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Genetic Tracing of Ca_v3.2 T-Type Calcium Channel Expression in the Peripheral Nervous System.

ABSTRACT: Characterizing the distinct functions of the T-type ion channel subunits Ca_v3.1, 3.2 or 3.3 has proven difficult due to their highly conserved amino-acid sequences and the lack of pharmacological blockers specific for each subunit. To precisely determine the expression pattern of the Ca_v3.2 channel in the nervous system we generated two knock-in mouse strains that express EGFP or Cre recombinase under the control of the Ca_v3.2 gene promoter. We show that in the brains of these animals, the Ca_v3.2 channel is predominantly expressed in the dentate gyrus of the hippocampus. In the peripheral nervous system, the activation of the promoter starts at E9.5 in neural crest cells that will give rise to dorsal root ganglia (DRG) neurons, but not sympathetic neurons. As development progresses the number of DRG cells expressing the Ca_v3.2 channel reaches around 7% of the DRG at E16.5, and remains constant until E18.5. Characterization of sensory neuron subpopulations at E18.5 showed that EGFP⁺ cells are a heterogeneous population consisting mainly of TrkB⁺ and TrkC⁺ cells, while only a small percentage of DRG cells were TrkA⁺. Genetic tracing of the sensory nerve end-organ innervation of the skin showed that the activity of the Ca_v3.2 channel promoter in sensory progenitors marks many mechanoreceptor and nociceptor endings, but spares slowly adapting mechanoreceptors with endings associated with Merkel cells. Our genetic analysis reveals for the first time that progenitors that express the Ca_v3.2 T-type calcium channel, defines a sensory specific lineage that populates a large proportion of the DRG. Using our Ca_v3.2-Cre mice together with AAV viruses containing a conditional fluorescent reporter (tdTomato) we could also show that Cre expression is largely restricted to two functionally distinct sensory neuron types in the adult ganglia. Ca_v3.2 positive neurons innervating the skin were found to only form lanceolate endings on hair follicles and are probably identical to D-hair receptors. A second population of nociceptive sensory neurons expressing the Ca_v3.2 gene was found to be positive for the calcitonin-gene related peptide but these neurons are deep tissue nociceptors that do not innervate the skin.

SUBMITTER: Bernal Sierra YA

PROVIDER: S-EPMC5350092 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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Genetic Tracing of Ca_v3.2 T-Type Calcium Channel Expression in the Peripheral Nervous System.

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Dataset Information

Genetic Tracing of Cav3.2 T-Type Calcium Channel Expression in the Peripheral Nervous System.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Genetic Tracing of Ca_v3.2 T-Type Calcium Channel Expression in the Peripheral Nervous System.