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1. We studied the gating kinetics of Kv4.2 channels, the molecular substrate of neuronal somatodendritic A-type currents. For this purpose wild-type and mutant channels were transiently expressed in the human embryonic kidney (HEK) 293 cell line and currents were measured in the whole-cell patch-clamp configuration. 2. Kv4.2 channels inactivated from pre-open closed state(s) with a mean time constant of 959 ms at -50 mV. This closed-state inactivation was not affected by a deletion of the Kv4.2 N-terminus (Delta2-40). 3. Kv4.2 currents at +40 mV inactivated with triple-exponential kinetics. A fast component (tau = 11 ms) accounted for 73 %, an intermediate component (tau = 50 ms) for 23 % and a slow component (tau = 668 ms) for 4 % of the total decay. 4. Both the fast and the intermediate components of inactivation were slowed by a deletion of the Kv4.2 N-terminus (tau = 35 and 111 ms) and accounted for 33 and 56 %, respectively, of the total decay. The slow component was moderately accelerated by the truncation (tau = 346 ms) and accounted for 11 % of the total Kv4.2 current inactivation. 5. Recovery from open-state inactivation and recovery from closed-state inactivation occurred with similar kinetics in a strongly voltage-dependent manner. Neither recovery reaction was affected by the N-terminal truncation. 6. Kv4.2 Delta2-40 channels displayed slowed deactivation kinetics, suggesting that the N-terminal truncation leads to a stabilization of the open state. 7. Simulations with an allosteric model of inactivation, supported by the experimental data, suggested that, in response to membrane depolarization, Kv4.2 channels accumulate in the closed-inactivated state(s), from which they directly recover, bypassing the open state.

1. Currents through voltage-activated Ca2+ channels in rat dorsal root ganglion (DRG) x mouse neuroblastoma hybrid (F-11) cells were studied using the whole-cell patch clamp technique with 30 mM-Ba2+ as charge carrier. Two components of the inward Ba2+ current were distinguished on the basis of voltage dependence and time course. Each component could be further subdivided based on pharmacology. 2. A transient inward current activated at test potentials positive to -40 mV, peaked within 20 ms and then decayed during a 200 ms depolarization. The peak amplitude of the transient current occurred between -10 and +10 mV. With a 300 ms conditioning pulse, half-inactivation of the transient current occurred at -30 mV. A sustained inward current activated at test potentials positive to -30 mV and reached a maximum at +20 to +30 mV. The sustained current showed little voltage-dependent inactivation over 200 ms. The amplitudes of both the transient and sustained currents were increased by perfusing with Ba2+ instead of Ca2+. 3. Most F-11 cells had both the transient and sustained Ba2+ currents although the relative amount of the two currents varied with culture conditions. The transient current was more prominent in cells fed with a 'growth' medium (15-20% serum) whereas the sustained current was increased in cells fed with a 'differentiation' medium (1% serum plus growth factors). F-11 cells can be used to study transient current in relative isolation from sustained Ca2+ current under certain culture conditions. The neuroblastoma parent of the F-11 cell line, N18TG-2 cells, exhibited little or no voltage-dependent Ba2+ current. 4. Brief application of omega-conotoxin fraction GVIA (10 microM) produced a long-lasting block of 81% of the sustained current and 27% of the transient current. 5. The transient and sustained Ba2+ currents in F-11 cells were reversibly blocked by brief exposure to Cd2+ or Ni2+. Block of the sustained current was evident with 100 nM-Cd2+ whereas the threshold concentration for Ni2+ block was 1 microM. Cd2+ and Ni2+ were equipotent blockers of the transient current. Dose-response curves for Cd2+ and Ni2+ block of both sustained and transient currents had shallow slopes suggesting that the block was more complex than a simple bimolecular interaction between blocker and one blocking site. Dose-response curves were fitted by a model that included two binding sites for each divalent blocker.(ABSTRACT TRUNCATED AT 400 WORDS)