Limits...
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 cells have endogenous Ca2+ currents but are capable of expressing recombinant current.(A-B) Currents were recorded from round (undifferentiated, no processes) or differentiated (having processes) endogenous ST14A cells. Note: round cells from ST14A cells grown at either 33°C or 37°C exhibited little to no current and were pooled. Currents were recorded from round ST14A cells grown at 33°C, transfected and grown at 37°C for at least 24 hours before recording. Each set of cells were recorded in the absence (white bars) and presence of 1 μM FPL (black bars). Summary of peak Ca2+ currents measured from a holding potential of -90 mV to a test potential of +10 mV (A) and then to a tail potential of -40 mV (B). Endogenous current from differentiated cells was significantly larger than endogenous current from round cells (†; p < 0.05); Transfected current was significantly larger than endogenous current from round cells (‡; p < 0.05); FPL significantly increased differentiated endogenous and transfected tail current (*; p < 0.05), n = 7–15. (C) Transfected ST14A cells expressed GFP throughout the cell soma and in a small percentage of differentiated cells, in the processes. Images (20X magnification) were captured ~24 hours post-transfection. The transfection rate for these cells was ~50%. (D) Top: Protocol for eliciting currents from a holding potential of -90 mV to the test potential of +10 mV for 100 ms before repolarizing to -40 ms. Individual traces from round endogenous (middle) or round transfected (bottom) cells. Dashed lines indicate where peak and tail current were measured 65 ms after depolarization and 15 ms after repolarization, respectively. In the presence of LTC agonist, 1 μM FPL, both the peak and long-lasting tail current increase (gray trace) and display slowed activation and deactivation kinetics, characteristic of FPL-induced L-current.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492559&req=5

pone.0132469.g001: ST14A cells have endogenous Ca2+ currents but are capable of expressing recombinant current.(A-B) Currents were recorded from round (undifferentiated, no processes) or differentiated (having processes) endogenous ST14A cells. Note: round cells from ST14A cells grown at either 33°C or 37°C exhibited little to no current and were pooled. Currents were recorded from round ST14A cells grown at 33°C, transfected and grown at 37°C for at least 24 hours before recording. Each set of cells were recorded in the absence (white bars) and presence of 1 μM FPL (black bars). Summary of peak Ca2+ currents measured from a holding potential of -90 mV to a test potential of +10 mV (A) and then to a tail potential of -40 mV (B). Endogenous current from differentiated cells was significantly larger than endogenous current from round cells (†; p < 0.05); Transfected current was significantly larger than endogenous current from round cells (‡; p < 0.05); FPL significantly increased differentiated endogenous and transfected tail current (*; p < 0.05), n = 7–15. (C) Transfected ST14A cells expressed GFP throughout the cell soma and in a small percentage of differentiated cells, in the processes. Images (20X magnification) were captured ~24 hours post-transfection. The transfection rate for these cells was ~50%. (D) Top: Protocol for eliciting currents from a holding potential of -90 mV to the test potential of +10 mV for 100 ms before repolarizing to -40 ms. Individual traces from round endogenous (middle) or round transfected (bottom) cells. Dashed lines indicate where peak and tail current were measured 65 ms after depolarization and 15 ms after repolarization, respectively. In the presence of LTC agonist, 1 μM FPL, both the peak and long-lasting tail current increase (gray trace) and display slowed activation and deactivation kinetics, characteristic of FPL-induced L-current.

Mentions: To determine if ST14A cells express endogenous Ca2+ current, we measured whole-cell currents from cells grown at 33 or 37°C. Two populations of cells were distinguished based on morphology: round or differentiated, with the definition of having neuron-like projections. From a holding potential of -90 mV, round cells exhibited zero to little endogenous peak or tail current measured at +10 mV or -40 mV respectively, regardless of the temperature at which the cells were grown (Fig 1A). Differentiated cells had significantly more endogenous current than round cells at both potentials (Fig 1B). Application of the LTC agonist, FPL 64176 (FPL; 1 μM) enhanced endogenous currents at both potentials (Fig 1A and 1B), indicating that at least a component of the endogenous current was LTC. Since round cells showed little to no LTC current, we transfected the ST14A cells with LTC channel subunits, CaV1.3, β2a, and α2δ-1, along with green fluorescent protein (GFP) and recorded whole-cell currents from green fluorescing round cells (Fig 1C). Peak and tail currents from round transfected cells were significantly larger than those recorded from round untransfected cells (Fig 1A and 1B). FPL significantly enhanced recombinant current at -40 mV. Fig 1D shows representative individual traces of round endogenous or round recombinant current before and after application of FPL. The recombinant current shows little to no inactivation with 20 mM Ba2+ as the charge carrier [35], typical of LTC current coexpressed with the accessory subunit, β2a [36].


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

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

ST14A cells have endogenous Ca2+ currents but are capable of expressing recombinant current.(A-B) Currents were recorded from round (undifferentiated, no processes) or differentiated (having processes) endogenous ST14A cells. Note: round cells from ST14A cells grown at either 33°C or 37°C exhibited little to no current and were pooled. Currents were recorded from round ST14A cells grown at 33°C, transfected and grown at 37°C for at least 24 hours before recording. Each set of cells were recorded in the absence (white bars) and presence of 1 μM FPL (black bars). Summary of peak Ca2+ currents measured from a holding potential of -90 mV to a test potential of +10 mV (A) and then to a tail potential of -40 mV (B). Endogenous current from differentiated cells was significantly larger than endogenous current from round cells (†; p < 0.05); Transfected current was significantly larger than endogenous current from round cells (‡; p < 0.05); FPL significantly increased differentiated endogenous and transfected tail current (*; p < 0.05), n = 7–15. (C) Transfected ST14A cells expressed GFP throughout the cell soma and in a small percentage of differentiated cells, in the processes. Images (20X magnification) were captured ~24 hours post-transfection. The transfection rate for these cells was ~50%. (D) Top: Protocol for eliciting currents from a holding potential of -90 mV to the test potential of +10 mV for 100 ms before repolarizing to -40 ms. Individual traces from round endogenous (middle) or round transfected (bottom) cells. Dashed lines indicate where peak and tail current were measured 65 ms after depolarization and 15 ms after repolarization, respectively. In the presence of LTC agonist, 1 μM FPL, both the peak and long-lasting tail current increase (gray trace) and display slowed activation and deactivation kinetics, characteristic of FPL-induced L-current.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132469.g001: ST14A cells have endogenous Ca2+ currents but are capable of expressing recombinant current.(A-B) Currents were recorded from round (undifferentiated, no processes) or differentiated (having processes) endogenous ST14A cells. Note: round cells from ST14A cells grown at either 33°C or 37°C exhibited little to no current and were pooled. Currents were recorded from round ST14A cells grown at 33°C, transfected and grown at 37°C for at least 24 hours before recording. Each set of cells were recorded in the absence (white bars) and presence of 1 μM FPL (black bars). Summary of peak Ca2+ currents measured from a holding potential of -90 mV to a test potential of +10 mV (A) and then to a tail potential of -40 mV (B). Endogenous current from differentiated cells was significantly larger than endogenous current from round cells (†; p < 0.05); Transfected current was significantly larger than endogenous current from round cells (‡; p < 0.05); FPL significantly increased differentiated endogenous and transfected tail current (*; p < 0.05), n = 7–15. (C) Transfected ST14A cells expressed GFP throughout the cell soma and in a small percentage of differentiated cells, in the processes. Images (20X magnification) were captured ~24 hours post-transfection. The transfection rate for these cells was ~50%. (D) Top: Protocol for eliciting currents from a holding potential of -90 mV to the test potential of +10 mV for 100 ms before repolarizing to -40 ms. Individual traces from round endogenous (middle) or round transfected (bottom) cells. Dashed lines indicate where peak and tail current were measured 65 ms after depolarization and 15 ms after repolarization, respectively. In the presence of LTC agonist, 1 μM FPL, both the peak and long-lasting tail current increase (gray trace) and display slowed activation and deactivation kinetics, characteristic of FPL-induced L-current.
Mentions: To determine if ST14A cells express endogenous Ca2+ current, we measured whole-cell currents from cells grown at 33 or 37°C. Two populations of cells were distinguished based on morphology: round or differentiated, with the definition of having neuron-like projections. From a holding potential of -90 mV, round cells exhibited zero to little endogenous peak or tail current measured at +10 mV or -40 mV respectively, regardless of the temperature at which the cells were grown (Fig 1A). Differentiated cells had significantly more endogenous current than round cells at both potentials (Fig 1B). Application of the LTC agonist, FPL 64176 (FPL; 1 μM) enhanced endogenous currents at both potentials (Fig 1A and 1B), indicating that at least a component of the endogenous current was LTC. Since round cells showed little to no LTC current, we transfected the ST14A cells with LTC channel subunits, CaV1.3, β2a, and α2δ-1, along with green fluorescent protein (GFP) and recorded whole-cell currents from green fluorescing round cells (Fig 1C). Peak and tail currents from round transfected cells were significantly larger than those recorded from round untransfected cells (Fig 1A and 1B). FPL significantly enhanced recombinant current at -40 mV. Fig 1D shows representative individual traces of round endogenous or round recombinant current before and after application of FPL. The recombinant current shows little to no inactivation with 20 mM Ba2+ as the charge carrier [35], typical of LTC current coexpressed with the accessory subunit, β2a [36].

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