Association of the T-Type Cav3.2 (α1H) VSCC with the Auxiliary α2δ1 Subunit in MLO-Y4 Cells

Date
2009-05
Authors
Majid, Amber S.
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University of Delaware
Abstract
Bone contains three major cell types: bone-forming osteoblasts, boneresorbing osteoclasts, and osteocytes, which are terminally differentiated osteoblasts embedded in mineralized matrix. Osteocytes communicate with one another through dendrite-like processes extending from cell bodies. While their function is not completely understood, osteocytes sense mechanical signals within bone and translate these signals into responses by effector cells. Voltage sensitive calcium channels (VSCCs) span the cell membrane and open in response to external stimuli resulting in altered Ca^2+ permeability. Once inside the cell, Ca^2+ acts as a second messenger eliciting specific cellular responses. L-type VSCCs are composed of a complex of polypeptide units consisting of a pore forming α1 subunit, an intracellular β subunit, a dimer of disulfide linked α2δ subunits, and a transmembrane γ subunit in some tissues, but not in osteoblasts. While osteoblasts predominantly express L-type Cav1.2 (α1C) VSCCs, osteocytes have been shown to express mainly T-type Cav3.2 (α1H) subunit. Expression of auxiliary VSCC subunits never has been studied in osteocytes, and furthermore, the association of auxiliary subunits with T-type VSCC subunits never has been demonstrated in bone or any other tissue. The goal of this study was to elucidate the expression profile of VSCC subunits in osteocytes and to demonstrate an association of the extracellular auxiliary α2δ subunit and the Cav3.2 subunit. Using RT-PCR, Westernblot, and immunostaining, I demonstrated that the T-type Cav3.2 (α1H) subunit is the predominant α1 subunit of MLO-Y4 cells. Additionally, MLO-Y4 cells express α2δ1, γ7, and β1-3 subunits at the transcript level. Co-immunoprecipitation assays demonstrated the presence of an association between Cav3.2 (α1H) and the α2δ1 auxiliary subunit. This association can stabilize the functional channel, and provide a mechanism of interaction with the extracellular environment. This key protein complex may provide a crucial function for mechnanosensitive osteocytes in bone, having implications in skeletal remodeling and osteoporosis.
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