Characterization of ST14A cells for studying modulation of voltage-gated calcium channels

Abstract

In medium spiny neurons (MSNs) of the striatum, dopamine D 2 receptors (D 2 Rs) specifically inhibit the Ca v 1.3 subtype of L-type Ca 2+ channels (LTCs). MSNs are heterogeneous in their expression of dopamine receptors making the study of D 2 R pathways difficult in primary neurons. Here, we employed the ST14A cell line, derived from embryonic striatum and characterized to have properties of MSNs, to study Ca v 1.3 current and its modulation by neurotransmitters. Round, undifferentiated ST14A cells exhibited little to no endogenous Ca 2+ current while differentiated ST14A cells expressed endogenous Ca 2+ current. Transfection with LTC subunits produced functional Ca v 1.3 current from round cells, providing a homogeneous model system compared to native MSNs for studying D 2 R pathways. However, neither endogenous nor recombinant Ca v 1.3 current was modulated by the D 2 R agonist quinpirole. We confirmed D 2 R expression in ST14A cells and also detected D 1 Rs, D 4 Rs, D 5 Rs, G q, calcineurin and phospholipase A 2 using RT-PCR and/or Western blot analysis. Phospholipase C β-1 (PLCβ-1) expression was not detected by Western blot analysis which may account for the lack of LTC modulation by D 2 Rs. These findings raise caution about the assumption that the presence of G-protein coupled receptors in cell lines indicates the presence of complete signaling cascades. However, exogenous arachidonic acid inhibited recombinant Ca v 1.3 current indicating that channels expressed in ST14A cells are capable of modulation since they respond to a known signaling molecule downstream of D 2 Rs. Thus, ST14A cells provide a MSN-like cell line for studying channel modulation and signaling pathways that do not involve activation of PLCβ-1. Copyright: