Exploring the Shaker K+ channel S3b segment using deletion mutants

Abstract

To asses the role of S3 region during the activation of the Shaker K+ channels we constructed Shaker K+ channel deletion mutants. A complete deletion of S3-S4 linker and part of the S3b segment Shaker Δ(6-46)Δ(327-360) produced a dramatic decrease in the maximum open probability. If we continue eliminating residues from the S3b segment, the resultant proteins do not express functional channels. Surprisingly, channel expression and functionality are recovered when both the S3-S4 linker and the entire S3b segment have been eliminated (Shaker Δ(6-46)Δ(312-360)). This result indicates that neither the S3-S4 linker nor the S3b segment is essential for the activation of Shaker K+ channels. When characterizing the S3b deletion mutants, we found a periodic relationship of the activation time constants and the number of the residues in this region of the protein Shaker Δ(6-46)Δ(326-360) through Shaker Δ(6-46)Δ(333-360). This periodic behavior can be represented by two sine functions with an angular frequency of 100o per residue, typical of α-helices but with an angular phase difference of 160o. This result suggests a break in the α-helix that occurs at threonine 329. The secondary structure break at Thr 329 was also found on a molecular dynamics simulation of this region (Iso 321 -Asp 336) done in H2O and for 1 ns time duration also showed a break point of the helix at threonine-329. These results are consistent with an extracellular location of segment S3b. Supported by Fondecyt 103-0830 and Fundacion Andes. The CECS is a Millenium Institute