Association of the α2δ1 subunit with Cav3.2 enhances membrane expression and regulates mechanically induced ATP release in MLO-Y4 osteocytes
✍ Scribed by William R Thompson; Amber S Majid; Kirk J Czymmek; Albert L Ruff; Jesús García; Randall L Duncan; Mary C Farach-Carson
- Publisher
- American Society for Bone and Mineral Research
- Year
- 2011
- Tongue
- English
- Weight
- 958 KB
- Volume
- 26
- Category
- Article
- ISSN
- 0884-0431
- DOI
- 10.1002/jbmr.437
No coin nor oath required. For personal study only.
✦ Synopsis
Voltage-sensitive calcium channels (VSCCs) mediate signaling events in bone cells in response to mechanical loading. Osteoblasts predominantly express L-type VSCCs composed of the a 1 pore-forming subunit and several auxiliary subunits. Osteocytes, in contrast, express T-type VSCCs and a relatively small amount of L-type a 1 subunits. Auxiliary VSCC subunits have several functions, including modulating gating kinetics, trafficking of the channel, and phosphorylation events. The influence of the a 2 d auxiliary subunit on T-type VSCCs and the physiologic consequences of that association are incompletely understood and have yet to be investigated in bone. In this study we postulated that the auxiliary a 2 d subunit of the VSCC complex modulates mechanically regulated ATP release in osteocytes via its association with the T-type Ca v 3.2 (a 1H ) subunit. We demonstrated by reverse-transcriptase polymerase chain reaction, Western blotting, and immunostaining that MLO-Y4 osteocyte-like cells express the T-type Ca v 3.2 (a 1H ) subunit more abundantly than the Ltype Ca v 1.2 (a 1C ) subunit. We also demonstrated that the a 2 d 1 subunit, previously described as an L-type auxiliary subunit, complexes with the T-type Ca v 3.2 (a 1H ) subunit in MLO-Y4 cells. Interestingly, siRNA-mediated knockdown of a 2 d 1 completely abrogated ATP release in response to membrane stretch in MLO-Y4 cells. Additionally, knockdown of the a 2 d 1 subunit resulted in reduced ERK1/2 activation. Together these data demonstrate a functional VSCC complex. Immunocytochemistry following a 2 d 1 knockdown showed decreased membrane localization of Ca v 3.2 (a 1H ) at the plasma membrane, suggesting that the diminished ATP release and ERK1/2 activation in response to membrane stretch resulted from a lack of Ca v 3.2 (a 1H ) at the cell membrane.