R-type Calcium Channel Isoform in Rat Dorsal Root Ganglion Neurons

Zhi Fang; Jae Hong Hwang; Joong Soo Kim; Sung Jun Jung; Seog Bae Oh

doi:10.4196/kjpp.2010.14.1.45

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Fang, Hwang, Kim, Jung, and Oh: R-type Calcium Channel Isoform in Rat Dorsal Root Ganglion Neurons

Original Article

Korean J Physiol Pharmacol 2010;14(1):45-49.

Published online: 18 February 2010

DOI: https://doi.org/10.4196/kjpp.2010.14.1.45

R-type Calcium Channel Isoform in Rat Dorsal Root Ganglion Neurons

Zhi Fang^1,^∗, Jae Hong Hwang^1,^∗, Joong Soo Kim¹, Sung Jun Jung², Seog Bae Oh^1,

¹National Research Laboratory for Pain, Dental Research Institute and Department of Physiology School of Dentistry, Seoul National University, Seoul 110-749, Korea

²Department of Physiology, College of Medicine, Kangwon National University, Chunchon 200-710, Korea

Corresponding to: Seog Bae Oh, Department of Physiology, School of Dentistry, Seoul National University, 28-2, Yeongeon-dong, Chongno-gu, Seoul 110-749, Korea. (Tel) 82-2-740-8656, (Fax) 82-2-762-5107, (E-mail) odolbae@snu.ac.kr

^∗ These authors contributed equally to this work.

Received 4 January 2010 Revised 16 January 2010 Accepted 25 January 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

R-type Ca_v2.3 high voltage-activated Ca²⁺ channels in peripheral sensory neurons contribute to pain transmission. Recently we have demonstrated that, among the six Ca_v2.3 isoforms (Ca_v2.3a∼Ca_v2.3e), the Ca_v2.3e isoform is primarily expressed in trigeminal ganglion (TG) nociceptive neurons. In the present study, we further investigated expression patterns of Ca_v2.3 isoforms in the dorsal root ganglion (DRG) neurons. As in TG neurons, whole tissue RT-PCR analyses revealed the presence of two isoforms, Ca_v2.3a and Ca_v2.3e, in DRG neurons. Single-cell RT-PCR detected the expression of Ca_v2.3e mRNA in 20% (n=14/70) of DRG neurons, relative to Ca_v2.3a expression in 2.8% (n=2/70) of DRG neurons. Ca_v2.3e mRNA was mainly detected in small-sized neurons (n=12/14), but in only a few medium-sized neurons (n=2/14) and not in large-sized neurons, indicating the prominence of Ca_v2.3e in nociceptive DRG neurons. Moreover, Ca_v2.3e was preferentially expressed in tyrosine-kinase A (trkA)-positive, isolectin B4 (IB4)-negative and transient receptor potential vanilloid 1 (TRPV1)-positive neurons. These results suggest that Ca_v2.3e may be the main R-type Ca²⁺ channel isoform in nociceptive DRG neurons and thereby a potential target for pain treatment, not only in the trigeminal system but also in the spinal system.

Keywords: R-type calcium channels, Ca_v2.3, Voltage-activated calcium channels, DRG neurons

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Fig. 1.

(A) Schematic diagram of α_1E (Ca_v2.3) subunit. The structural variations cover two segments of 19 (insert I) and 7 amino acids (insert II) in the loop between domain II and III, and a third segment of 43 amino acids (insert III) in the proximal carboxy terminus. (B) Two Ca_v2.3 isoforms were found in DRG. Insert I, II and III was analyzed by whole tissue RT-PCR. Ca_v2.3 isoforms amplified from DRG neurons have insert II, but not insert I, and either lack or contain insert III depending on the isoform. Ca_v2.3a contains insert II, but not insert I and insert III, while Ca_v2.3e has insert II and insert III, but not insert I. (C) Illustrated are the locations of the primers designed to detect insert I, insert II and insert III for RT-PCR analysis in relation to the Ca_v2.3 subunit.

Fig. 2.

Expression of Ca_v2.3a and Ca_v2.3e isoforms from individual DRG neurons. (A) Single-cell RT-PCR products amplified with nested primer from DRG neurons. PCR products, with or without insert III (larger, 1 and smaller, 2), were produced in each DRG neuron. (B) Representative gels showing single-cell RT-PCR products amplified using insert III-specific primers. The numbers (1–5) indicate five different neurons examined from single-cell RT-PCR reaction. β-actin was used in each reaction as a positive control. Circle diagram shows the distribution of Ca_v2.3a and Ca_v2.3e in DRG neurons (total 70 neurons).

Fig. 3.

Expression pattern of Ca_v2.3e was analyzed in three groups: small-sized (<16μm), medium-sized (16∼20 μm) and large-sized (20∼30 μm) DRG neurons. Representative gels showing single-cell RT-PCR products obtained from six different neurons. White bars indicate the number of neurons in each group; black bars indicate the number of neurons with expression of Ca_v2.3e.

Fig. 4.

(A) Small-sized DRG neurons were divided into IB4-negative and IB4-positive neurons; expression of Ca_v2.3e was determined in each group with or without trkA and P2X₃ expression. The black bar in the graph shows that Ca_v2.3e is prominent in trkA+/IB4-/TRPV1+ neurons. (B) Expression of Ca_v2.3e in TRPV1-expressing nociceptive neurons. Representative gels showing RT-PCR products amplified with Ca_v2.3e, TRPV1, and β-actin-specific primers from six different neurons. The majority of small-sized Ca_v2.3e-expressing DRG neurons are also TRPV1-positive, though TRPV1 expression is not exclusive to DRG expressing the Ca_v2.3e subunit (graph; right).

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