Channel Through Modification of a Hydrophobic Seal

The primary activation gate in K channels is thought to reside near the intracellular entrance to the ion conduction pore. In a previous study of the S6 activation gate in Shaker (Hackos et al., 2002), we found that mutation of V478 to W results in a channel that cannot conduct ions even though the voltage sensors are competent to translocate gating charge in response to membrane depolarization. In the present study we explore the mechanism underlying the nonconducting phenotype in V478W and compare it to that of W434F, a mutation located in an extracellular region of the pore that is nonconducting because the channel is predominantly found in an inactivated state. We began by examining whether the intracellular gate moves using probes that interact with the intracellular pore and by studying the inactivation properties of heterodimeric channels that are competent to conduct ions. The results of these experiments support distinct mechanisms underlying nonconduction in W434F and V478W, suggesting that the gate in V478W either remains closed, or that the mutation has created a large barrier to ion permeation in the open state. Single channel recordings for heterodimeric and double mutant constructs in which ion conduction is rescued suggest that the V478W mutation does not dramatically alter unitary conductance. Taken together, our results suggest that the V478W mutation causes a profound shift of the closed to open equilibrium toward the closed state. This mechanism is discussed in the context of the structure of this critical region in K channels. key words: voltage-dependent gating • closed gate • Agitoxin • mutagenesis • potassium channel I N T R O D U C T I O N For most ion channel proteins, the ion conduction pathway is not always open, but is dynamically regulated so that the pore opens and closes in response to a particular stimulus (Hille, 2001). In voltage-gated potassium (K v ) channels, a K selective pore opens and closes in response to changes in membrane voltage (Yellen, 1998). These channels are tetramers, with each subunit containing six transmembrane (TM) segments, designated S1 through S6 (Fig. 1 B). Some of the earliest functional studies on the gating of K v channels in the squid giant axon suggest that the activation gate is located near the intracellular entrance to the ion conduction pore (Fig. 1 A) (Armstrong, 1969, 1971; Armstrong and Hille, 1972). More recent studies on the Shaker K v channel support the notion of an intracellular activation gate, pointing to a specific region within the S6 TM where the access of methanesulfonates and silver ions changes quite dramatically with opening of the gate (Holmgren et al., 1997, 1998; Liu et al., 1997; del Camino and Yellen, 2001; Zhou et al., 2001). A comparison between the X-ray structures of two prokaryotic K channels, the KcsA K channel (Doyle et al., 1998), which is thought to be closed (Roux et al., 2000; Zhou et al., 2001; Jiang et al., 2002a), and the Ca 2 -activated MthK channel, which is open (Jiang et al., 2002a), supports the notion of an intracellular gate that is formed by the TM2 helix, a region that corresponds to S6 in K v channels (Jiang et al., 2002b). In the prokaryotic channels, opening of the intracellular gate has been proposed to involve bending of the TM2 helices at a glycine residue that is conserved in K channels (red highlighting in Fig. 1 C), greatly widening the intracellular entrance to the ion conduction pathway. In contrast, several studies in K v channels point to a unique structure of the intracellular pore region and a significantly smaller movement during opening (del Camino et al., 2000; del Camino and Yellen, 2001; Swartz, 2004; Webster et al., 2004). Mutational studies on the intracellular gate region of K v channels show that amino acid substitutions most frequently shift the closed–open equilibrium in favor of the open state (Hackos et al., 2002; Yifrach and MacKinnon, 2002). This observation can be explained if the closed conformation of the channel is intrinsically Address correspondence to Kenton J. Swartz, Molecular Physiology and Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Porter Neuroscience Research Center, 3B-215, 35 Convent Dr., MSC 3701, Bethesda, MD 208923701. Fax: (301) 435-5666; email: [email protected] Abbreviations used in this paper: TEA, tetraethylammonium; TM, transmembrane. on Jne 0, 2017 D ow nladed fom Published September 13, 2004