Activation of Shaker Potassium Channels III. An Activation Gating Model for Wild-Type and V2 Mutant Channels

A functional kinetic model is developed to describe the activation gating process of the Shaker potassium channel. The modeling in this paper is constrained by measurements described in the preceding two papers, including macroscopic ionic and gating currents and single channel ionic currents. These data were obtained from the normally activating wild-type channel as well as a mutant channel V2, in which the leucine at position 382 has been mutated to a valine. Different classes of models that incorporate Shaker ’s symmetrical tetrameric structure are systematically examined. Many simple gating models are clearly inadequate, but a model that can account for all of the qualitative features of the data has the channel open after its four subunits undergo three transitions in sequence, and two final transitions that reflect the concerted action of the four subunits. In this model, which we call Scheme 3 1 2 9 , the channel can also close to several states that are not part of the activation path. Channel opening involves a large total charge movement (10.8 e 0 ), which is distributed among a large number of small steps each with rather small charge movements (between 0.6 and 1.05 e 0 ). The final two transitions are different from earlier steps by having slow backward rates. These steps confer a cooperative mechanism of channel opening at Shaker ’s activation voltages. In the context of Scheme 3 1 2 9 , significant effects of the V2 mutation are limited to the backward rates of the final two transitions, implying that L382 plays an important role in the conformational stability of the final two states. key words: ion channel • gating current • single-channel current • patch clamp • kinetic model i n t r o d u c t i o n Several functional kinetic models have been proposed that describe the activation gating process of Shaker potassium channels (Schoppa et al., 1992; Tytgat and Hess, 1992; Bezanilla et al., 1994; McCormack et al., 1994; Zagotta et al., 1994 b ). However, these models are fundamentally quite diverse. One of the reasons for the differences is that no single group has attempted to model all of the available data that reflect the activation gating process for Shaker channels; instead, different groups have modeled different subsets of the data. Another reason is that the activation process is likely to involve a very large number of gating transitions (Zagotta et al., 1994 a ), and data have not been available to constrain all of the transitions of appropriately complicated models. This paper is the last in a series of three papers in which we describe our efforts to produce a well-constrained functional gating model for the Shaker potassium channel. The specific channel that we have studied is the Shaker 29-4 channel (Iverson and Rudy, 1990), which has been truncated at the NH 2 terminus to remove rapid inactivation, and which has been expressed in Xenopus laevis oocytes. Our strategy in the first two papers (Schoppa and Sigworth, 1998 a , 1998 b ) has been to characterize in detail the electrophysiological properties of the Shaker channel, using a combination of measurements of macroscopic ionic and gating currents and single channel currents. We have obtained data from not only the normally activating (wild type, WT) 1 channel, but also from a mutant channel (V2) having a leucine to valine mutation at position L370 in the Shaker 29-4 sequence, corresponding to L382 in the better-known Sh B sequence. Data from these channels, taken together, have yielded starting estimates of rate constants for several gating transitions. Our strategy for the modeling here will be first to explore systematically several classes of gating models. All of these models invoke the tetrameric structure of Shaker channels (MacKinnon, 1991; Kavanaugh et al., 1992; Li et al., 1994), by having many of their transitions correspond to Shaker ’s four subunits moving one subunit at a time, and with the subunits acting equivalently. We will show that different lines of data rule out the most simple models, leading us to our first hypothesis for a gating model, which we call Scheme 2 1 2 9 . This model has the channel open after each of Shaker ’s four Address correspondence to Fred J. Sigworth, Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Fax: 203-785-4951; E-mail: [email protected] 1 Abbreviation used in this paper: WT, wild type. on A uust 0, 2017 jgp.rress.org D ow nladed fom