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

Abstract

R-type Ca(v)2.3 high voltage-activated Ca(2+) channels in peripheral sensory neurons contribute to pain transmission. Recently we have demonstrated that, among the six Ca(v)2.3 isoforms (Ca(v)2.3a~Ca(v)2.3e), the Ca(v)2.3e isoform is primarily expressed in trigeminal ganglion (TG) nociceptive neurons. In the present study, we further investigated expression patterns of Ca(v)2.3 isoforms in the dorsal root ganglion (DRG) neurons. As in TG neurons, whole tissue RT-PCR analyses revealed the presence of two isoforms, Ca(v)2.3a and Ca(v)2.3e, in DRG neurons. Single-cell RT-PCR detected the expression of Ca(v)2.3e mRNA in 20% (n=14/70) of DRG neurons, relative to Ca(v)2.3a expression in 2.8% (n=2/70) of DRG neurons. Ca(v)2.3e 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(v)2.3e in nociceptive DRG neurons. Moreover, Ca(v)2.3e 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(v)2.3e may be the main R-type Ca(2+) 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: Cav2.3; DRG neurons; R-type calcium channels; Voltage-activated calcium channels.

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 (16~20µm) and large-sized (20~30µm) 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).