Supplementary Material to Johansson, Wulfetange, Porée, Michard, Gajdanowicz, Lacombe, Sentenac, Thibaud, Mueller-Roeber, Blatt, Dreyer External [K + ] modulates the activity of the Arabidopsis potassium channel SKOR via an unusual mechanism

The physiological background

animated from Wegner & DeBoer, 1997, Plant Physiol. 115, Supplemental Material, Johansson et al. Long distance transport of K + in plants

animated from Wegner & DeBoer, 1997, Plant Physiol. 115, Supplemental Material, Johansson et al. Demand for K + in the shoot  Reduced re-circulation of K +

How does the plant sense the shoot’s K + -demand?

Supplemental Material, Johansson et al. How does the plant sense the shoot’s K + -demand? The outward-rectifying K + channel SKOR senses the K + concentration in the stelar apoplast and releases K + when [K + ] apoplast decreases (as a consequence of a reduced K + re-circulation) (Wegner and DeBoer, 1997, Plant Physiol. 115, ; Gaymard et al., Cell 94, ) SKOR

How does SKOR sense [K + ] apoplast ?

The K + -sensing problem How do the K + channels sense [K + ] ext and not the transmembrane K + gradient?

A model to explain [K + ] ext. -sensing of outward-rectifying plant K + channels

Supplemental Material, Johansson et al. In the following only those details of an outward-rectifying plant K + 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.

The main ideas in short…

Supplemental Material, Johansson et al. Upon closing the access of internal [K + ] to the pore is cut off. External [K + ] in the selectivity filter stabilizes via pore-S6 interactions a closed state. The Boltzmann function of this simple gating scheme is: with V ½ 0 : V ½ at [K + ] ext. =0 V s : slope factor K 0 : constant; K 0 >0 An open channel mediates K + efflux.  With increasing [K + ] ext. the Boltzmann curve shifts positive.

The detailed model for SKOR [K + ] ext. -sensing

Supplemental Material, Johansson et al. An open 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 stabilize 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. K + ions from inside 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 outward-rectifying K + channel mediates K + efflux again. open

Supplemental Material, Johansson et al. The Boltzmann function of this gating scheme is: with V ½ 0 : V ½ at [K + ] ext. =0 V s : slope factor K 0, K 1 : constants; K 0, K 1 >0  With increasing [K + ] ext. the Boltzmann curve of SKOR shifts positive.

Summary - The model explains several observations on SKOR and its mutants

(1)The model explains the insusceptibility to internal [K + ]: With the gate closed the selectivity filter should be accessible only from outside (compare S-Figure 1), and thus can serve as a K + binding site and sensor independent of [K + ] int. (2)The model explains the observation that increasing [K + ] ext introduces a substantial, long-lived closed state of the channel (C 2high ; compare Figure 2). Thus, the K + - sensitivity of SKOR appears in the closed state. (3)The model explains the observation that certain mutations affecting the pore-S6 interaction (M286L and D312N) eliminated the longer-lived channel closed state(s). In the model this removal of the pore-S6 interaction eliminates state C 2high which in effect reverses the [K + ] ext -dependency. (4)The model explains the parallels in cation selectivity between permeation and gating.