Association of the α2δ1 Subunit With Cav3.2 Enhances Membrane Expression and Regulates Mechanically Induced ATP Release in MLO-Y4 Osteocytes

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 alpha(1) pore-forming subunit and several auxiliary subunits. Osteocytes, in contrast, express T-type VSCCs and a relatively small amount of L-type alpha(1) subunits. Auxiliary VSCC subunits have several functions, including modulating gating kinetics, trafficking of the channel, and phosphorylation events. The influence of the alpha(2)delta 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 alpha(2)delta subunit of the VSCC complex modulates mechanically regulated ATP release in osteocytes via its association with the T-type Ca(v)3.2 (alpha(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 (alpha(1H)) subunit more abundantly than the L-type Ca(v)1.2 (alpha(1C)) subunit. We also demonstrated that the alpha(2)delta(1) subunit, previously described as an L-type auxiliary subunit, complexes with the T-type Ca(v)3.2 (alpha(1H)) subunit in MLO-Y4 cells. Interestingly, siRNA-mediated knockdown of alpha(2)delta(1) completely abrogated ATP release in response to membrane stretch in MLO-Y4 cells. Additionally, knockdown of the alpha(2)delta(1) subunit resulted in reduced ERK1/2 activation. Together these data demonstrate a functional VSCC complex. Immunocytochemistry following alpha(2)delta(1) knockdown showed decreased membrane localization of Ca(v)3.2 (alpha(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 (alpha(1H)) at the cell membrane. (C) 2011 American Society for Bone and Mineral Research.