The β4 subunit of the voltage-gated calcium channel (Cacnb4) regulates the rate of cell proliferation in Chinese Hamster Ovary cells

Abstract

The β subunits of Voltage-Gated Calcium Channel (VGCC) are cytosolic proteins that interact with the VGCC pore -forming subunit and participate in the trafficking of the channel to the cell membrane and in ion influx regulation. β subunits also exert functions independently of their binding to VGCC by translocation to the cell nucleus including the control of gene expression. Mutations of the neuronal Cacnb4 (β₄) subunit are linked to human neuropsychiatric disorders including epilepsy and intellectual disabilities. It is believed that the pathogenic phenotype induced by these mutations is associated with channel-independent functions of the β₄ subunit. In this report, we investigated the role of β₄ subunit in cell proliferation and cell cycle progression and examined whether these functions could be altered by a pathogenic mutation. To this end, stably transfected Chinese Hamster Ovary (CHO-K1) cells expressing either rat full-length β₄ or the rat C-terminally truncated epileptic mutant variant β_1-481 were generated. The subcellular localization of both proteins differed significantly. Full-length β₄ localizes almost exclusively in the cell nucleus and concentrates into the nucleolar compartment, while the C-terminal-truncated β_1-481 subunit was less concentrated within the nucleus and absent from the nucleoli. Cell proliferation was found to be reduced by the expression of β₄, while it was unaffected by the epileptic mutant. Also, full-length β₄ interfered with cell cycle progression by presumably preventing cells from entering the S-phase via a mechanism that partially involves endogenous B56δ, a regulatory subunit of the phosphatase 2A (PP2A) that binds β₄ but not β_1-481. Analysis of β₄ subcellular distribution during the cell cycle revealed that the protein is highly expressed in the nucleus at the G1/S transition phase and that it is translocated out of the nucleus during chromatin condensation and cell division. These results suggest that nuclear accumulation of β₄ at the G1/S transition phase affects the progression into S-phase resulting in a decrease in the rate of cell proliferation. Regulation of the cell cycle exit is a critical step in determining the number of neuronal precursors and neuronal differentiation suggesting that mutations of the β₄ subunit could affect neural development and formation of the mature central nervous system.