Skip to main content
. Author manuscript; available in PMC: 2017 Apr 1.
Published in final edited form as: Biochim Biophys Acta. 2015 Dec 23;1858(4):783–792. doi: 10.1016/j.bbamem.2015.12.024

Fig. 2.

Fig. 2

Alanine scan of S5 segment of Slo2.1. (A) Amino acid sequence alignment for the S4-S5 linker (S45L) and the S5 transmembrane segment (S5) of Slo2.1 and other K+ channels. The name of each channel is indicated to the left of each sequence. The position of the terminal residue in the channel subunit is indicated on the right of the sequence. Ala substitution of 6 residues (indicated by red text) caused a gain of function. Residues indicated by green text (Trp-211 in KCa3.1, Leu-273 in KCNQ1) have been reported to interact with the base of the pore helix, as described in the Discussion. (B) Representative traces of wt Slo2.1 channel currents. Top panel shows voltage pulse protocol. Currents were applied once every 10 s. Bottom panel shows Slo2.1 channel current traces recorded under control conditions and after peak responses to application of a solution containing 1 mM and 6 mM NFA. (C) Average current amplitudes (in µA) recorded at a Vt of 0 mV is plotted on the y-axis. The x-axis indicates the point mutation in Slo2.1. Currents recorded from uninjected oocytes (Uninj) and cells expressing wild-type channels (wt) are also plotted. The 25 residues within the S5 segment are underlined. Oocytes were injected with 0.4–10 ng cRNA and recorded by TEVC 1–4 days later (n = 4–14). (D) Data from panel C expressed as normalized current (Irel), defined as current magnitude under control conditions, or in the presence of 1 mM NFA, relative to current measured in the presence of 6 mM NFA. *, p < 0.01 compared to wt channels. (E) Helical wheel plot of the S5 segment of Slo2.1 from residue Val-199 to Gly-216 (indicated by single letter amino acid code), constructed using Wheel.pl program (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi ). Asterisks mark those residues that when mutated to Ala had both constitutive channel activity and responses to 1 mM NFA (dark and light bars, respectively in panel D) that were significantly larger (p < 0.01) than wt channels. (F) Homology model of the S5 segment, pore helix (PH) and S6 segment of Slo2.1 based on the structure of Slo2.2 channel. The side chains of Leu-202, Thr-205, Leu-209, Thr-212 and Gly-216 are labeled. Error bars in panels C and D indicate + S.E.