Activation Gate Opening Precedes Slow Inactivation in Shaker K+ Channels

Abstract

In the absence of N-type inactivation Shaker potassium channels display slow (C-type) inactivation. Functional and structural studies indicated that channel opening precedes slow inactivation in voltage-gated and KcsA K+ channels, whereas others argued for slow inactivation from the closed sate of the channel as well. None of the previous studies correlated the opening of the of the activation gate (A-gate), formed by the bundle crossing of the S6 segments, with the development of inactivation, which is associated with the structural rearrangement in the selectivity filter. These two gates are coupled and thus, the current experiments addressed the hypothesis that opening of the activation gate must precede slow inactivation.To address this hypothesis we compared the voltage dependence of A-gate opening and that of the development of inactivation in T449A/V474C Shaker-IR channels. Opening of the A-gate was monitored by the accessibility of 474C to Cd2+ from the intracellular side. The membrane potential was changed repeatedly from a holding potential of −120 mV to test potentials ranging from −110 mV to −60 mV in the presence or absence of Cd2+.Our results show that the function describing the voltage dependence of Cd2+ block is shifted toward the negative potentials compared to the voltage dependence of steady-state inactivation curve. This indicates that A-gate opening already occurs at such negative potentials where no inactivation can be detected. Furthermore, the curve representing Cd2+ block is also negative-shifted compared to the voltage dependence of steady-state activation (G-V) curve. This suggests that even at fairly negative holding potentials, at which no macroscopic current can be detected, rare channel openings occur yielding access to the channel cavity. Based on these results we suggest that A-gate opening always precedes structural changes associated with slow inactivation.Supported by OTKA K75904