a - Helical Structural Elements within the Voltage - sensing Domains of a K 1 Channel Yingying

Voltage-gated K 1 channels are tetramers with each subunit containing six (S1–S6) putative membrane spanning segments. The fifth through sixth transmembrane segments (S5–S6) from each of four subunits assemble to form a central pore domain. A growing body of evidence suggests that the first four segments (S1–S4) comprise a domain-like voltage-sensing structure. While the topology of this region is reasonably well defined, the secondary and tertiary structures of these transmembrane segments are not. To explore the secondary structure of the voltage-sensing domains, we used alanine-scanning mutagenesis through the region encompassing the first four transmembrane segments in the drk1 voltage-gated K 1 channel. We examined the mutation-induced perturbation in gating free energy for periodicity characteristic of a -helices. Our results are consistent with at least portions of S1, S2, S3, and S4 adopting a -helical secondary structure. In addition, both the S1–S2 and S3–S4 linkers exhibited substantial helical character. The distribution of gating perturbations for S1 and S2 suggest that these two helices interact primarily with two environments. In contrast, the distribution of perturbations for S3 and S4 were more complex, suggesting that the latter two helices make more extensive protein contacts, possibly interfacing directly with the shell of the pore domain. key words: secondary structure • scanning mutagenesis • amphipathic • Fourier transform • voltage-dependent gating I N T R O D U C T I O N The voltage-gated K 1 channels comprise a large family of tetrameric membrane proteins that open and close in response to changes in membrane voltage. Based on hydrophobicity analysis, each subunit in the tetramer contains six putative transmembrane segments, termed S1 through S6 (Fig. 1 A). The central pore domain, which contains the K 1 selective ion conduction pathway, is formed by the assembly of the S5 through S6 regions (MacKinnon and Miller, 1989; MacKinnon and Yellen, 1990; Hartmann et al., 1991; MacKinnon, 1991; Yellen et al., 1991; Yool and Schwarz, 1991; Liman et al., 1992; Heginbotham et al., 1994; Ranganathan et al., 1996; Armstrong and Hille, 1998). The KcsA K 1 channel, a tetrameric membrane protein of known three-dimensional structure, is a relatively simple prokaryotic K 1 channel with two transmembrane segments in each subunit that are homologous to S5–S6 in voltage-gated K 1 channels (Schrempf et al., 1995; Doyle et al., 1998). Both sequence homology and the conservation of poreblocking toxin receptors suggests that the structure of the pore domain of voltage-gated channels is likely to be similar to that of KcsA (Schrempf et al., 1995; Doyle et al., 1998; MacKinnon et al., 1998). Thus, both S5 and S6 are undoubtedly membrane spanning a -helices with the S5–S6 linker, the most conserved region of all K 1 channels, forming a short pore helix and the selectivity filter. The first four transmembrane segments (S1–S4) of voltage-gated K 1 channels are not present in the simple pore K 1 channels, like KcsA and the inward rectifier K 1 channels, and appear to underlie their unique voltagesensing capabilities (Armstrong and Hille, 1998). However, when compared with the pore domain, much less is known about the structure of this voltage-sensing region of the channel. The presence of four transmembrane segments outside the pore domain is supported by hydrophobicity analysis (Schwarz et al., 1988; Butler et al., 1989; Frech et al., 1989; Stühmer et al., 1989; Durell et al., 1998) and sequence comparisons showing that highly conserved residues tend to cluster into four main groups (Fig. 1, bold residues). In addition, the numerous topological constraints that support the membrane-folding model shown in Fig. 1 A argue for the presence of four transmembrane segments in this region (Hoshi et al., 1990; Zagotta et al., 1990; Santacruz-Toloza et al., 1994; Holmgren et al., 1996; Swartz and MacKinnon, 1997a,b; Larsson et al., 1996; Yang et al., 1996; Yusaf et al., 1996). There is considerable evidence suggesting that most, if not all, of the first four transmembrane segments participate in sensing changes in membrane voltage. This is particularly true for S4, an unusual transmembrane segment that conAddress correspondence to Kenton J. Swartz, Molecular Physiology and Biophysics Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36 Room 2C19, 36 Convent Dr., MSC 4066, Bethesda, MD 20892. Fax: 301-435-5666; E-mail: [email protected] on M arch 1, 2013 jgp.rress.org D ow nladed fom Published December 28, 1999