1. Suspended Swiss 3T3 fibroblasts were voltage clamped using the whole-cell technique. 2. Passage from the cell-attached to the whole-cell mode was accompanied by only a minor decrease in input resistance. Direct measurement of resting potential gave values between O and -15 mV. 3. In order to account for the effects of leak on the membrane potential measurements, I-V curves were obtained immediately before and after patch rupture by applying voltage ramps. After subtraction of the cell-attached current from the whole-cell current, the true membrane potential was estimated as the zero-current potential in the I-V curve. An average value of -8.2 +/- 0.9 mV in 8 mM-Ca2+ was obtained in this way. 4. In 2 mM-Ca2+, step depolarizations 100 ms long from holding potentials (Vh) more negative than -60 mV caused a transient inward current to appear. From Vh greater than -60 mV only a linear leakage component was apparent. 5. In 2 mM-Ca2+ depolarizations to potentials greater than +40 mV (from Vh = -100 mV) generated transient, outwardly directed currents. 6. Increasing extracellular Ca2+ up to 32 mM shifted the peak current vs. voltage curve and the reversal potential (Erev) towards more positive potentials, and caused an increase of the peak current. 7. The steady-state inactivation curve was the same for both inward and outward currents, indicating that they flow through the same channels. The currents are completely inactivated at V = -60 mV. 8. Recovery of the fully inactivated current upon hyperpolarization had an exponential time course with tau = 0.22 s at V = -80 mV and tau = 0.18 s at V = -100 mV. 9. In the absence of Ca2+ (but with Mg2+ present) the inward current disappeared but a large, inactivating outward current appeared when V greater than 0 mV. The current was strongly reduced by Cd2+ (1 mM) or Co2+ (10 mM). 10. Complete removal of divalent cations from the external solution caused the channel to become highly permeable to monovalent cations. 11. Nitrendipine (10 microM) and verapamil (5 microM) were unable to block the current. 12. On the whole the present results indicate that voltage-dependent Ca2+ channels are present in these cells. Their sensitivity to divalent cations, to organic blockers and to potential is similar to that of the low-voltage-activated, or 'T' type, Ca2+ channels described in other cells.
Properties of the voltage-dependent calcium channel of mouse Swiss 3T3 fibroblasts.
PERES, ANTONIO;
1988-01-01
Abstract
1. Suspended Swiss 3T3 fibroblasts were voltage clamped using the whole-cell technique. 2. Passage from the cell-attached to the whole-cell mode was accompanied by only a minor decrease in input resistance. Direct measurement of resting potential gave values between O and -15 mV. 3. In order to account for the effects of leak on the membrane potential measurements, I-V curves were obtained immediately before and after patch rupture by applying voltage ramps. After subtraction of the cell-attached current from the whole-cell current, the true membrane potential was estimated as the zero-current potential in the I-V curve. An average value of -8.2 +/- 0.9 mV in 8 mM-Ca2+ was obtained in this way. 4. In 2 mM-Ca2+, step depolarizations 100 ms long from holding potentials (Vh) more negative than -60 mV caused a transient inward current to appear. From Vh greater than -60 mV only a linear leakage component was apparent. 5. In 2 mM-Ca2+ depolarizations to potentials greater than +40 mV (from Vh = -100 mV) generated transient, outwardly directed currents. 6. Increasing extracellular Ca2+ up to 32 mM shifted the peak current vs. voltage curve and the reversal potential (Erev) towards more positive potentials, and caused an increase of the peak current. 7. The steady-state inactivation curve was the same for both inward and outward currents, indicating that they flow through the same channels. The currents are completely inactivated at V = -60 mV. 8. Recovery of the fully inactivated current upon hyperpolarization had an exponential time course with tau = 0.22 s at V = -80 mV and tau = 0.18 s at V = -100 mV. 9. In the absence of Ca2+ (but with Mg2+ present) the inward current disappeared but a large, inactivating outward current appeared when V greater than 0 mV. The current was strongly reduced by Cd2+ (1 mM) or Co2+ (10 mM). 10. Complete removal of divalent cations from the external solution caused the channel to become highly permeable to monovalent cations. 11. Nitrendipine (10 microM) and verapamil (5 microM) were unable to block the current. 12. On the whole the present results indicate that voltage-dependent Ca2+ channels are present in these cells. Their sensitivity to divalent cations, to organic blockers and to potential is similar to that of the low-voltage-activated, or 'T' type, Ca2+ channels described in other cells.
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