Ablation of α2δ-1 inhibits cell-surface trafficking of endogenous N-type calcium channels in the pain pathway in vivo

Manuela Nieto-Rostro,Krishma Ramgoolam,Wendy S Pratt,Akos Kulik,Annette C Dolphin

doi:10.1073/pnas.1811212115

Abstract

The auxiliary α2δ calcium channel subunits play key roles in voltage-gated calcium channel function. Independent of this, α2δ-1 has also been suggested to be important for synaptogenesis. Using an epitope-tagged knockin mouse strategy, we examined the effect of α2δ-1 on CaV2.2 localization in the pain pathway in vivo, where CaV2.2 is important for nociceptive transmission and α2δ-1 plays a critical role in neuropathic pain. We find CaV2.2 is preferentially expressed on the plasma membrane of calcitonin gene-related peptide-positive small nociceptors. This is paralleled by strong presynaptic expression of CaV2.2 in the superficial spinal cord dorsal horn. EM-immunogold localization shows CaV2.2 predominantly in active zones of glomerular primary afferent terminals. Genetic ablation of α2δ-1 abolishes CaV2.2 cell-surface expression in dorsal root ganglion neurons and dramatically reduces dorsal horn expression. There was no effect of α2δ-1 knockout on other dorsal horn pre- and postsynaptic markers, indicating the primary afferent pathways are not otherwise affected by α2δ-1 ablation.

Highlights

The auxiliary α2δ calcium channel subunits play key roles in voltage-gated calcium channel function
A key role for N-type calcium channels was identified in primary afferent neurotransmission in the dorsal horn of the spinal cord, and these toxins were pursued as a therapeutic target in the alleviation of chronic pain [7, 8]
Nuclei were stained with DAPI. (Scale bars: 20 μm.) (E) Surface CaV2.2_HA intensity in α2δ-1KO/KO dorsal root ganglion (DRG) neurons for small, medium, and large DRG neurons that are either CGRP-positive or CGRP-negative. n = 198, 197, 70, 134, 97, and 109 DRG neurons, respectively, from sections from at least three mice. ****P < 0.0001, **P = 0.0022. (F) Surface CaV2.2_HA intensity

Summary

Introduction

The auxiliary α2δ calcium channel subunits play key roles in voltage-gated calcium channel function. A key role for N-type calcium channels was identified in primary afferent neurotransmission in the dorsal horn of the spinal cord, and these toxins were pursued as a therapeutic target in the alleviation of chronic pain [7, 8]. 11 and 12), these have not shown plasma membrane localization of the endogenous channel in neurons and have not been rigorously examined against knockout tissue For this reason, we developed a CaV2.2 construct with an exofacial epitope tag to detect its cell-surface expression and trafficking [13]. We took advantage of our finding that the presence of the epitope tag did not affect function [13] to generate a knockin (KI) mouse line containing the hemagglutinin (HA) tag in the same position in the Cacna1b gene This has allowed us to examine the distribution of native CaV2.2 protein in the intact nervous system.

Methods

Results

Conclusion