Inactivation Gating of Kv4 Potassium Channels Molecular Interactions Involving the Inner Vestibule of the Pore

Kv4 channels represent the main class of brain A-type K 1 channels that operate in the subthreshold range of membrane potentials (Serodio, P., E. Vega-Saenz de Miera, and B. Rudy. 1996. J. Neurophysiol. 75:2174– 2179), and their function depends critically on inactivation gating. A previous study suggested that the cytoplasmic NH 2 and COOH-terminal domains of Kv4.1 channels act in concert to determine the fast phase of the complex time course of macroscopic inactivation (Jerng, H.H., and M. Covarrubias. 1997. Biophys. J . 72:163–174). To investigate the structural basis of slow inactivation gating of these channels, we examined internal residues that may affect the mutually exclusive relationship between inactivation and closed-state blockade by 4-aminopyridine (4-AP) (Campbell, D.L., Y. Qu, R.L. Rasmussen, and H.C. Strauss. 1993. J. Gen. Physiol. 101:603–626; Shieh, C.-C., and G.E. Kirsch. 1994. Biophys. J. 67:2316–2325). A double mutation V[404,406]I in the distal section of the S6 region of the protein drastically slowed channel inactivation and deactivation, and significantly reduced the blockade by 4-AP. In addition, recovery from inactivation was slightly faster, but the pore properties were not significantly affected. Consistent with a more stable open state and disrupted closed state inactivation, V[404,406]I also caused hyperpolarizing and depolarizing shifts of the peak conductance–voltage curve ( z 5 mV) and the prepulse inactivation curve ( . 10 mV), respectively. By contrast, the analogous mutations (V[556,558]I) in a K 1 channel that undergoes Nand C-type inactivation (Kv1.4) did not affect macroscopic inactivation but dramatically slowed deactivation and recovery from inactivation, and eliminated open-channel blockade by 4-AP. Mutation of a Kv4specifc residue in the S4–S5 loop (C322S) of Kv4.1 also altered gating and 4-AP sensitivity in a manner that closely resembles the effects of V[404,406]I. However, this mutant did not exhibit disrupted closed state inactivation. A kinetic model that assumes coupling between channel closing and inactivation at depolarized membrane potentials accounts for the results. We propose that components of the pore’s internal vestibule control both closing and inactivation in Kv4 K 1 channels. key words: Shal channels • inactivation kinetics • A-type currents • 4-aminopyridine i n t r o d u c t i o n Voltage-gated K 1 channels (Kv channels) 1 activate and open upon membrane depolarization. Most Kv channels, however, do not remain open during a sustained depolarization. Instead, they adopt a nonconducting conformation by a process known as inactivation. Kv channels may exhibit multiple mechanisms of inactivation that involve fast and slow processes. A great deal has been learned about the mechanisms of inactivation of Shaker K 1 channels. These channels exhibit two clearly distinct forms of inactivation: Nand C-type (Choi et al., 1991). N-type inactivation is fast and involves a “ball and chain”–type mechanism (Armstrong and Bezanilla, 1977), where approximately the first 20 amino acids at the NH 2 terminus of the Shaker subunit act as a tethered internal particle capable of blocking the open pore (Hoshi et al., 1990; Demo and Yellen, 1991; Ruppersberg et al., 1991; Murrell-Lagnado and Aldrich, 1993; Tseng-Crank et al., 1993; MacKinnon et al., 1993; Gomez-Lagunas and Armstrong, 1995). The cytoplasmic loop between the S4 and S5 seqments (the S4–S5 loop) contributes to the putative receptor for the inactivation particle in the pore of the channel (Isacoff et al., 1991; Holmgren et al., 1996). C-type inactivation is typically slower than N-type and depends on residues in the pore region (the S5–S6 loop) and the external section of the S6 region (Hoshi et al., 1991; LopezBarneo et al., 1993). The rate of C-type inactivation is slower in the absence of N-type inactivation, suggesting that the two processes are coupled (Hoshi et al., 1991; Baukrowitz and Yellen, 1995). A current hypothesis proposes that C-type inactivation involves a rearrangement of the extracellular mouth of the pore resulting from a cooperative conformational change of the channel subunits (Ogielska et al., 1995; Panyi et al., 1995; Portions of this work were previously published in abstract form (Jerng, H.H. , M. Shahidullah, and M. Covarrubias. 1998. Biophys. J.