A model to explain K + -sensing of plant K out channels Supplemental Material to Johansson, Wulfetange, Porée, Michard, Lacombe, Sentenac, Thibaud, Mueller-Roeber, Blatt, Dreyer “ K + dependence of the Arabidopsis K + channel SKOR indicates an unique gating mechanism coupled to ion permeation ”

Supplemental Material, Johansson et al. In the following only those details of a plant K out channel are displayed which are important to explain the K + -sensing mechanism: The P-domain and the S6 segment of 2 (out of 4) subunits.

Supplemental Material, Johansson et al. An open K out channel mediates K + efflux.

open Supplemental Material, Johansson et al. Spontaneous (voltage-dependent) closing occurs via rearrangements of C-terminal parts of the S6 segment. closed

openclosed Supplemental Material, Johansson et al. The ionic compositions of the pore and the cavity equilibrate with the external solution. When the external K + concentration is low also the occupancy of the pore by K + is low. low K + ext. high K + ext. When the external K + concentration is high also the occupancy of the pore by K + is high.

openclosed low K + ext. high K + ext. Supplemental Material, Johansson et al. When the external K + concentration is high also the occupancy of the pore by K + is high.  The pore is not flexible but rather rigid. Interactive forces between the pore (SKOR-M286) and S6 (SKOR-D312) induce then further conformational changes in S6 which stabilise the closed conformation.

openclosed low K + ext. high K + ext. Supplemental Material, Johansson et al. These rearrangements reduce the interactive forces between the pore and S6.  No further conformational changes in S6 are induced. When K + ext. is low  the pore is not crowded with K + ions  the pore is flexible and undergoes structural rearrangements (low-K + structure, Zhou et al., 2001, Nature 414, ).

openclosed low K + ext. high K + ext. Supplemental Material, Johansson et al. Spontaneous rearrangements of the C-terminal part of S6 “re-open” the channel. However, the “low-K + structure” of the pore does not allow K + flux.

openclosed low K + ext. high K + ext. Supplemental Material, Johansson et al. The cavity is flushed by internal K +.

openclosed low K + ext. high K + ext. Supplemental Material, Johansson et al. When the internal K + concentration is large enough, K + ions can alter the conformation of the pore (high-K + structure, Zhou et al., 2001, Nature 414, ).

openclosed low K + ext. high K + ext. Supplemental Material, Johansson et al. The K out channel mediates K + efflux again. open

Summary

Extracellular K + stabilises a closed conformation of plant K out channels. Supplemental Material, Johansson et al.

The model explains mutant phenotypes: SKOR-M286L

closed low K + ext. Supplemental Material, Johansson et al. open high K + ext. : C 2high low K + ext. : C 2low high K + ext. Prediction for the mutant SKOR-M286L The SKOR-mutation M286  L eliminates the interactive pore-S6 forces. The stabilised closed state C 2high at high K + ext. does not exist anymore. At high K + ext. the channel remains in state C 1. In contrast, C 2low at low K + ext. (low-K + pore structure) still exists. As a consequence, the channel SKOR-M286L appears to be K + ext. -activated.

Supplemental Material, Johansson et al.SKOR-M286L: elimination of the K + -induced stabilisation of the closed state C 2high  Number of open channels increases with increasing K + ext.

The model explains mutant phenotypes: SKOR-D312N

closed low K + ext. Supplemental Material, Johansson et al. Prediction for the mutant SKOR-D312N open high K + ext. high K + ext. : C 2high low K + ext. : C 2low The SKOR-mutation D312  N eliminates the interactive pore-S6 forces. The stabilised closed state C 2high at high K + ext. does not exist anymore. At high K + ext. the channel remains in state C 1. In contrast, C 2low at low K + ext. (low-K + pore structure) still exists. As a consequence, the channel SKOR-D312N appears to be K + ext. -activated.

Supplemental Material, Johansson et al.SKOR-D312N: elimination of the K + -induced stabilisation of the closed state C 2high  Number of open channels increases with increasing K + ext.

Mathematical Appendix An Analytical Consideration of the Model

The transitions O  O* and C 2low  C 1low are strongly energetically disfavoured. The only [K + ext. ]-dependent transition is C 1low  C 1. d and a are voltage-dependent rate-constants. h 1, h 2, k d, k a, L, and r can be voltage-dependent or voltage-independent. Supplemental Material, Johansson et al.

Experimentally accessible is p O : Some further simplifications of the equation: 

Supplemental Material, Johansson et al. Wild-type SKOR : Increasing [K + ] (=increasing x)  decreasing p O Mutants SKOR-M286L and SKOR-D312N :  p O increases with increasing [K + ] (=increasing x) Predictions for the K + -dependence at a fixed voltage