title: 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.
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.
authors.affiliation: Biomechanics and Movement Science Program, University of Delaware, Newark, DE 19716, USA. wthomp@udel.edu
authors.forename: William R
authors.initials: WR
authors.lastname: Thompson
authors.forename: Amber S
authors.initials: AS
authors.lastname: Majid
authors.forename: Kirk J
authors.initials: KJ
authors.lastname: Czymmek
authors.forename: Albert L
authors.initials: AL
authors.lastname: Ruff
authors.forename: Jesús
authors.initials: J
authors.lastname: García
authors.forename: Randall L
authors.initials: RL
authors.lastname: Duncan
authors.forename: Mary C
authors.initials: MC
authors.lastname: Farach-Carson
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D020747:Calcium Channels, T-Type / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D010011:Osteocytes / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats
chemical_list: C512066:CACNA2D1 protein, mouse; C504883:Cacna1h protein, mouse; D015220:Calcium Channels; D020747:Calcium Channels, T-Type; D021122:Protein Subunits; D012333:RNA, Messenger; D000255:Adenosine Triphosphate; D048049:Extracellular Signal-Regulated MAP Kinases