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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

ST14A endogenous or recombinant CaV1.3 current is not modulated by GPCR activation.(A) Time course (left) and representative sweeps (right) of recombinant CaV1.3 current before (FPL), 1 min following 10 μM quinpirole application (Quin) and after removing Quin (Wash). (B–C) Endogenous, differentiated current traces in the presence of FPL across a range of test potentials (-50 mV to 0 mV) before (left) and after (right) application of (B) 10 μM Quin or (C) 10 μM oxotremorine M (Oxo-M). (D) Summary of percent inhibition of peak (left) and long-lasting tail (right) recombinant CaV1.3 or endogenous currents by D2R or M1R agonist; n = 3–7.
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pone.0132469.g003: ST14A endogenous or recombinant CaV1.3 current is not modulated by GPCR activation.(A) Time course (left) and representative sweeps (right) of recombinant CaV1.3 current before (FPL), 1 min following 10 μM quinpirole application (Quin) and after removing Quin (Wash). (B–C) Endogenous, differentiated current traces in the presence of FPL across a range of test potentials (-50 mV to 0 mV) before (left) and after (right) application of (B) 10 μM Quin or (C) 10 μM oxotremorine M (Oxo-M). (D) Summary of percent inhibition of peak (left) and long-lasting tail (right) recombinant CaV1.3 or endogenous currents by D2R or M1R agonist; n = 3–7.

Mentions: To determine whether D2R activation by the agonist quinpirole (Quin) inhibits CaV1.3 currents in ST14A cells, we recorded recombinant currents in the presence of FPL to enhance current amplitude. At a concentration of 10 μM, Quin had no significant effect on peak or tail current amplitude over time, (Fig 3Aleft) or in individual traces (Fig 3Aright). To determine if only recombinant CaV1.3 current was insensitive to D2R activation, we tested whether endogenous ST14A current from differentiated cells could undergo modulation. Again 10 μM Quin had no significant effect on endogenous peak or long-lasting tail current amplitude. Fig 3B shows representative current traces in the presence of 1 μM FPL from a range of voltages before and after Quin. Since a majority of MSNs express muscarinic M1 receptors (M1Rs) as well as dopamine receptors [37], we tested whether activation of this receptor would inhibit endogenous current. The muscarinic agonist oxotremorine-M (Oxo-M; 10 μM) had no effect on endogenous peak or long-lasting tail current amplitude. Fig 3C shows representative current traces in the presence of FPL from a range of voltages (-60 mV to -10 mV) tested before and after Oxo-M. Fig 3D summarizes the effect of Quin and Oxo-M on peak (left) and long-lasting tail current (right) from recombinant CaV1.3 versus endogenous ST14A current. These results suggest that the D2R and M1R signaling pathways, which inhibit LTC current in MSNs, are not intact in ST14A cells. However, application of dopamine or Quin, increases CREB phosphorylation in ST14A cells, indicating that D2Rs do couple to intact signaling cascades such as the adenylyl cyclase and MAPK pathways [29].


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

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

ST14A endogenous or recombinant CaV1.3 current is not modulated by GPCR activation.(A) Time course (left) and representative sweeps (right) of recombinant CaV1.3 current before (FPL), 1 min following 10 μM quinpirole application (Quin) and after removing Quin (Wash). (B–C) Endogenous, differentiated current traces in the presence of FPL across a range of test potentials (-50 mV to 0 mV) before (left) and after (right) application of (B) 10 μM Quin or (C) 10 μM oxotremorine M (Oxo-M). (D) Summary of percent inhibition of peak (left) and long-lasting tail (right) recombinant CaV1.3 or endogenous currents by D2R or M1R agonist; n = 3–7.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132469.g003: ST14A endogenous or recombinant CaV1.3 current is not modulated by GPCR activation.(A) Time course (left) and representative sweeps (right) of recombinant CaV1.3 current before (FPL), 1 min following 10 μM quinpirole application (Quin) and after removing Quin (Wash). (B–C) Endogenous, differentiated current traces in the presence of FPL across a range of test potentials (-50 mV to 0 mV) before (left) and after (right) application of (B) 10 μM Quin or (C) 10 μM oxotremorine M (Oxo-M). (D) Summary of percent inhibition of peak (left) and long-lasting tail (right) recombinant CaV1.3 or endogenous currents by D2R or M1R agonist; n = 3–7.
Mentions: To determine whether D2R activation by the agonist quinpirole (Quin) inhibits CaV1.3 currents in ST14A cells, we recorded recombinant currents in the presence of FPL to enhance current amplitude. At a concentration of 10 μM, Quin had no significant effect on peak or tail current amplitude over time, (Fig 3Aleft) or in individual traces (Fig 3Aright). To determine if only recombinant CaV1.3 current was insensitive to D2R activation, we tested whether endogenous ST14A current from differentiated cells could undergo modulation. Again 10 μM Quin had no significant effect on endogenous peak or long-lasting tail current amplitude. Fig 3B shows representative current traces in the presence of 1 μM FPL from a range of voltages before and after Quin. Since a majority of MSNs express muscarinic M1 receptors (M1Rs) as well as dopamine receptors [37], we tested whether activation of this receptor would inhibit endogenous current. The muscarinic agonist oxotremorine-M (Oxo-M; 10 μM) had no effect on endogenous peak or long-lasting tail current amplitude. Fig 3C shows representative current traces in the presence of FPL from a range of voltages (-60 mV to -10 mV) tested before and after Oxo-M. Fig 3D summarizes the effect of Quin and Oxo-M on peak (left) and long-lasting tail current (right) from recombinant CaV1.3 versus endogenous ST14A current. These results suggest that the D2R and M1R signaling pathways, which inhibit LTC current in MSNs, are not intact in ST14A cells. However, application of dopamine or Quin, increases CREB phosphorylation in ST14A cells, indicating that D2Rs do couple to intact signaling cascades such as the adenylyl cyclase and MAPK pathways [29].

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