Tuning the temperature dependence of Shaker potassium channel with mutations at S4-S5 linker.

Abstract

We investigated the effects of temperature on mutations that uncouple the voltage sensor (VSD) from the pore (PD) in the Shaker-IR potassium channel using temperature step (Tstep). We utilized a setup that achieves fast temperature changes (millisecond) and allows the recording of the cell membrane temperature with sub-millisecond resolution (https://doi.org/10.1101/2022.07.11.499616). We used the mutants V369I, I372L, S376T (ILT), in the N-terminus of the S4-S5, that produces a split in the components of the gating charge movement, and I384N, near the S4-S5 C-terminus, that uncouples the VSD charge movement from the PD. From a bath temperature of 17 ºC, we used a Tstep of around ∼9 ºC with rising phase of 1.5 ms. In ILT, applying a Tstep, after the gating currents have subsided, promotes a right shift in the first component of gating charge and a left shift of the second component (the last concerted step associated with PD opening). Consistently, Tstep applied after the onset of a depolarizing voltage pulse produces a ∼5 mV left shift in the conductance-voltage curve (G-V). The entropy, calculated from G-V, changes from 24 for WT to −42 J/K for ILT. In I384N, applying a Tstep reduces the ionic current indicating the closure of the pore. We hypothesize that the temperature dependence of the ILT G-V arises from the changes in the last step of the charge movement. On the other hand, since the VSD and PD are loosely coupled in I384N, the increase in temperature is likely to further decrease the VSD-PD coupling, thus decreasing the measured conductance. (Support: NIH: GM030376, Fondecyt: 1190203)