Two-suction-electrode voltage-clamp analysis of the sustained calcium current in cat sensory neurones

William Taylor

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Abstract

1. The kinetics of the sustained calcium current were examined in cat dorsal root ganglion (DRG) neurones, using a two-suction-electrode voltage clamp. It was shown that this current could be examined with minimal contamination from other ionic currents. Experiments were performed at 20°C, with a concentration of 10 mM-calcium externally. 2. The transient calcium current was eliminated by using a holding potential of -50 mV. The sustained calcium current showed no evidence of steady-state inactivation at potentials between -90 and -30 mV. 3. The activation and deactivation time course of the calcium current was described by a double-exponential function. The activation process was examined without interference from significant inactivation under the conditions used. 4. The steady-state activation of the calcium channels was approximated by a two-step activation process. Both reactions were voltage sensitive, the first having an equivalent valency of 3.4 ± 0.1 electronic charges (e-), and the second with an equivalent valency of 1.0 ± 0.2 e-. On average, half-maximal channel activation occurred at +10 mV. 5. The fast and the slow time constants of the exponential relaxations differed by a factor of 4-10 and both showed significant voltage dependence. Both the fast and slow time constants were greatest at potentials where approximately half the available channels were activated in the steady state. The slow time constant measured from activation and deactivation appeared to be independent of the starting potential. 6. The fractional amplitude of the slow exponential component of the tail currents was 0.09 ± 0.01 at -70 mV and increased steadily at more positive potentials passing through a clear maximum of 0.59 ± 0.03 at -10 mV. 7. Reducing the temperature decreased the magnitude of the peak inward current, with an apparent activation energy (E(a)) of 67 kJ/mol. The slow time constants measured from activation and deactivation were also reduced at lower temperatures. The slow activation time constant had a higher temperature sensitivity (E(a) = 85 kJ/mol) than the slow tail current time constant (E(a) = 29 kJ/mol). The fast tail current time constant was reduced at lower temperatures with an apparent E(a) of 79 kJ/mol. 8. Application of 10 μM-(±)Bay K 8644 prolonged the tail currents and shortened the activation time constant. The calcium current was activated at more negative potentials and the slope of the steady-state activation curve was reduced. 9. Two sequential rate theory models were fifted to the data, one having two closed states preceding an open state (three-state model), and the second having three closed states preceding an open state (four-state model). The three-state model did not describe the steady-state and kinetic data accurately, while the four-state model provided an adequate description of the data. 10. The four-state model predicted a mean open time for the calcium channel of 0.5 ms at 0 mV, which depended on the membrane potential with an equivalent dipole moment of 0.56 e-. It also predicted the presence of a short-lived intermediate closed state with a mean lifetime of 40 μs at 0 mV. The model predicted that the equivalent of 5.65 e- move from the inside to the outside of the membrane during channel activation. The predicted gating charges were unevenly distributed across the three reaction steps.

Original languageEnglish (US)
Pages (from-to)405-432
Number of pages28
JournalJournal of Physiology
Volume407
StatePublished - 1988
Externally publishedYes

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Suction
Sensory Receptor Cells
Electrodes
Cats
Calcium
Temperature
Calcium Channels
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester
Spinal Ganglia
Ion Channels
Membrane Potentials
Neurons

ASJC Scopus subject areas

  • Physiology

Cite this

Two-suction-electrode voltage-clamp analysis of the sustained calcium current in cat sensory neurones. / Taylor, William.

In: Journal of Physiology, Vol. 407, 1988, p. 405-432.

Research output: Contribution to journalArticle

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N2 - 1. The kinetics of the sustained calcium current were examined in cat dorsal root ganglion (DRG) neurones, using a two-suction-electrode voltage clamp. It was shown that this current could be examined with minimal contamination from other ionic currents. Experiments were performed at 20°C, with a concentration of 10 mM-calcium externally. 2. The transient calcium current was eliminated by using a holding potential of -50 mV. The sustained calcium current showed no evidence of steady-state inactivation at potentials between -90 and -30 mV. 3. The activation and deactivation time course of the calcium current was described by a double-exponential function. The activation process was examined without interference from significant inactivation under the conditions used. 4. The steady-state activation of the calcium channels was approximated by a two-step activation process. Both reactions were voltage sensitive, the first having an equivalent valency of 3.4 ± 0.1 electronic charges (e-), and the second with an equivalent valency of 1.0 ± 0.2 e-. On average, half-maximal channel activation occurred at +10 mV. 5. The fast and the slow time constants of the exponential relaxations differed by a factor of 4-10 and both showed significant voltage dependence. Both the fast and slow time constants were greatest at potentials where approximately half the available channels were activated in the steady state. The slow time constant measured from activation and deactivation appeared to be independent of the starting potential. 6. The fractional amplitude of the slow exponential component of the tail currents was 0.09 ± 0.01 at -70 mV and increased steadily at more positive potentials passing through a clear maximum of 0.59 ± 0.03 at -10 mV. 7. Reducing the temperature decreased the magnitude of the peak inward current, with an apparent activation energy (E(a)) of 67 kJ/mol. The slow time constants measured from activation and deactivation were also reduced at lower temperatures. The slow activation time constant had a higher temperature sensitivity (E(a) = 85 kJ/mol) than the slow tail current time constant (E(a) = 29 kJ/mol). The fast tail current time constant was reduced at lower temperatures with an apparent E(a) of 79 kJ/mol. 8. Application of 10 μM-(±)Bay K 8644 prolonged the tail currents and shortened the activation time constant. The calcium current was activated at more negative potentials and the slope of the steady-state activation curve was reduced. 9. Two sequential rate theory models were fifted to the data, one having two closed states preceding an open state (three-state model), and the second having three closed states preceding an open state (four-state model). The three-state model did not describe the steady-state and kinetic data accurately, while the four-state model provided an adequate description of the data. 10. The four-state model predicted a mean open time for the calcium channel of 0.5 ms at 0 mV, which depended on the membrane potential with an equivalent dipole moment of 0.56 e-. It also predicted the presence of a short-lived intermediate closed state with a mean lifetime of 40 μs at 0 mV. The model predicted that the equivalent of 5.65 e- move from the inside to the outside of the membrane during channel activation. The predicted gating charges were unevenly distributed across the three reaction steps.

AB - 1. The kinetics of the sustained calcium current were examined in cat dorsal root ganglion (DRG) neurones, using a two-suction-electrode voltage clamp. It was shown that this current could be examined with minimal contamination from other ionic currents. Experiments were performed at 20°C, with a concentration of 10 mM-calcium externally. 2. The transient calcium current was eliminated by using a holding potential of -50 mV. The sustained calcium current showed no evidence of steady-state inactivation at potentials between -90 and -30 mV. 3. The activation and deactivation time course of the calcium current was described by a double-exponential function. The activation process was examined without interference from significant inactivation under the conditions used. 4. The steady-state activation of the calcium channels was approximated by a two-step activation process. Both reactions were voltage sensitive, the first having an equivalent valency of 3.4 ± 0.1 electronic charges (e-), and the second with an equivalent valency of 1.0 ± 0.2 e-. On average, half-maximal channel activation occurred at +10 mV. 5. The fast and the slow time constants of the exponential relaxations differed by a factor of 4-10 and both showed significant voltage dependence. Both the fast and slow time constants were greatest at potentials where approximately half the available channels were activated in the steady state. The slow time constant measured from activation and deactivation appeared to be independent of the starting potential. 6. The fractional amplitude of the slow exponential component of the tail currents was 0.09 ± 0.01 at -70 mV and increased steadily at more positive potentials passing through a clear maximum of 0.59 ± 0.03 at -10 mV. 7. Reducing the temperature decreased the magnitude of the peak inward current, with an apparent activation energy (E(a)) of 67 kJ/mol. The slow time constants measured from activation and deactivation were also reduced at lower temperatures. The slow activation time constant had a higher temperature sensitivity (E(a) = 85 kJ/mol) than the slow tail current time constant (E(a) = 29 kJ/mol). The fast tail current time constant was reduced at lower temperatures with an apparent E(a) of 79 kJ/mol. 8. Application of 10 μM-(±)Bay K 8644 prolonged the tail currents and shortened the activation time constant. The calcium current was activated at more negative potentials and the slope of the steady-state activation curve was reduced. 9. Two sequential rate theory models were fifted to the data, one having two closed states preceding an open state (three-state model), and the second having three closed states preceding an open state (four-state model). The three-state model did not describe the steady-state and kinetic data accurately, while the four-state model provided an adequate description of the data. 10. The four-state model predicted a mean open time for the calcium channel of 0.5 ms at 0 mV, which depended on the membrane potential with an equivalent dipole moment of 0.56 e-. It also predicted the presence of a short-lived intermediate closed state with a mean lifetime of 40 μs at 0 mV. The model predicted that the equivalent of 5.65 e- move from the inside to the outside of the membrane during channel activation. The predicted gating charges were unevenly distributed across the three reaction steps.

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