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Reversal of neuropathic pain in diabetes by targeting glycosylation of Ca(V)3.2 T-type calcium channels.

Diabetes (2013-07-10)
Peihan Orestes, Hari Prasad Osuru, William E McIntire, Megan O Jacus, Reza Salajegheh, Miljen M Jagodic, Wonjoo Choe, Jeonghan Lee, Sang-Soo Lee, Kirstin E Rose, Nathan Poiro, Michael R Digruccio, Katiresan Krishnan, Douglas F Covey, Jung-Ha Lee, Paula Q Barrett, Vesna Jevtovic-Todorovic, Slobodan M Todorovic
ABSTRACT

It has been established that Ca(V)3.2 T-type voltage-gated calcium channels (T-channels) play a key role in the sensitized (hyperexcitable) state of nociceptive sensory neurons (nociceptors) in response to hyperglycemia associated with diabetes, which in turn can be a basis for painful symptoms of peripheral diabetic neuropathy (PDN). Unfortunately, current treatment for painful PDN has been limited by nonspecific systemic drugs with significant side effects or potential for abuse. We studied in vitro and in vivo mechanisms of plasticity of Ca(V)3.2 T-channel in a leptin-deficient (ob/ob) mouse model of PDN. We demonstrate that posttranslational glycosylation of specific extracellular asparagine residues in Ca(V)3.2 channels accelerates current kinetics, increases current density, and augments channel membrane expression. Importantly, deglycosylation treatment with neuraminidase inhibits native T-currents in nociceptors and in so doing completely and selectively reverses hyperalgesia in diabetic ob/ob mice without altering baseline pain responses in healthy mice. Our study describes a new mechanism for the regulation of Ca(V)3.2 activity and suggests that modulating the glycosylation state of T-channels in nociceptors may provide a way to suppress peripheral sensitization. Understanding the details of this regulatory pathway could facilitate the development of novel specific therapies for the treatment of painful PDN.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-Asparagine, ≥98% (HPLC)
Sigma-Aldrich
L-Asparagine, BioReagent, suitable for cell culture, suitable for insect cell culture
Sigma-Aldrich
PNGase F from Elizabethkingia meningoseptica, BioReagent, ≥95% (SDS-PAGE), for proteomics
Sigma-Aldrich
PNGase F from Elizabethkingia miricola, buffered aqueous solution
Sigma-Aldrich
PNGase F from Elizabethkingia meningoseptica, lyophilized powder, recombinant, expressed in E. coli
Supelco
L-Asparagine, certified reference material, TraceCERT®
Sigma-Aldrich
Glycopeptidase A from almonds, buffered aqueous glycerol solution, ≥0.05 unit/mL
Sigma-Aldrich
PNGase F from Elizabethkingia meningoseptica, buffered aqueous solution, recombinant, expressed in E. coli
Sigma-Aldrich
PNGase F from Elizabethkingia meningoseptica, recombinant, expressed in E. coli, set of 100 units nanomolar unit