Dynamic interactions between L-type voltage-sensitive calcium channel Cav1.2 subunits and ahnak in osteoblastic cells.
Voltage-sensitive Ca(2+) channels (VSCCs) mediate Ca(2+) permeability in osteoblasts. Association between VSCC alpha(1)- and beta-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 beta(2)-subunit of the L-type Ca(v)1.2 (alpha(1C)) VSCC complex. Ca(v)1.2 is the major alpha(1)-subunit in osteoblasts, but the cytoskeletal complex and subunit composition are unknown. Among the four beta-subtypes, the beta(2)-subunit and, to a lesser extent, the beta(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 beta(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 beta(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 beta(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 beta(2)-subunits and ahnak form a stable complex in osteoblastic cells that permits Ca(2+) signaling independently of association with the actin cytoskeleton.
Shao
Ying
Y
Dept. of Biological Sciences, University of Delaware, Newark, DE 19716, USA.
Czymmek
Kirk J
KJ
Jones
Patricia A
PA
Fomin
Victor P
VP
Akanbi
Kamil
K
Duncan
Randall L
RL
Farach-Carson
Mary C
MC
D016475:3T3 Cells; D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D055785:Gene Knockdown Techniques; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology / Q000502:physiology*; D034741:RNA, Small Interfering
C489572:Ahnak protein, mouse; C514350:CACNA1C protein, mouse; D020746:Calcium Channels, L-Type; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D002118:Calcium