Gating currents in Shaker K+ channels Implications for activation and inactivation models Eduardo Peraza, Diane M. Papazian, Enrico Stefani,* and Francisco Bezanilla Department of Physiology, University of California at Los Angeles, Los Angeles, California 90024; and *Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030 USA ABSTRACT We have studied ionic and gating currents in mutant and wild-type Shaker K+ channels to investigate the mechanisms of channel activation and the relationship between the voltage sensor of the channel and its inactivation particle. The turn on of the gating current shows a rising phase, indicating that the hypothetical identical activation subunits are not independent. Hyperpolarizing prepulses indicate that most of the voltage-dependence occurs in the transitions between closed states. The open-to-closed transition is voltage independent, as suggested by the presence of a rising phase in the off gating currents. In Shaker channels showing fast inactivation, the off gating charge is partially immobilized as a result of depolarizing pulses that elicit inactivation. In mutant channels lacking inactivation, the charge is recovered quickly at the end of the pulse. Internal TEA mimics the inactivation particle in its behavior but the charge immobilization is established faster and is complete. We conclude that the activation mechanism cannot be due to the movement of identical independent gating subunits, each undergoing first order transitions, and that the inactivation particle is responsible for charge immobilization in this channel. INTRODUCTION Voltage-dependent ion channels respond to changes in the electric field across the membrane by rearrange- ments of charges or dipoles within the transmembrane segments of the channel molecule. These intramolecular charge displacements, which can be measured as gating currents, are thought to induce a series of conforma- tional changes that convert the closed channel into a conducting pore (Armstrong, 1981, Bezanilla, 1985). Channel gating has been interpreted as a series of transitions between kinetically distinct closed states that must be populated before the channel opens. According to this view, depolarizations shift the equilibrium to- wards the open state whereas hyperpolarizations shift the equilibrium towards the first closed state. Gating currents are particularly sensitive to the voltage- dependent transitions between closed states because most of the charge seems to move between these states. Therefore gating current information is crucial for the establishment of working kinetic models for activation of ion channels. On sustained depolarization, several types of channels enter a long-lived nonconducting state known as the inactivated state. In Na channels, it has been observed that the inactivation process is associated with a partial immobilization of the gating charge transferred during channel activation (Armstrong and Bezanilla, 1974). Dr. Perozo's present address is Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90024. Charge immobilization has been defined as the failure to quickly recover the on gating charge at the end of a depolarizing pulse. In fact, if the membrane is repolar- ized long enough, all the charge must move back because a subsequent depolarizing pulse moves the same amount of charge. The kinetics and extent of charge immobiliza- tion closely resemble the properties of ionic current inactivation, which has led to the proposal that these two phenomena represent the same mechanism. Based on gating charge immobilization and the effect of internally perfused proteases on the squid Na chan- nel, a mechanistic model of the inactivation process was advanced by Armstrong and Bezanilla (1977). It was proposed that a positively charged domain of the chan- nel, located intracellularly, enters into the open pore in a voltage-independent manner and effectively blocks ion flow. Support for this proposal has come from elegant experiments using mutant Shaker channels by Hoshi et ai. (1990). These authors have identified a portion of the NH z terminus of the molecule as the putative inactiva- tion particle. Additionally, synthetic peptides based on sequence of this region induce time-dependent inactiva- tion in noninactivating mutants (Zagotta et aI., 1990). We have measured ionic and gating currents from Shaker channels and a mutant lacking inactivation (Beza- nilla et aI., 1991), and showed a close correlation between the presence of the inactivating particle and immobilization of the gating charge. It was also shown that an open pore blocker, tetraethylammonium, is able to induce charge immobilization in noninactivating chan- 160 0006-3495/92/04/160/12 $2.00 Biophys. J eJ Biophysical Society Volume 62 Discussions 1992 160-171