Kinetic characterization of rat brain type IIA sodium channel alpha-subunit stably expressed in a somatic cell line.
1. The rat brain type IIA Na+ channel alpha-subunit was stably expressed in Chinese hamster ovary (CHO) cells. Current through the expressed Na+ channels was studied using the whole-cell configuration of the patch clamp technique. The transient Na+ current was sensitive to TTX and showed a bell-shaped peak current vs. membrane potential relation. 2. Na+ current inactivation was better described by the sum of two exponentials in the potential range -30 to + 40 mV, with a dominating fast component and a small slower component. 3. The steady-state inactivation, h infinity, was related to potential by a Boltzmann distribution, underlying three states of the inactivation gate. 4. Recovery of the channels from inactivation at different potentials in the range -70 to -120 mV were characterized by an initial delay which decreased with hyperpolarization. The time course was well fitted by the sum of two exponentials. In this case the slower exponential was the major component, and both time constants decreased with hyperpolarization. 5. For a working description of the Na+ channel inactivation in this preparation, with a minimal deviation from the Hodgkin-Huxley model, a three-state scheme of the form O<-->I1<-->I2 was proposed, replacing the original two-state scheme of the Hodgkin-Huxley model, and the rate constants are reported. 6. The instantaneous current-voltage relationship showed marked deviation from linearity and was satisfactorily fitted by the constant-field equation. 7. The time course of activation was described by an m chi model. However, the best-fitted value of chi varied with the membrane potential and had a mean value of 2. 8. Effective gating charge was determined to be 4.7e from the slope of the activation plot, plotted on a logarithmic scale. 9. The rate constants of activation, alpha m and beta m, were determined. Their functional dependence on the membrane potential was investigated.
Sarkar
S N
SN
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
Adhikari
A
A
Sikdar
S K
SK
D000818:Animals; D001923:Brain Chemistry / Q000502:physiology; D016466:CHO Cells / Q000502:physiology*; D006224:Cricetinae; D015640:Ion Channel Gating / Q000502:physiology; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D051381:Rats; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000502:physiology* / Q000648:ultrastructure; D013997:Time Factors; D014981:Xenopus
D015222:Sodium Channels; D012964:Sodium