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

Department of Biological Sciences, University of Delaware, Newark, Delaware Submitted 14 August 2008 ; accepted in final form 3 March 2009 Voltage-sensitive Ca 2+ channels (VSCCs) mediate Ca 2+ permeability in osteoblasts. Association between VSCC 1 - 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 β 2 -subunit of the L-type Ca v 1.2 ( 1C ) VSCC complex. Ca v 1.2 is the major 1 -subunit in osteoblasts, but the cytoskeletal complex and subunit composition are unknown. Among the four β-subtypes, the β 2 -subunit and, to a lesser extent, the β 3 -subunit coimmunoprecipitated with the Ca v 1.2 subunit in MC3T3-E1 preosteoblasts. Fluorescence resonance energy transfer revealed a complex between Ca v 1.2 and β 2 -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 β 2 -subunit. Cytochalasin D exposure disrupted the actin cytoskeleton but did not disassemble or disrupt the function of the complex of L-type VSCC Ca v 1.2 and β 2 -subunits and ahnak. Similarly, small interfering RNA knockdown of ahnak did not disrupt the actin cytoskeleton but significantly impaired Ca 2+ influx. Collectively, we showed that Ca v 1.2 and β 2 -subunits and ahnak form a stable complex in osteoblastic cells that permits Ca 2+ signaling independently of association with the actin cytoskeleton. osteoblasts; fluorescence resonance energy transfer; cytoskeleton Address for reprint requests and other correspondence: M. C. Farach-Carson, Dept. of Biological Sciences, Univ. of Delaware, Newark, DE 19716 (e-mail: farachca{at}udel.edu )