Voltage-dependent structural interactions in the Shaker K+ channel

Using a strategy related to intragenic suppression, we previously obtained evidence for structural interactions in the voltage sensor of Shaker K+ cha nnels between residues E283 in S2 and R368 and R371 in S4 (Ti-wari-Woodruff , S.K, C.T. Schulteis, A.F. Mock, and D.M. Papazian. 1997. Biophys. J. 72:1 489-1500). 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 conformation al states that make up the activation pathway in Shaker channels. First, th e location of residue 283 at hyperpolarized and depolarized potentials was inferred by substituting a cysteine at that position and determining its re activity 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 co nclude that E283 is located near the extracellular surface of the protein i n both resting and activated conformations. Second, we studied the function al phenotypes of double charge reversal mutations between positions 283 and 368 and between 283 and 371 to gain insight into the conformations in whic h 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 chan nel, 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 re arrangement of structural interactions of the voltage sensor during activat ion of Shaker K+ channels.