Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and beta(3) Subunit in Osteoblasts

• Authors: Xuemei Zhang, Fudan University; Fangping Li, Fudan University; Lin Guo, Fudan University; Hongya Hei, Fudan University; Lulu Tian, Fudan University; Wen Peng, Shanghai University; Hui Cai, Emory University
• Journal Title: PLoS ONE
• Volume: Volume 10, Number 4
• Publisher: Public Library of Science | 2015-04-22, Pages e0124274-e0124274
• Type of Work: Article | Final Publisher PDF
• Publisher DOI: http://doi.org/10.1371/journal.pone.0124274
• Subjects: Chemistry, Biochemistry; Health Sciences, Pharmacology; Health Sciences, Pharmacy
• Keywords: Science & Technology; Multidisciplinary Sciences; Science & Technology - Other Topics; DEPENDENT PROTEIN-KINASE; INDUCED BONE-FORMATION; CA2+ CHANNELS; ALPHA-ACTININ; PARATHYROID-HORMONE; CELLS; CYTOSKELETON; MODULATION; DYSTROPHIN; REQUIRES; Calcium channel; Actins; Osteoblasts; Cytoskeleton; Protein interactions; Immunoprecipitation; Cell membranes; Calcium signaling

Abstract

Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α<inf>1c</inf> subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β<inf>3</inf> subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells.