Voltage - dependent Structural Interactions in the Shaker K 1 Channel

Using a strategy related to intragenic suppression, we previously obtained evidence for structural interactions in the voltage sensor of Shaker K 1 channels between residues E283 in S2 and R368 and R371 in S4 (Tiwari-Woodruff, S.K., C.T. Schulteis, A.F. Mock, and D.M. Papazian. 1997. Biophys . J . 72:1489–1500). Because R368 and R371 are involved in the conformational changes that accompany voltage-dependent activation, we tested the hypothesis that these S4 residues interact with E283 in S2 in a subset of the conformational states that make up the activation pathway in Shaker channels. First, the location of residue 283 at hyperpolarized and depolarized potentials was inferred by substituting a cysteine at that position and determining its reactivity with hydrophilic, sulfhydryl-specific probes. The results indicate that position 283 reacts with extracellularly applied sulfhydryl reagents with similar rates at both hyperpolarized and depolarized potentials. We conclude that E283 is located near the extracellular surface of the protein in both resting and activated conformations. Second, we studied the functional phenotypes of double charge reversal mutations between positions 283 and 368 and between 283 and 371 to gain insight into the conformations in which these positions approach each other most closely. We found that combining charge reversal mutations at positions 283 and 371 stabilized an activated conformation of the channel, and dramatically slowed transitions into and out of this state. In contrast, charge reversal mutations at positions 283 and 368 stabilized a closed conformation, which by virtue of the inferred position of 368 corresponds to a partially activated (intermediate) closed conformation. From these results, we propose a preliminary model for the rearrangement of structural interactions of the voltage sensor during activation of Shaker K 1 channels. key words: conformational changes • transmembrane segment • voltage clamp • cysteine mutagenesis I N T R O D U C T I O N Voltage-dependent cation channels control the excitability of nerve and muscle (Hille, 1992). Normally closed at the hyperpolarized resting potentials found in cells, these channels open in response to membrane depolarization. The control of activity by voltage is due to the presence of an intrinsic voltage sensor in the channel protein (Papazian and Bezanilla, 1997). In the voltagedependent Shaker K 1 channel, four conserved charged residues in transmembrane segments are essential components of the voltage sensor (Seoh et al., 1996). The fourth putative transmembrane segment, S4, contains three positively charged voltage-sensing residues, R365, R368, and R371 (Aggarwal and MacKinnon, 1996; Seoh et al., 1996). During voltage-dependent activation, these residues traverse a large fraction or all of the transmembrane electric field (Larsson et al., 1996; Starace et al., 1997). E293, a negatively charged residue found in the S2 transmembrane segment, is also crucial for the voltage sensor (Seoh et al., 1996). Whether this residue traverses the field or instead contributes to the electric field detected by the moving S4 residues has not yet been determined (Papazian and Bezanilla, 1997). Other charged residues, including E283 in S2 and K374 in S4, do not contribute to the channel’s gating charge and therefore do not move significantly relative to the transmembrane electric field during voltage-dependent activation (Seoh et al., 1996). Biophysical analysis indicates that activation gating is a dynamic process involving a number of different conformational changes. In Shaker channels, several models for activation gating have been presented (Bezanilla et al., 1994; Zagotta et al., 1994; Schoppa and Sigworth, 1998). These models posit a minimum of two voltagedependent conformational changes per subunit in the tetrameric channel. These steps prime the channel for opening. Entry into the conducting state involves a less voltage-dependent step that may be cooperative (SmithMaxwell et al., 1998; Ledwell and Aldrich, 1999). To understand gating in molecular terms, it is important to identify the structural changes underlying voltage-dependent activation. We have used a strategy related to intragenic suppression to provide some constraints on the packing of the voltage sensor (Papazian et al., 1995; Tiwari-Woodruff et al., 1997). In this apDrs. Tiwari-Woodruff and Lin contributed equally to the work and should be considered co-first authors. Dr. Tiwari-Woodruff’s present address is Department of Neurology, UCLA School of Medicine, Los Angeles, CA 90095. Dr. Schulteis’ present address is Molecular Neurobiology Laboratory, The Salk Institute, La Jolla, CA 92037. Address correspondence to Diane M. Papazian, Ph.D., Department of Physiology, Box 951751, UCLA School of Medicine, Los Angeles, CA 90095-1751. Fax: 310-206-5661; E-mail: [email protected] on July 1, 2017 jgp.rress.org D ow nladed fom