1. 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 dreyer@rz.uni-potsdam.de

2. The physiological background

3. animated from Wegner & DeBoer, 1997, Plant Physiol. 115, 1707-1719. Supplemental Material, Johansson et al. Long distance transport of K + in plants

4. animated from Wegner & DeBoer, 1997, Plant Physiol. 115, 1707-1719. Supplemental Material, Johansson et al. Demand for K + in the shoot  Reduced re-circulation of K +

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

6. 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, 1707-1719; Gaymard et al., Cell 94, 647-655) SKOR

7. How does SKOR sense [K + ] apoplast ?

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

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

10. 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.

11. The main ideas in short…

12. 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.

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

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

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

16. 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.

17. 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.

18. 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, 43-48 ).

19. 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.

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

21. 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, 43-48 ).

22. openclosed low K + ext. high K + ext. Supplemental Material, Johansson et al. The outward-rectifying K + channel mediates K + efflux again. open

23. 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.

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

25. (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.