 20 ], and that T-channel blockers can successfully alleviate both thermal and mechanical hyperalgesia after surgical incision in rats [ 19 , 20 , 26 ].…”

“…We have previously established that Ca V 3.2 channels play an important role in the development of both thermal and mechanical hyperalgesia in the incisional pain model in rats and mice [ 19 , 20 , 26 ]. We also demonstrated that hyperalgesia develops to a lesser extent in Ca V 3.2 knock-out (KO) mice as compared to WT mice [ 20 ], and that T-channel blockers can successfully alleviate both thermal and mechanical hyperalgesia after surgical incision in rats [ 19 , 20 , 26 ]. Our earlier study [ 25 ] demonstrated that ALA reduced T-currents in freshly dissociated cell bodies of sensory neurons in dorsal root ganglia of rats.…”

“…Each animal was allowed to recover individually in a cage, and all experiments were initiated as early as 2 hours after incision. Full details are provided in our recent publications [ 19 , 26 ].…”

The Mechanisms of Plasticity of Nociceptive Ion Channels in Painful Diabetic Neuropathy

“…Selective in vivo knock-down of the Ca V 3.2 isoform of T-channels in DRG neurons reduced both hyperalgesia in vivo , and T-currents in dissociated DRG neurons of small diameter in vitro , suggesting a correlation between hyperglycemia and increased T-currents with the appearance of mechanical and thermal sensitivity in diabetic STZ-treated rats ( 59 , 61 ). In vivo application of glycosylation inhibitors, such as NEU both peripherally and centrally, ameliorated hyperalgesia in a model of type I diabetes in rodents, except in Ca V 3.2 knock-out mice ( 62 ). Similarly, recent study by ( 63 ) revealed that NEU exhibits desensitizing effects in terms of spontaneous activity and stimulated release of CGRP (calcitonin gene related peptide).…”

The life cycle of voltage-gated Ca2+ channels in neurons: an update on the trafficking of neuronal calcium channels

“…However, further investigations focusing on the net forward trafficking of Ca V 3.2 will be needed to ascertain a role of N-glycosylation of Ca V 3.2 on its recycling or forward trafficking. As Ca V 3.2 channel up-regulation is a common feature in the development and maintenance of multiple pain processes [ 48 ], and alterations of Ca V 3.2 channels glycosylation have been associated with the development of pain related to diabetes [ 43 , 45 , 49 ], understanding Ca V 3.2 trafficking and the impact of its glycosylation are of significant therapeutic relevance [ 50 ].…”