Association of the α(2)δ(1) subunit with Ca(v)3.2 enhances membrane expression and regulates mechanically induced ATP release in MLO-Y4 osteocytes

J Bone Miner Res. 2011 Sep;26(9):2125-39. doi: 10.1002/jbmr.437.

Abstract

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 α(1) pore-forming subunit and several auxiliary subunits. Osteocytes, in contrast, express T-type VSCCs and a relatively small amount of L-type α(1) subunits. Auxiliary VSCC subunits have several functions, including modulating gating kinetics, trafficking of the channel, and phosphorylation events. The influence of the α(2)δ 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 α(2) δ subunit of the VSCC complex modulates mechanically regulated ATP release in osteocytes via its association with the T-type Ca(v) 3.2 (α(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(α(1H)) subunit more abundantly than the L-type Ca(v)1.2 (α(1C)) subunit. We also demonstrated that the α(2) δ(1) subunit, previously described as an L-type auxiliary subunit, complexes with the T-type Ca(v)3.2 (α(1H)) subunit in MLO-Y4 cells. Interestingly, siRNA-mediated knockdown of α(2) δ(1) completely abrogated ATP release in response to membrane stretch in MLO-Y4 cells. Additionally, knockdown of the α(2)δ(1) subunit resulted in reduced ERK1/2 activation. Together these data demonstrate a functional VSCC complex. Immunocytochemistry following α(2)δ(1) knockdown showed decreased membrane localization of Ca(v) 3.2 (α(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 (α(1H)) at the cell membrane.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Animals
  • Calcium Channels / genetics
  • Calcium Channels / metabolism*
  • Calcium Channels, T-Type / genetics
  • Calcium Channels, T-Type / metabolism*
  • Cell Line
  • Cell Membrane / metabolism*
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • Gene Expression Regulation
  • Gene Knockdown Techniques
  • Mice
  • Mice, Inbred C57BL
  • Osteocytes / cytology
  • Osteocytes / enzymology
  • Osteocytes / metabolism*
  • Phosphorylation
  • Protein Binding
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Protein Transport
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Rats

Substances

  • CACNA2D1 protein, mouse
  • Cacna1h protein, mouse
  • Calcium Channels
  • Calcium Channels, T-Type
  • Protein Subunits
  • RNA, Messenger
  • Adenosine Triphosphate
  • Extracellular Signal-Regulated MAP Kinases