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Coupling between charge movement and pore opening in voltage dependent potassium channels.

Ionic and gating currents from Shaker K+ channels were characterized with the cut-open oocyte voltage clamp (COVG) technique. Experiments were performed in normally conducting channels and in channels with the W434F mutation which completely abolished ion conduction without affecting the voltage dependence of gating charge. Subtracted and unsubtracted gating currents with the COVG technique, and gating currents recorded in cell attached macro-patches had the same properties and time course. However, OFF gating currents and ionic deactivation tails became slower after patch excision. Gating currents had the following salient properties: 1) the turn-on of the gating current shows a rising phase, 2) the more negative position of the charge-voltage curve (Q-V) vs. the conductance-voltage (G-V) curve and the charge displacement by hyperpolarizing prepulses indicate that a large fraction of the voltage-dependence occurs in the transitions between closed states, 3) the Q-V relationship showed two component with different half activation potential and effective valence; the one appearing at more negative potential had a shallower voltage dependence, while the one at more depolarized potentials had a larger effective valence and correlated with channel opening, 4) ionic and gating currents were similarly time shifted by preceding hyperpolarizing and depolarizing pulses which substantiates the relationship between charge movement and channel opening, 5) in the wild type Shaker K+ clone with fast inactivation, the OFF gating charge is partially immobilized for large depolarizing pulses, while in the mutant channel lacking inactivation the charge is recovered quickly at the end of the pulse, indicating that the channel blockade by the inactivating particle slows down charge recovery, 6) the OFF gating current rapidly returns for small depolarizations, while for larger pulses which open the channel the OFF gating current return is delayed suggesting that the closed to open transitions carry little charge and 7) in the W434F mutant with the conserved amino terminus large depolarizations that would have opened the channel induced OFF charge immobilization, indicating that although the conduction pathway was not functional, the channel can still undergo the closed-open conformation in response to voltage changes. In conclusion, the kinetic properties of gating currents discard equal and independent gating subunits with two positions. They favor the presence of interactions among the gating subunits and are consistent with a sequential model for K+ channel activation in which an early less voltage-dependent transition between very deep closed states was followed by more voltage-dependent transitions between later closed states, and finally by a much less voltage dependent transition between the last closed state and the open state.