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Characterization of ST14A Cells for Studying Modulation of Voltage-Gated Calcium Channels.

Roberts-Crowley ML, Rittenhouse AR - PLoS ONE (2015)

Bottom Line: 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.Thus, ST14A cells provide a MSN-like cell line for studying channel modulation and signaling pathways that do not involve activation of PLCβ-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Program in Neuroscience, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
In medium spiny neurons (MSNs) of the striatum, dopamine D2 receptors (D2Rs) 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 D2R 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 A2 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 D2Rs. 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.

No MeSH data available.


Related in: MedlinePlus

Whole-cell currents from transfected ST14A cells have biophysical and pharmacological properties of CaV1.3 LTCs.(A) The current-voltage (I-V) relationship shows that recombinant CaV1.3 current activates at approximately -60 mV; n = 15. (B) Example time course of peak current at -10 mV. 1 μM ω-conotoxin GVIA (CTX, an N-type Ca2+ channel antagonist) was added for 2 min. Bath solution was exchanged with 1 μM nimodipine (NIM, an LTC antagonist) for 2 min or until a new stable baseline was reached and then washed off to show reversibility. FPL (1 μM) was added at the end of the recording. (C) Summary of pharmacological inhibition of transfected current; n = 3–6.
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pone.0132469.g002: Whole-cell currents from transfected ST14A cells have biophysical and pharmacological properties of CaV1.3 LTCs.(A) The current-voltage (I-V) relationship shows that recombinant CaV1.3 current activates at approximately -60 mV; n = 15. (B) Example time course of peak current at -10 mV. 1 μM ω-conotoxin GVIA (CTX, an N-type Ca2+ channel antagonist) was added for 2 min. Bath solution was exchanged with 1 μM nimodipine (NIM, an LTC antagonist) for 2 min or until a new stable baseline was reached and then washed off to show reversibility. FPL (1 μM) was added at the end of the recording. (C) Summary of pharmacological inhibition of transfected current; n = 3–6.

Mentions: We characterized recombinant CaV1.3 current further to determine whether transfected channel activity in ST14A cells exhibits biophysical and pharmacological properties of CaV1.3 observed in oocytes and HEK 293 cells [30]. First, we measured peak current across a range of voltages to show that channels open at relatively negative voltages compared to CaV1.2 [30]. Indeed, in 20 mM Ba2+, CaV1.3 currents activated at a test potential of -60 mV and peaked at -10 mV to 0 mV (Fig 2A). Recombinant current in ST14A cells exhibited a CaV1.3 LTC pharmacological profile. Fig 2B shows a time course of the effects of Ca2+ channel ligands on CaV1.3 current. The current was insensitive to the N-type Ca2+ channel antagonist, ω-conotoxin GVIA (1 μM; CTX) but was inhibited in a concentration-dependent manner by the LTC antagonist, nimodipine (0.1–3.0 μM; NIM). The inhibition produced by 1.0 μM NIM (51.8 ± 4%) is characteristic of the low-voltage sensitivity of CaV1.3 compared to CaV1.2, which would be fully blocked by 1.0 μM NIM [30]. Inhibition fully reversed by washing with bath solution. After wash out, channels remained sensitive to FPL. The antagonist data are summarized in the bar graph in Fig 2C. These findings show that recombinant current in ST14A cells displayed both current-voltage and pharmacological profiles specific to CaV1.3 channels.


Characterization of ST14A Cells for Studying Modulation of Voltage-Gated Calcium Channels.

Roberts-Crowley ML, Rittenhouse AR - PLoS ONE (2015)

Whole-cell currents from transfected ST14A cells have biophysical and pharmacological properties of CaV1.3 LTCs.(A) The current-voltage (I-V) relationship shows that recombinant CaV1.3 current activates at approximately -60 mV; n = 15. (B) Example time course of peak current at -10 mV. 1 μM ω-conotoxin GVIA (CTX, an N-type Ca2+ channel antagonist) was added for 2 min. Bath solution was exchanged with 1 μM nimodipine (NIM, an LTC antagonist) for 2 min or until a new stable baseline was reached and then washed off to show reversibility. FPL (1 μM) was added at the end of the recording. (C) Summary of pharmacological inhibition of transfected current; n = 3–6.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492559&req=5

pone.0132469.g002: Whole-cell currents from transfected ST14A cells have biophysical and pharmacological properties of CaV1.3 LTCs.(A) The current-voltage (I-V) relationship shows that recombinant CaV1.3 current activates at approximately -60 mV; n = 15. (B) Example time course of peak current at -10 mV. 1 μM ω-conotoxin GVIA (CTX, an N-type Ca2+ channel antagonist) was added for 2 min. Bath solution was exchanged with 1 μM nimodipine (NIM, an LTC antagonist) for 2 min or until a new stable baseline was reached and then washed off to show reversibility. FPL (1 μM) was added at the end of the recording. (C) Summary of pharmacological inhibition of transfected current; n = 3–6.
Mentions: We characterized recombinant CaV1.3 current further to determine whether transfected channel activity in ST14A cells exhibits biophysical and pharmacological properties of CaV1.3 observed in oocytes and HEK 293 cells [30]. First, we measured peak current across a range of voltages to show that channels open at relatively negative voltages compared to CaV1.2 [30]. Indeed, in 20 mM Ba2+, CaV1.3 currents activated at a test potential of -60 mV and peaked at -10 mV to 0 mV (Fig 2A). Recombinant current in ST14A cells exhibited a CaV1.3 LTC pharmacological profile. Fig 2B shows a time course of the effects of Ca2+ channel ligands on CaV1.3 current. The current was insensitive to the N-type Ca2+ channel antagonist, ω-conotoxin GVIA (1 μM; CTX) but was inhibited in a concentration-dependent manner by the LTC antagonist, nimodipine (0.1–3.0 μM; NIM). The inhibition produced by 1.0 μM NIM (51.8 ± 4%) is characteristic of the low-voltage sensitivity of CaV1.3 compared to CaV1.2, which would be fully blocked by 1.0 μM NIM [30]. Inhibition fully reversed by washing with bath solution. After wash out, channels remained sensitive to FPL. The antagonist data are summarized in the bar graph in Fig 2C. These findings show that recombinant current in ST14A cells displayed both current-voltage and pharmacological profiles specific to CaV1.3 channels.

Bottom Line: 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.Thus, ST14A cells provide a MSN-like cell line for studying channel modulation and signaling pathways that do not involve activation of PLCβ-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Program in Neuroscience, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
In medium spiny neurons (MSNs) of the striatum, dopamine D2 receptors (D2Rs) 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 D2R 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 A2 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 D2Rs. 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.

No MeSH data available.


Related in: MedlinePlus