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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

Transfected CaV1.3 currents in ST14A cells are inhibited by AA.(A) Individual sweeps with 1 μM FPL (black trace), and 1 min after 10 μM AA (dotted trace) in the continued presence of FPL. AA inhibited both peak and tail currents; n = 4. (B) I-V relationship of recombinant current in the presence of FPL (●), FPL + AA (○), and after washing off AA with bath solution containing FPL (Δ). (C) Time course of CaV1.3 current at -10 mV with 10 μM AA. After 4 min, AA was washed off with 1 mg/ml BSA. (D) Summary of CaV1.3 current inhibition by AA over time (open bars) and its recovery following BSA (Black bar); *p < 0.001; n = 5–7. (E) Time course of peak CaV1.3 current with 10 μM oleic acid (OA); n = 3.
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pone.0132469.g006: Transfected CaV1.3 currents in ST14A cells are inhibited by AA.(A) Individual sweeps with 1 μM FPL (black trace), and 1 min after 10 μM AA (dotted trace) in the continued presence of FPL. AA inhibited both peak and tail currents; n = 4. (B) I-V relationship of recombinant current in the presence of FPL (●), FPL + AA (○), and after washing off AA with bath solution containing FPL (Δ). (C) Time course of CaV1.3 current at -10 mV with 10 μM AA. After 4 min, AA was washed off with 1 mg/ml BSA. (D) Summary of CaV1.3 current inhibition by AA over time (open bars) and its recovery following BSA (Black bar); *p < 0.001; n = 5–7. (E) Time course of peak CaV1.3 current with 10 μM oleic acid (OA); n = 3.

Mentions: To circumvent the absence of a key signaling molecule, and determine whether CaV1.3 could be modulated, we directly applied exogenous AA (10 μM) to the bath and measured CaV1.3 recombinant current over several minutes. In the presence of FPL, AA inhibited CaV1.3 peak and long-lasting tail currents by 40 ± 12% and 29 ± 25% respectively after 1 min (n = 4). Fig 6A shows representative sweeps before (FPL) and after AA application (FPL + AA). The large variability in tail current inhibition by AA suggested that voltage may be important for this modulation. However, AA inhibited CaV1.3 current at all voltages when tested over a range of test potentials as shown in the I-V plot in Fig 6B. Bovine serum albumin (BSA), which binds free fatty acids [43], reversed inhibition at all test potentials. To show that inhibition was not due to AA competing with FPL, we measured CaV1.3 current in the absence of FPL prior to and after application of AA (Fig 6C) and still observed inhibition that could be reversed after adding BSA. Inhibition of CaV1.3 over time by AA (open bars) and recovery by BSA (solid bar) is summarized in Fig 6D. Conversely, oleic acid (10 μM) enhanced current by 7.9 ± 0.7% after 1 minute (Fig 6E; p < 0.001; n = 3), suggesting that inhibition by AA is not simply the result of a nonspecific fatty acid effect. Moreover, inhibition of CaV1.3 by AA demonstrates that the transfected CaV1.3 LTCs in ST14A cells are capable of modulation.


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

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

Transfected CaV1.3 currents in ST14A cells are inhibited by AA.(A) Individual sweeps with 1 μM FPL (black trace), and 1 min after 10 μM AA (dotted trace) in the continued presence of FPL. AA inhibited both peak and tail currents; n = 4. (B) I-V relationship of recombinant current in the presence of FPL (●), FPL + AA (○), and after washing off AA with bath solution containing FPL (Δ). (C) Time course of CaV1.3 current at -10 mV with 10 μM AA. After 4 min, AA was washed off with 1 mg/ml BSA. (D) Summary of CaV1.3 current inhibition by AA over time (open bars) and its recovery following BSA (Black bar); *p < 0.001; n = 5–7. (E) Time course of peak CaV1.3 current with 10 μM oleic acid (OA); n = 3.
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pone.0132469.g006: Transfected CaV1.3 currents in ST14A cells are inhibited by AA.(A) Individual sweeps with 1 μM FPL (black trace), and 1 min after 10 μM AA (dotted trace) in the continued presence of FPL. AA inhibited both peak and tail currents; n = 4. (B) I-V relationship of recombinant current in the presence of FPL (●), FPL + AA (○), and after washing off AA with bath solution containing FPL (Δ). (C) Time course of CaV1.3 current at -10 mV with 10 μM AA. After 4 min, AA was washed off with 1 mg/ml BSA. (D) Summary of CaV1.3 current inhibition by AA over time (open bars) and its recovery following BSA (Black bar); *p < 0.001; n = 5–7. (E) Time course of peak CaV1.3 current with 10 μM oleic acid (OA); n = 3.
Mentions: To circumvent the absence of a key signaling molecule, and determine whether CaV1.3 could be modulated, we directly applied exogenous AA (10 μM) to the bath and measured CaV1.3 recombinant current over several minutes. In the presence of FPL, AA inhibited CaV1.3 peak and long-lasting tail currents by 40 ± 12% and 29 ± 25% respectively after 1 min (n = 4). Fig 6A shows representative sweeps before (FPL) and after AA application (FPL + AA). The large variability in tail current inhibition by AA suggested that voltage may be important for this modulation. However, AA inhibited CaV1.3 current at all voltages when tested over a range of test potentials as shown in the I-V plot in Fig 6B. Bovine serum albumin (BSA), which binds free fatty acids [43], reversed inhibition at all test potentials. To show that inhibition was not due to AA competing with FPL, we measured CaV1.3 current in the absence of FPL prior to and after application of AA (Fig 6C) and still observed inhibition that could be reversed after adding BSA. Inhibition of CaV1.3 over time by AA (open bars) and recovery by BSA (solid bar) is summarized in Fig 6D. Conversely, oleic acid (10 μM) enhanced current by 7.9 ± 0.7% after 1 minute (Fig 6E; p < 0.001; n = 3), suggesting that inhibition by AA is not simply the result of a nonspecific fatty acid effect. Moreover, inhibition of CaV1.3 by AA demonstrates that the transfected CaV1.3 LTCs in ST14A cells are capable of modulation.

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