CaV 3.1 and CaV 3.3 account for T-type Ca2+ current in GH3 cells

T-type Ca2+ channels are important for cell signaling by a variety of cells. We report here the electrophysiological and molecular characteristics of the whole-cell Ca2+ current in GH3 clonal pituitary cells. The current inactivation at 0 mV was described by a single exponential function with a time constant of 18.32 ± 1.87 ms (N = 16). The I-V relationship measured with Ca2+ as a charge carrier was shifted to the left when we applied a conditioning pre-pulse of up to -120 mV, indicating that a low voltage-activated current may be present in GH3 cells. Transient currents were first activated at -50 mV and peaked around -20 mV. The half-maximal voltage activation and the slope factors for the two conditions are -35.02 ± 2.4 and 6.7 ± 0.3 mV (pre-pulse of -120 mV, N = 15), and -27.0 ± 0.97 and 7.5 ± 0.7 mV (pre-pulse of -40 mV, N = 9). The 8-mV shift in the activation mid-point was statistically significant (P < 0.05). The tail currents decayed bi-exponentially suggesting two different T-type Ca2+ channel populations. RT-PCR revealed the presence of a1G (CaV3.1) and a1I (CaV3.3) T-type Ca2+ channel mRNA transcripts.

Calcium channel; T-type; Electrophysiology; RT-PCR; GH3 cells

Authorship

M.A. Mudado

Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas , Departamento de Bioquímica e Imunologia, Minas Gerais, BrazilUniversidade Federal de Minas GeraisBrazilMinas Gerais, BrazilUniversidade Federal de Minas Gerais, Instituto de Ciências Biológicas , Departamento de Bioquímica e Imunologia, Minas Gerais, Brazil

A.L. Rodrigues

V.F. Prado

Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas , Departamento de Bioquímica e Imunologia, Belo Horizonte, Minas Gerais, BrazilUniversidade Federal de Minas GeraisBrazilBelo Horizonte, Minas Gerais, BrazilUniversidade Federal de Minas Gerais, Instituto de Ciências Biológicas , Departamento de Bioquímica e Imunologia, Belo Horizonte, Minas Gerais, Brazil

P.S.L. Beirão

J.S. Cruz

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (6)
Tables (1)

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Figure 1.
Whole-cell inward Ca²⁺ currents evoked by a double-pulse protocol in GH3 neuroendocrine cells. A, Representative traces in response to step depolarization to 0 mV for 120 ms from a holding potential of -80 mV. The first current trace corresponds to the activation of both T-type (transient) and L-type Ca²⁺ (sustained) currents. The second trace was recorded after a brief time at -80 mV (10 ms) and then a return to 0 mV. The recorded current trace represents only L-type Ca²⁺ current (sustained) because T-type channels were inactivated. B, Current trace obtained by digitally subtracting the current evoked after the pre-pulse from that before the pre-pulse as in A.

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Figure 2.
Current-voltage relationships (I-V) in GH3 cells. A, Current traces evoked by depolarizations from -60 mV to -20 mV (holding potential -80 mV, pre-pulse -120 mV). B, Current traces elicited by depolarizations from -50 mV to -10 mV (holding potential -80 mV, pre-pulse -40 mV). C, Averaged density current-voltage relationship obtained for GH3 Ca²⁺ currents (filled circles, N = 15). Currents were normalized to cell capacitance to avoid variability due to cell size. D, Averaged current density-voltage relationships for peak currents obtained using a pre-pulse at -120 mV (continuous line), and after subtracting current traces between the two different holding potentials (Figure 2D, open circles, N = 7). All recordings were done using 5 mM calcium as the charge carrier. Data in panels C and D are reported as means ± SEM.

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Figure 3.
Voltage-dependent activation of GH3 Ca²⁺ currents. The peak current at each potential was normalized to the maximum peak current and was averaged for each potential. The symbols indicate composite data obtained from a pre-pulse set at -120 mV (squares, N = 15) and from a pre-pulse at -40 mV (circles, N = 9). Solid lines represent the fitting of Boltzmann equations with half-activation voltages of -35.02 mV (pre-pulse at -120 mV) and -27.04 mV (pre-pulse at -40 mV) and slope factors of 6.65 (pre-pulse at -120 mV) and 7.51 (pre-pulse at -40 mV).

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Figure 4.
Voltage dependence of deactivation kinetics. A, Representative calcium current tail traces evoked by different repolarizing potentials between -120 and -30 mV. The pipette solution contained a high chloride concentration. B, Representative calcium tail currents. The voltage protocol was the same as in A. The pipette solution had a high concentration of fluoride ion. C, Plot of mean deactivation time constants (t) against repolarization potentials. Data points are the two time constants, which represent the fast (open circles, N = 8) and slow components of the tail (filled circles, N = 8) current decay shown in panel A. Also shown is the weighted t (t_w, triangles) that was derived using the equation: t_w = A₁t_fast + A₂t_slow, where A₁ and A₂are the normalized amplitudes of the fast and slow components. D, Deactivation time constants plotted as in C. The fast (open circles, N = 4) and slow (filled circles, N = 4) components are represented and were taken from current traces as shown in panel B. The triangles indicate the weighted time constant.

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Figure 5.
Exponential fits to tail currents. Records are from Figure 4B on an expanded time scale. The thicker smooth gray lines are fits to a single exponential (A, left) or the sum of two exponentials (B, right).

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Figure 6.
T-type Ca²⁺ channel subtypes in rat GH3 cells. RT-PCR performed to identify the presence of mRNA for the a1G and a1I subunits in GH3 cells. The positive control contained primers specific for ß-actin (lane 2). Negative controls did not include RNA or reverse transcriptase (RT) in the tubes (lanes 3, 4, 6, 7, 9 and 10).

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Figure 1. Whole-cell inward Ca²⁺ currents evoked by a double-pulse protocol in GH3 neuroendocrine cells. A, Representative traces in response to step depolarization to 0 mV for 120 ms from a holding potential of -80 mV. The first current trace corresponds to the activation of both T-type (transient) and L-type Ca²⁺ (sustained) currents. The second trace was recorded after a brief time at -80 mV (10 ms) and then a return to 0 mV. The recorded current trace represents only L-type Ca²⁺ current (sustained) because T-type channels were inactivated. B, Current trace obtained by digitally subtracting the current evoked after the pre-pulse from that before the pre-pulse as in A.

Figure 2. Current-voltage relationships (I-V) in GH3 cells. A, Current traces evoked by depolarizations from -60 mV to -20 mV (holding potential -80 mV, pre-pulse -120 mV). B, Current traces elicited by depolarizations from -50 mV to -10 mV (holding potential -80 mV, pre-pulse -40 mV). C, Averaged density current-voltage relationship obtained for GH3 Ca²⁺ currents (filled circles, N = 15). Currents were normalized to cell capacitance to avoid variability due to cell size. D, Averaged current density-voltage relationships for peak currents obtained using a pre-pulse at -120 mV (continuous line), and after subtracting current traces between the two different holding potentials (Figure 2D, open circles, N = 7). All recordings were done using 5 mM calcium as the charge carrier. Data in panels C and D are reported as means ± SEM.

Figure 3. Voltage-dependent activation of GH3 Ca²⁺ currents. The peak current at each potential was normalized to the maximum peak current and was averaged for each potential. The symbols indicate composite data obtained from a pre-pulse set at -120 mV (squares, N = 15) and from a pre-pulse at -40 mV (circles, N = 9). Solid lines represent the fitting of Boltzmann equations with half-activation voltages of -35.02 mV (pre-pulse at -120 mV) and -27.04 mV (pre-pulse at -40 mV) and slope factors of 6.65 (pre-pulse at -120 mV) and 7.51 (pre-pulse at -40 mV).

Figure 4. Voltage dependence of deactivation kinetics. A, Representative calcium current tail traces evoked by different repolarizing potentials between -120 and -30 mV. The pipette solution contained a high chloride concentration. B, Representative calcium tail currents. The voltage protocol was the same as in A. The pipette solution had a high concentration of fluoride ion. C, Plot of mean deactivation time constants (t) against repolarization potentials. Data points are the two time constants, which represent the fast (open circles, N = 8) and slow components of the tail (filled circles, N = 8) current decay shown in panel A. Also shown is the weighted t (t_w, triangles) that was derived using the equation: t_w = A₁t_fast + A₂t_slow, where A₁ and A₂are the normalized amplitudes of the fast and slow components. D, Deactivation time constants plotted as in C. The fast (open circles, N = 4) and slow (filled circles, N = 4) components are represented and were taken from current traces as shown in panel B. The triangles indicate the weighted time constant.

Figure 5. Exponential fits to tail currents. Records are from Figure 4B on an expanded time scale. The thicker smooth gray lines are fits to a single exponential (A, left) or the sum of two exponentials (B, right).

Figure 6. T-type Ca²⁺ channel subtypes in rat GH3 cells. RT-PCR performed to identify the presence of mRNA for the a1G and a1I subunits in GH3 cells. The positive control contained primers specific for ß-actin (lane 2). Negative controls did not include RNA or reverse transcriptase (RT) in the tubes (lanes 3, 4, 6, 7, 9 and 10).

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CaV 3.1 and CaV 3.3 account for T-type Ca2+ current in GH3 cells

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