supplementary material to johansson, wulfetange, porée, michard, gajdanowicz, lacombe, sentenac,...

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

The physiological background

animated from Wegner & DeBoer, 1997, Plant Physiol. 115, 1707-1719.

Supplemental Material, Johansson et al.

Long distance transport of K+ in plants

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+

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

Supplemental Material, Johansson et al.

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

The outward-rectifying KThe outward-rectifying K++ channel SKOR senses the K channel SKOR senses the K++ concentration in the stelar apoplast concentration in the stelar apoplast

and releases Kand releases K++ when [K when [K++]]apoplastapoplast decreases (as a consequence of a reduced K decreases (as a consequence of a reduced K++ re-circulation) re-circulation)(Wegner and DeBoer, 1997, Plant Physiol. 115, 1707-1719; Gaymard et al., Cell 94, 647-655)(Wegner and DeBoer, 1997, Plant Physiol. 115, 1707-1719; Gaymard et al., Cell 94, 647-655)

SKORSKOR

How does SKOR sense [K+]apoplast?

The K+-sensing problemHow 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 KIn the following only those details of an outward-rectifying plant K++ channel channel are displayed which are important to explain the Kare displayed which are important to explain the K++-sensing mechanism:-sensing mechanism:

The P-domain and the S6 segment of 2 (out of 4) subunits.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 Upon closing the access of internal [Kof internal [K++] to the pore ] to the pore

is cut off.is cut off.

External [KExternal [K++] in the ] in the selectivity filter stabilizes via selectivity filter stabilizes via

pore-S6 interactions a pore-S6 interactions a closed state.closed state.

The Boltzmann function of this simple gating scheme is:The Boltzmann function of this simple gating scheme is:

withwithV½

0 : V½ at [K+]ext.=0Vs : slope factorK0 : constant; K0>0

An open channelAn open channelmediates Kmediates K++ efflux. efflux.

With increasing [KWith increasing [K++]]ext.ext. the Boltzmann curve shifts positive. the Boltzmann curve shifts positive.

The detailed model for

SKOR [K+]ext.-sensing

Supplemental Material, Johansson et al.

An open channel mediates KAn open channel mediates K++ efflux. efflux.

open

Supplemental Material, Johansson et al.

Spontaneous (voltage-dependent) closing occursSpontaneous (voltage-dependent) closing occursvia rearrangements of C-terminal parts of the S6 segment.via rearrangements of C-terminal parts of the S6 segment.

closed

open closed

Supplemental Material, Johansson et al.

The ionic compositions of the pore and the cavity equilibrate with the external solution. The ionic compositions of the pore and the cavity equilibrate with the external solution. When the external KWhen the external K++ concentration is low also the occupancy of the pore by K concentration is low also the occupancy of the pore by K++ is low. is low.

lowK+

ext.

highK+

ext.

When the external KWhen the external K++ concentration is high also the occupancy of the pore by K concentration is high also the occupancy of the pore by K++ is high. is high.

open closed

lowK+

ext.

highK+

ext.

Supplemental Material, Johansson et al.

When the external KWhen the external K++ concentration is high also the occupancy of the pore by K concentration is high also the occupancy of the pore by K++ is high. is high. The pore is not flexible but rather rigid.The pore is not flexible but rather rigid.Interactive forces between the pore (SKOR-M286) and S6 (SKOR-D312) induce then Interactive forces between the pore (SKOR-M286) and S6 (SKOR-D312) induce then further conformational changes in S6 which stabilize the closed conformation.further conformational changes in S6 which stabilize the closed conformation.

open closed

lowK+

ext.

highK+

ext.

Supplemental Material, Johansson et al.

These rearrangements reduce the interactive forces between the pore and S6.These rearrangements reduce the interactive forces between the pore and S6.No further conformational changes in S6 are induced.No further conformational changes in S6 are induced.

When KWhen K++ext.ext. is low is low the pore is not crowded with Kthe pore is not crowded with K++ ions ionsthe pore is flexible and the pore is flexible and

undergoes structural rearrangements (low-Kundergoes structural rearrangements (low-K++ structure, structure, Zhou et al., 2001, Nature 414, 43-48Zhou et al., 2001, Nature 414, 43-48).).

open closed

lowK+

ext.

highK+

ext.

Supplemental Material, Johansson et al.

Spontaneous rearrangements of the C-terminal part of S6 “re-open” the channel. Spontaneous rearrangements of the C-terminal part of S6 “re-open” the channel. However, the “low-KHowever, the “low-K++ structure” of the pore does not allow K structure” of the pore does not allow K++ flux. flux.

open closed

lowK+

ext.

highK+

ext.

Supplemental Material, Johansson et al.

The cavity is flushed by internal KThe cavity is flushed by internal K++..

open closed

lowK+

ext.

highK+

ext.

Supplemental Material, Johansson et al.

KK++ ions from inside can alter the conformation of the pore ions from inside can alter the conformation of the pore (high-K(high-K++ structure, structure, Zhou et al., 2001, Nature 414, 43-48Zhou et al., 2001, Nature 414, 43-48).).

open closed

lowK+

ext.

highK+

ext.

Supplemental Material, Johansson et al.

The outward-rectifying KThe outward-rectifying K++ channel mediates K channel mediates K++ efflux again. efflux again.

open

Supplemental Material, Johansson et al.

The Boltzmann function of this gating scheme is:The Boltzmann function of this gating scheme is:

withwithV½

0 : V½ at [K+]ext.=0Vs : slope factorK0, K1 : constants; K0, K1 >0

With increasing [KWith increasing [K++]]ext.ext. the Boltzmann curve of SKOR shifts positive. the Boltzmann curve of SKOR shifts positive.

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

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

(1)(1) The model explains the insusceptibility to internal [KThe model explains the insusceptibility to internal [K++]: With the gate closed the ]: With the gate closed the

selectivity filter should be accessible only from outside (compare S-Figure 1), and selectivity filter should be accessible only from outside (compare S-Figure 1), and

thus can serve as a Kthus can serve as a K++ binding site and sensor binding site and sensor inindependent of [Kdependent of [K++]]intint. .

(2)(2) The model explains the observation that increasing [KThe model explains the observation that increasing [K++]]extext introduces a substantial, introduces a substantial,

long-lived closed state of the channel (Clong-lived closed state of the channel (C2high2high; compare Figure 2). Thus, the K; compare Figure 2). Thus, the K++--

sensitivity of SKOR appears in the closed state. sensitivity of SKOR appears in the closed state.

(3)(3) The model explains the observation that certain mutations affecting the pore-S6 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 interaction (M286L and D312N) eliminated the longer-lived channel closed state(s). In

the model this removal of the pore-S6 interaction eliminates state Cthe model this removal of the pore-S6 interaction eliminates state C2high2high which in effect which in effect

reverses the [Kreverses the [K++]]extext-dependency.-dependency.

(4)(4) The model explains the parallels in cation selectivity between permeation and gating.The model explains the parallels in cation selectivity between permeation and gating.