Dynamic interactions between L-type voltage-sensitive calcium channel Cav1.2 subunits and ahnak in osteoblastic cells

Abstract

Voltage-sensitive Ca2+channels (VSCCs) mediate Ca2+permeability in osteoblasts. Association between VSCC α1- and β-subunits targets channel complexes to the plasma membrane and modulates function. In mechanosensitive tissues, a 700-kDa ahnak protein anchors VSCCs to the actin cytoskeleton via the β2-subunit of the L-type Cav1.2 (α1C) VSCC complex. Cav1.2 is the major α1-subunit in osteoblasts, but the cytoskeletal complex and subunit composition are unknown. Among the four β-subtypes, the β2-subunit and, to a lesser extent, the β3-subunit coimmunoprecipitated with the Cav1.2 subunit in MC3T3-E1 preosteoblasts. Fluorescence resonance energy transfer revealed a complex between Cav1.2 and β2-subunits and demonstrated their association in the plasma membrane and secretory pathway. Western blot and immunohistochemistry showed ahnak association with the channel complex in the plasma membrane via the β2-subunit. Cytochalasin D exposure disrupted the actin cytoskeleton but did not disassemble or disrupt the function of the complex of L-type VSCC Cav1.2 and β2-subunits and ahnak. Similarly, small interfering RNA knockdown of ahnak did not disrupt the actin cytoskeleton but significantly impaired Ca2+influx. Collectively, we showed that Cav1.2 and β2-subunits and ahnak form a stable complex in osteoblastic cells that permits Ca2+signaling independently of association with the actin cytoskeleton.