Mechanosensitive Ion Channels in Plants

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Washington University in St. Louis

Washington University Open Scholarship Biology Faculty Publications

Biology

2015

United in Diversity: Mechanosensitive Ion Channels in Plants Eric S. Hamilton Angela M. Schlegel Elizabeth S. Haswell Washington University in St Louis, [email protected]

Follow this and additional works at: http://openscholarship.wustl.edu/bio_facpubs Part of the Biology Commons, and the Plant Biology Commons Recommended Citation Hamilton, Eric S.; Schlegel, Angela M.; and Haswell, Elizabeth S., "United in Diversity: Mechanosensitive Ion Channels in Plants" (2015). Biology Faculty Publications. Paper 55. http://openscholarship.wustl.edu/bio_facpubs/55

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UNITED IN DIVERSITY: MECHANOSENSITIVE ION CHANNELS IN PLANTS

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Eric S. Hamilton, Angela M. Schlegel & Elizabeth S. Haswell*

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Department of Biology, MC1137

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Washington University in Saint Louis

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Saint Louis, MO 63130

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314-935-9223

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[email protected]

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[email protected]

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[email protected]

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*Corresponding author

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TABLE OF CONTENTS

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1. Introduction

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2. Mechanosensitive Ion Channels: Transducing Force into Current

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2.1 Models for MS Channel Gating

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3. Physiological Roles for MS Channels

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3.1 Animal MS Channels

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3.2 Plant MS Channels

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4. Approaches Used to Study Plant MS Ion Channels

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4.1 Pharmacological Inhibition and Activation

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4.2 Electrophysiology

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4.3 Genetics

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5. Criteria for Assignment as a Mechanosensitive Ion Channel

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6. MSL Channels

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6.1 A Diverse Family of MS Channels

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6.2 Evidence that MSLs are MS Ion Channels

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6.3 MSLs Serve as Osmotic Conduits in the Plastid Envelope

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6.4 MSLs at the Plant Plasma Membrane

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6.5 Beyond the Paradigm of Emergency Release Valves

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7. MCA Channels

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7.1 MCA Proteins Modulate Ca2+ Influx in Response to Mechanical Stimuli

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7.2 Structural Features of MCAs

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8. TPK Channels

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9. Other Activities Yet to be Identified

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KEYWORDS

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Mechanotransduction, MscS, MSL, MCA, TPK1

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41

ABSTRACT

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Mechanosensitive (MS) ion channels are a commonly used mechanism for the

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perception and response to mechanical force. This class of mechanoreceptors is

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capable of transducing membrane tension directly into ion flux. In plant systems, MS ion

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channels have been proposed to play a wide array of roles, from the perception of touch

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and gravity to osmotic homeostasis of intracellular organelles. Three families of plant

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MS channels have been identified: the MscS-Like (MSL), Mid1-Complementing Activity

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(MCA), and Two-Pore Potassium (TPK) families. Channels from these three families

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vary widely in terms of structure and function, localize to multiple cellular compartments,

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and conduct chloride, calcium, and/or potassium ions. However, they are still likely to

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represent only a fraction of the MS ion channel diversity present in plant systems.

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1. INTRODUCTION

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How cells sense mechanical force is a long-standing question in biology. Mechanical

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signals such as touch, gravity, and osmotic pressure are critical to proper development,

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environmental stress response, and overall cellular health in a wide variety of

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prokaryotic and eukaryotic organisms and cell types. The perception of force can be

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mediated

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reorganization, or nuclear deformation (reviewed in (32)).

through

the

actions

of

integrins

or

focal

adhesions,

cytoskeletal

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Alternatively, the application of intracellular or extracellular force can result in the

64

deformation of cellular membranes, where it is perceived by a specialized class of ion

65

channels. Ion channels, membrane-spanning protein complexes that facilitate the flux of

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ions across the lipid bilayer, are responsible for a wide range of functions across all of

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life, including the production of action potentials in nerve cells (102), maintaining the

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ionic conditions required for metabolism in plants (132) and Ca2+ signaling in all cells

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(19). Interested readers are referred to two recent reviews on plant ion channel function

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(48, 121).

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The flux of ions through a channel can be regulated by a variety of stimuli, including

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transmembrane voltage (10), ligand binding (57), light (23), and mechanical force (78). It

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is the latter stimulus that defines a diverse group of channels known as

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mechanosensitive (MS) ion channels, also referred to as stretch-activated or force-

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gated channels. MS ion channels are found in all three domains of life (5, 58, 61),

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pointing to the fundamental requirement for mechanosensation in all cells.

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Several recent reviews describe the wide variety of mechanical stimuli that land plants

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must sense and respond to during their lifespan (21, 85, 114). In addition, as many as

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18 distinct MS ion channel activities have been identified in plant membranes by patch-

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clamp electrophysiology (these are described in detail below), implying that

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mechanically gated ion channels play an important role in plant systems. In this review

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we provide a general introduction to MS ion channel structure and function, outline the

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approaches used to study plant MS ion channels, and summarize what is currently

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known about three families of plant MS ion channels, emphasizing their diverse

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structure, evolutionary history, and physiological roles.

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2. MECHANOSENSITIVE ION CHANNELS: TRANSDUCING FORCE INTO CURRENT

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The electrical excitability of cells was first studied in the giant cells of Characean algae,

92

prior to the adoption of the giant axons of squid as a model system in the 1930s

93

(reviewed in (122)). The advent of the patch-clamp technique, which permitted the study

94

of individual channels in isolated cell membranes (see Section 4.2 below), made

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possible the first identification of MS ion channel activities in animal skeletal cells (41,

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43). Shortly thereafter, MS ion channel activities were detected in tobacco, broad bean

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and giant Escherichia coli protoplasts (33, 80, 106). The structures of two of the MS

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channels identified in those E. coli protoplasts have been solved at atomic resolution,

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providing the foundation for many elegant experimental and theoretical investigations

100

into the molecular mechanism of MS channel activity (reviewed in (111)).

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2.1 Models for MS Channel Gating

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An ion channel can be idealized as a two-state system, where it exists in either a closed

104

(or non-conducting) or an open (conducting) state. The transition from a closed to an

105

open state is referred to as “gating”. Once gated, an ion channel does not require

106

additional energy to conduct a current; rather, ions move down their electrochemical

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gradient in either direction across the membrane through the channel pore (49).

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For some classes of MS ion channels, increased membrane tension leads directly to

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gating. This behavior has been described by a number of biophysical models that

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address the energetic interactions at the membrane-protein interface (reviewed in (47)).

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One proposed mechanism is the lipid disordering model illustrated in Figure 1A. An ion

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channel increases the free energy of the membrane in which it is embedded, as the

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lipids in that membrane must disorder to conform to the shape imposed by the

115

boundaries of the protein. With the addition of potential energy in the form of membrane

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tension, a conformational change in the channel that reduces the local deformation

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imposed on the membrane, while opening the channel pore, is favored (79, 115).

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119

Another driving force for MS channel gating may be the thinning of the lipid bilayer

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under increased membrane tension (Figure 1B). According to this model, membrane

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thinning results in a mismatch between the height of the channel’s hydrophobic

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transmembrane (TM) domain and the profile of the lipid bilayer, leading to the exposure

123

of nonpolar side chains to the aqueous intra- or extra-cellular environment. A

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conformational change in the channel that maintains energetically favorable interactions

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between the TM domain and the lipid bilayer (such as rotating a TM helix within the

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plane of the membrane) is then coupled to the opening of the channel pore (77, 81). It is

127

worth noting that the lipid disordering and hydrophobic mismatch mechanisms are not

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mutually exclusive, and that there are likely many other mechanisms capable of driving

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intrinsic mechanosensitivity in ion channels (96, 98).

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In the two models shown in Figure 1, membrane tension is transmitted directly to the

132

channel through the lipid bilayer. Alternatively, some MS ion channels—including those

133

proposed to mediate hearing in the vertebrate inner ear hair cells and gentle touch in

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Caenorhabditis elegans—are likely to be gated indirectly by tethering to other cellular

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components (29). Tension applied to a physical link between a channel and the

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extracellular matrix or the intracellular cytoskeletal system could directly stretch open

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the channel, reorient the channel in the lipid bilayer, or lead to lipid raft reorganization

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(3, 13, 47). A unifying theme in all of these models, however, is that the responsiveness

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of a MS ion channel to force depends on highly dynamic interactions with the lipid

140

bilayer.

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3. PHYSIOLOGICAL ROLES FOR MS CHANNELS

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There are many ways in which an organism might employ a mechanosensor capable of

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transducing force into ion flux; many MS channels from animals have been studied in

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detail and shown to improve fitness during development or in a changeable

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environment. Here we briefly summarize what is known about the physiological roles of

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MS channels in C. elegans, D. melanogaster, and other metazoans—as these studies

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inform our understanding of MS channels in plants, whether or not the channels are

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evolutionarily related—and then address their potential functions in plants.

150 151

3.1 MS Ion Channels in Animals

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Several distinct families of MS channels are thought to underlie the senses of touch,

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pain, hearing, proprioception, and gravity sensation in animal systems. For example,

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response to light touch is mediated by the Degenerin/Epithelial Sodium Channel

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(Deg/ENaC) family in C. elegans. The Transient Receptor Potential (TRP) family

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mediates nose touch and proprioception in C. elegans and hearing, nociception, and

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bristle touch in Drosophila melanogaster (reviewed in (5)). Two-Pore Potassium (TPK)

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MS ion channels are required for pressure-responsive vasodilation and also appear to

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regulate the pain threshold for cold and heat in mice (reviewed in (50)). The recently

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identified Piezo family mediates diverse mechanosensory events in animals, from touch

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and pain in sensory neurons to intercellular communication and osmotic control

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(reviewed in (119), see sidebar: Piezo).

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3.2 MS Ion Channels in Plants

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Plants sense and respond to many of the same mechanical stimuli as animals, including

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touch, gravity, and osmotic stress (16, 85, 114). They also respond to unique signals

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associated with developmental processes such as lateral root emergence, pollen tube

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growth, cell wall damage, and plant-pathogen interactions (4, 52, 72). In many of these

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cases, applying a mechanical stimulus leads to a rapid burst of ion flux, and it has long

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been speculated that this correlation may be attributed to the action of MS ion channels

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in the stimulated cells, in part because of the speed of the response (reviewed in (34,

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51, 85)).

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The flux of Ca2+ in particular has been implicated in various mechanosensory pathways.

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For example, gravity stimulation (introduced by rotating a root or shoot 90 degrees) is

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associated with membrane depolarization, the rapid influx of Ca2+ ions and subsequent

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alkalinization of the cells in the root cap (reviewed in (114)). Ca2+ influx is also

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associated with touch stimulus (reviewed in (34)), osmotic stress (108), and bending

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(84), consistent with the action of a mechanically gated calcium channel in these

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processes. After influx, Ca2+ could serve as a second messenger in a large number of

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downstream events—some of which, such as the activation of calmodulin and

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calmodulin-like proteins, are also implicated in mechanotransduction (16).

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4. APPROACHES USED TO STUDY MS ION CHANNELS IN PLANTS

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4.1 Pharmacological Inhibition or Activation

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Evidence that MS ion channels are an integral part of a particular mechanosensory

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process can be obtained by pharmacological treatments with known inhibitors or

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activators of MS ion channels. The Ca2+ influx associated with mechanical stimulation

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can be inhibited by lanthanides (26, 83, 89), ruthenium red (67) or cytoskeletal inhibitors

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(25). However, these treatments are often non-specific. For example, the commonly

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used lanthanide Gd3+ blocks a wide variety of channels, not only Ca2+-selective or

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mechanosensitive (69). Furthermore, Gd3+ can indirectly inhibit the action of non-

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selective MS ion channels by reducing overall membrane fluidity (31, 74). On the other

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hand, the chemical trinitrophenol (TNP), which increases curvature and tension when

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applied to membranes (79), behaves as a MS channel activator and can induce Ca2+

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flux or lower the threshold for mechanical stimulation (37, 89, 103). While sensitivity to

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one of these pharmacological agents can provide evidence that MS ion channels are

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involved in a particular response, confirmation will likely require knowing the molecular

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identity of the channels involved and characterization of their channel properties through

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the methods described below.

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4.2 Electrophysiology

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The gold standard technique for the analysis of MS ion channels is patch-clamp

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electrophysiology. Patch-clamping involves the production of a high resistance seal

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between the glass of a micropipette tip and a small patch of membrane containing the

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channels of interest (105). The pipette can either remain attached to the cell with an

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intact patch (cell-attached), remain attached to the cell with a ruptured patch (whole-

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cell), or be completely removed from the cell along with the patch (excised). In all of

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these configurations, membrane tension is increased by introducing positive or negative

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pressure through the patch pipette, and the resulting current across the membrane is

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recorded over time. This technique has been used to identify and characterize plant MS

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channels in their native membranes or heterologously expressed in Xenopus oocytes,

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as described in more detail below. While patch clamping allows the identification of

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individual MS ion channels—and in the excised patch configuration, control over the

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ionic conditions on both sides of the membrane—a drawback especially relevant to

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plant systems is that it requires isolation of cells from the tissue and the removal of the

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cell wall.

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4.3 Genetics

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Molecular genetic approaches in Arabidopsis thaliana and other model plant systems

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have added another dimension to the study of MS channels in recent years. Although a

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forward genetic screen has not yet successfully been used to identify a MS channel,

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reverse genetics motivated by either phylogenetics or a functional assay has identified

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several candidates (45, 73, 89). Once a candidate gene is identified, the protein can be

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heterologously expressed and tested for MS channel activity in cell survival or

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electrophysiological assays. Genetic ablation or overexpression of candidate MS

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channel genes in planta can be powerful tools for characterizing channels in their native

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systems.

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5. CRITERIA FOR ASSIGNMENT AS A MECHANOSENSITIVE ION CHANNEL

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Establishing that a particular gene encodes the primary force-transducer in a MS

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response, as opposed to an accessory or downstream component of the MS response,

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is a challenging endeavor. The following criteria have previously been established: 1)

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proper expression and localization for the observed MS response; 2) the channel is

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required for the response, but not for the normal development of the cell or tissue in

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which the response occurs (unless the MS response being measured is the

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development of the tissue itself); 3) evidence of MS gating in isolation or in heterologous

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systems; and 4) structural alterations of the protein produce changes in the MS

241

response and channel behavior (5, 18, 87). Assembling all four criteria to definitively

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categorize MS ion channels can be difficult, especially if MS ion channels function as

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heteromultimers or with other cellular structures.

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Although none of the MS ion channel candidates so far identified in plants fulfill all four

246

of these criteria, we refer to them here as MS channels in consideration of the strong

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evidence that does exist in favor of this interpretation in each case. Table 1 summarizes

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relevant information about the three families of plant MS channels that have so far been

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identified: the MscS-Like (MSL), Mid1-Complementing Activity (MCA), and Two-Pore

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Potassium (TPK) channels. As summarized below, channels from these three families

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vary widely in terms of structure and function, localize to multiple cellular compartments,

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and conduct diverse subsets of ions.

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6. MSCS-LIKE CHANNELS

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The first family of putative plant MS ion channels were identified based on their similarity

257

to the E. coli Mechanosensitive channel of Small conductance (MscS), a well-

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established model system for the study of MS ion channels ((12, 82), see sidebar:

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MscS). MscS serves as an “emergency release valve” under conditions of hypoosmotic

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shock in E. coli (12, 68). Genes predicted to encode homologs of MscS are found

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throughout bacterial and archaeal genomes, in some protist genomes—including

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pathogenic protozoa—and in all plant genomes so far examined (7, 44, 59, 60, 76, 100,

263

101, 126). MscS homologs have not yet been identified in animal genomes.

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The predicted evolutionary relationship among representative members of the MscS

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superfamily is presented in Figure 2A, using the ~100 amino acid domain conserved

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among the MscS superfamily (99). This sequence maps to the pore-lining helix and the

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upper part of the cytoplasmic vestibule of MscS and is marked in cyan in Figure 2B (8).

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A number of highly conserved motifs within this domain are important for function in

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bacterial and plant channels (7, 22, 53). Land plant MscS homologs fall into three

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phylogenetic groups, I-III, which also correspond to three different subcellular

272

localizations (see below).

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6.1 A Diverse Family of MS Channels

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Outside of the conserved MscS domain, MscS family members are highly divergent in

276

their topology and domain structure. Among others, domains associated with cyclic

277

nucleotide, Ca2+ or K+ binding are found appended to the basic MscS topology (76, 116,

278

126). In addition, plant MSL proteins localize to multiple cellular compartments,

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providing further evidence that they serve a diverse set of functions within the cell

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(Figure 2B).

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Group I and Group II MSL proteins are predicted (and in some cases, have been

283

shown) to localize to mitochondria and to plastids, respectively (44, 45). Both groups are

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predicted to contain five TM helices, the last helix corresponding to the pore-lining

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domain of MscS, and a C-terminus that is located in the stroma or matrix. Group III MSL

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proteins are predicted or shown to localize to the plasma membrane (44, 46) and to

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contain six TM helices, again with the most C-terminal TM segment corresponding to

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the pore-lining domain of MscS. They also have a large cytoplasmic N-terminus, a

289

cytoplasmic loop of variable length between TM regions four and five, and a cytoplasmic

290

C-terminus.

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The A. thaliana genome encodes ten MSL genes (Table 1), and they show a range of

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expression patterns including root- and flower-specific expression (44). At the protein

294

level, most A. thaliana MSLs can be grouped into pairs based on sequence homology.

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MSL2 and MSL3 are 50% identical at the amino acid level; MSL4 and MSL5 are 68%

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identical; and MSL7 and MSL8 are 71% identical and located in tandem on the

297

chromosome (44). The existence of highly similar pairs of MSLs in the A. thaliana

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genome may indicate functional redundancy (as with MSL2 and MSL3, see below), but

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may also have permitted the evolution of unique characteristics (as with MSL9 and

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MSL10, see below).

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6.2 Evidence that MSLs are MS Ion Channels

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It is currently accepted that MS ion channel activity has been retained among most

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members of the MscS superfamily (however, for one exception see (15)). All six MscS

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family members in E. coli are capable of producing tension-gated activities in giant

306

spheroplasts (28, 68, 70, 107), as is MSC1 from Chlamydomonas reinhardtii, (91), and

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MSY1 from fission yeast (92).

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Several lines of evidence support the hypothesis that MSLs form functional MS ion

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channels in A. thaliana. Plastid-localized MSL3 was able to partially rescue the

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susceptibility of an E. coli strain missing three major MS ion channels to hypoosmotic

312

shock (45). More direct evidence was obtained for the endoplasmic reticulum (ER)- and

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plasma membrane-localized channels MSL9 and MSL10, which are genetically required

17

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for the primary MS ion channel activity detected by whole-cell electrophysiology in root

315

protoplasts (46). A characterization of the conductance of MS ion channel activities

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present in root protoplasts from single msl9, single msl10 or double msl9 msl10 mutants

317

suggests that MSL9 and MSL10 can form a heteromeric channel with a conductance of

318

~50 pS, while MSL9 and MSL10 homomeric channels have conductances of ~45 pS

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and ~140 pS, respectively (46, 97). The ability to form homo- and heteromeric channels

320

with distinct properties, in combination with overlapping tissue-specific expression

321

patterns for multiple MSL genes, could produce a range of MS responses across

322

different tissues in plants.

323 324

In agreement with the in planta electrophysiology described above, it was recently

325

shown that MSL10 is associated with a ~100 pS MS ion channel activity when

326

expressed heterologously in Xenopus oocytes (75). MSL10 channel activity in ooctyes

327

has a slight (6-fold) preference for anions, and closes at lower tensions than it opens.

328

MSL10 meets three of the four criteria for a bona fide MS ion channel: 1) MSL10 is

329

expressed in root cells, where 2) it is required for the wild type MS ion currents but not

330

for the normal development of the tissue, and 3) expression of MSL10 in Xenopus

331

oocytes confirms it can form a functional MS ion channel in a heterologous system.

332

However, it has not yet been determined that structural changes (such as point

333

mutations in the putative pore-lining domain) alter its mechanosensitivity.

334 335

6.3 MSLs Serve as Osmotic Conduits in the Plastid Envelope

18

336

Of all MscS homologs in plants, we know the most about those that localize to the

337

plastid envelope. Originally, it was surprising to find homologs of a protein known to

338

protect a bacterial cell from environmental osmotic shock targeted to intracellular

339

organelles. However, all evidence now suggests that Group II MSLs serve a role related

340

to (but distinct from) function as an emergency release valve. Consistent with

341

bioinformatic predictions, A. thaliana MSL2 and MSL3 localize to the plastid envelope,

342

likely to the inner membrane, and are observed in foci at the plastid poles (45, 125).

343

Immunofluorescence of algal MSC1 similarly revealed a complex localization pattern of

344

punctate spots both within the chloroplast and the cytoplasm (91).

345 346

Plants harboring lesions in MSL2 and MSL3 show numerous whole-plant and

347

subcellular defects, but the most striking phenotype is the presence of greatly enlarged

348

and spherical non-green plastids in the epidermis and root (small, ovoid plastids are

349

seen in wild type epidermal cells) (45). These defects in plastid size and shape can be

350

rescued by increasing the osmolarity of the cytoplasm relative to the plastid by a variety

351

of genetic and environmental manipulations (117), strongly suggesting that non-green

352

plastids experience hypoosmotic stress under normal conditions within the cytoplasm,

353

and that MSL2 and MSL3 function redundantly to relieve this stress.

354 355

In photosynthetic tissues, msl2 msl3 mutants have fewer and larger chloroplasts than

356

the wild type, possibly as a result of the multiple FtsZ rings observed in the chloroplasts

357

of this mutant (125). As MSL2 and MSL3-GFP fusion proteins co-localize with the

19

358

plastid division protein AtMinE (45), it is possible that MSL2 and MSL3 interact with the

359

plastid division machinery to influence division site selection; it is equally feasible that

360

the defect in plastid division in msl2 msl3 mutants derives from altered stromal ion

361

homeostasis or a mechanical inability to constrict the FtsZ ring (124). A function for MS

362

channels in division is evolutionarily conserved, as E. coli mutants lacking several key

363

MS channels also exhibit defects in division site selection when exposed to the division

364

inhibitor cephalexin (125). Chloroplast-localized MSC1 of Chlamydomonas is required

365

for chloroplast integrity; whether the mechanism behind this defect is the same as in

366

msl2 msl3 mutants is not clear (91).

367 368

Single msl2 and double msl2 msl3 mutants also exhibit a number of whole-plant

369

phenotypes, including dwarfing, rumpled leaf surfaces, thicker leaf lamina, and

370

variegation (45, 53, 125). While the source of these phenotypes remains under

371

investigation, at least some of them are likely to be developmental responses to plastid

372

osmotic stress. Plastid osmotic stress in these mutants leads to the activation of

373

dehydration stress responses such as the accumulation of proline and the production of

374

ABA, even in the absence of any extracellular osmotic stress (123). MSL2 and MSL3

375

appear to function partially redundantly to relieve plastid osmotic stress. While a null

376

msl2 allele produces developmental defects even in the wild type MSL3 background

377

(53), all mutant phenotypes are exacerbated in the msl2 msl3 double mutant (45, 117,

378

123, 125). There is currently no null msl3 allele, and it remains to be established exactly

379

how the functions of MSL2 and MSL3 overlap and diverge.

20

380 381

6.4 MSLs at the Plant Plasma Membrane

382

In contrast to the Group II MSLs, establishing a role for Group III MSLs in plants has

383

been a challenge. None of the obvious assays (touch, gravity, osmotic shock, etc.)

384

produce phenotypes distinguishable from the wild type, even in a msl4 msl5 msl6 msl9

385

msl10 quintuple null mutant (46, 114). This may be surprising, given that MSY1 and

386

MSY2, which localize to the ER of Schizosaccharomyces pombe, play an essential role

387

in protecting cells from hypoosmotic shock (92). This could be due to redundant

388

mechanosensory pathways, or because this class of MSLs is required for plants to

389

survive stressful conditions not easily replicated in the laboratory. Consistent with the

390

latter interpretation, recent evidence points to a role for MSL10 in one or more stress-

391

induced cell death signaling pathways. Both transient and stable MSL10 overexpression

392

leads to dwarfing, H2O2-associated cell death, and the induction of cell death-associated

393

gene expression (116).

394 395

6.5 Beyond the Paradigm of Emergency Release Valves

396

While MscS functions as an emergency release valve in E. coli, it has become clear that

397

it and other members of the MscS superfamily serve multiple and complex roles in both

398

prokaryotes and eukaryotes (reviewed in (11, 22, 76, 126)). Based on the diverse

399

localization, topology, and domain structure within the MscS superfamily, we have

400

previously suggested that 1) some MscS-like channels may respond to osmotic stress

401

other than that provided by the extracellular environment, 2) some may be regulated by

21

402

mechanisms other than membrane tension, and 3) some might even have functions that

403

are completely separable from their role in mediating ion flux (47).

404 405

Experimental support for these three ideas in A. thaliana MSLs has accumulated over

406

the past decade. For example, 1) the osmotic swelling of msl2 msl3 mutant plastids

407

illustrates that the environment of the cytoplasm can be as osmotically stressful to

408

organelles as the extracellular environment is to a bacterial cell, and that MscS-like

409

channels can serve to protect organellar membranes this stress during normal growth

410

and development (45, 117). Furthermore, 2) there is evidence that Group I, II, and III

411

MSLs may be regulated by phosphorylation in addition to membrane tension. Multiple

412

proteomic studies have identified phosphopeptides that map to MSL1, MSL3, MSL4,

413

MSL5, MSL6, MSL9 and MSL10 (summarized at http://phosphat.uni-hohenheim.de/). At

414

least some of these modifications are likely to be functionally relevant, as the cell death

415

signaling function of MSL10 can be controlled by mutating the phosphorylated residues

416

in its soluble N-terminal domain (116), and MSL9 is a direct target of the drought-

417

associated kinase SnRK2.6 (120). Finally, 3) at least one MSL does indeed have a

418

function that is separable from ion flux, as the cell death signaling function of MSL10

419

requires only its soluble N-terminal domain, which is unique to MSL10 and its orthologs

420

in other plant species and does not form a channel on its own (116). We anticipate that

421

future studies in A. thaliana and other model systems will uncover a multiplicity of

422

physiological functions and regulatory mechanisms for MSL channels.

423

22

424

7. MID1-COMPLEMENTING ACTIVITY CHANNELS

425 426

While MSLs are essential to organelle osmoregulation and likely play complex roles at

427

the plasma membrane and ER, they are essentially non-selective ion channels. Thus,

428

their discovery and characterization still left open the identity of the elusive calcium

429

channels thought to be associated with mechanical signaling, as reviewed above. Soon,

430

however, candidates for such channels were provided by the discovery of the novel land

431

plant-specific family of membrane-associated proteins called Mid1-Complementing

432

Activity (MCA). Only one or two family members are found in each plant genome, and

433

homologs have not been found in algae or animals (64). Sequence conservation is not

434

restricted to a single domain, but is distributed along the length of the protein.

435 436

7.1 MCA Protein Function is Tightly Correlated with Ca2+ influx

437

The founding member of the family, A. thaliana MCA1, was identified in a functional

438

screen for cDNAs capable of rescuing the mid1 mutant strain of Saccharomyces

439

cerevisiae (89). Mid1 is a stretch-activated MS ion channel required for Ca2+ influx and

440

cell survival after exposure to mating pheromone (56). A. thaliana MCA2 and Nicotiana

441

tabacum MCA1 and MCA2 were identified based on homology to AtMCA1 and are also

442

capable of promoting the survival of mid1 yeast in the mating factor assay (64, 129).

443 444

MCA proteins from A. thaliana, rice, and tobacco appear to serve similar roles in all

445

three organisms; for an overview of these genes and their characteristics, see Table 1.

23

446

MCA-mediated activity responds to stimuli associated with increased membrane

447

tension, but also appears to contribute to Ca2+ homeostasis in the absence of stress

448

(63, 64, 89, 129). Taken together, the current data support a model wherein MCA

449

proteins either are themselves MS calcium channels, or are closely associated with the

450

activity of a MS calcium channel.

451 452

Most MCA-GFP fusion proteins localize to the plasma membrane of plant cells, often in

453

a punctate pattern (63, 89, 90, 129). When expressed in yeast, MCA1 fractionates with

454

plasma membrane proteins and behaves like an intrinsic membrane protein in solubility

455

tests (89, 90). The overexpression of MCA proteins is associated with increased influx

456

of Ca2+ into plant roots, plant tissue culture cells, CHO cells, or yeast cells, either in the

457

absence of stimulus or transiently in response to hypoosmotic shock, cell stretching, or

458

treatment with the membrane-distorting lipid TNP (63, 64, 89, 129). Additionally,

459

increased expression of the touch-inducible genes TCH3 (in A. thaliana) and ERF3 (in

460

N. tabacum) is correlated with the overexpression of MCA proteins (14, 64, 89, 95).

461 462

MCA genes are expressed broadly in a variety of tissues (63, 89, 129) and MCA T-DNA

463

insertion mutants in A. thaliana and OsMCA1-silenced lines in rice show growth defects

464

and late flowering (63, 129). AtMCA1 and AtMCA2 have partially divergent functions;

465

mca1 but not mca2 mutants show defects in root entry into hard agar (89, 129), while

466

mca2 but not mca1 mutants are defective in Ca2+ uptake in A. thaliana roots (129). The

467

mca1 mca2 double mutant exhibits both of these phenotypes, as well as growth that is

24

468

hypersensitive to Mg2+ (probably due to competition with Ca2+ for uptake) (129). Further

469

evidence that MCAs are involved in signaling in response to membrane tension comes

470

from studies on the cellular response to treatment with the cell wall biosynthesis inhibitor

471

isoxaben, which leads to cellular swelling (66). MCA1 is required for the accumulation of

472

lignin and altered expression pattern of carbohydrate metabolism genes that are

473

observed in wild type cells treated with isoxaben (24, 42, 127).

474 475

7.2 Structural Features of MCAs

476

Surprisingly, MCAs do not resemble Mid1, nor any known ion channels or membrane-

477

bound transporters. They do encode three recognizable motifs, including an EF-hand-

478

like motif at the N-terminus, a coiled-coil motif, and a Plac8 motif at the C-terminus (62).

479

The membrane-spanning domains and topology of MCAs is still under investigation.

480

Unpublished data indicate that MCAs harbor a single TM helix at the extreme N-

481

terminus (H. Iida, personal communication). Deleting this TM helix disrupts MCA1 and

482

MCA2 function in yeast cells, as does changing a single conserved aspartic acid within

483

it to asparagine (D21N) (90). Gel migration, gel filtration and crosslinking studies

484

indicate that MCA1 and MCA2 form homotetramers (90, 109). Cryo-electron microscopy

485

followed by single particle reconstruction of purified MCA2 complexes revealed a tear-

486

shaped structure, consistent with the complex forming a single narrow TM spanning

487

region and a larger cytoplasmic domain (109).

488

25

489

Like MSL10, MCA-associated channel activity has been characterized in Xenopus

490

oocytes. Using the cell-attached patch clamp method, a statistically significant increase

491

in current in response to negative pressure was observed in oocytes expressing MCA1

492

or MCA2, compared to those expressing the plant potassium channel KAT1 or those

493

injected with water (36). In addition, single channel activities of ~15 pS and ~35 pS were

494

occasionally detected in MCA1-expressing (but not in water-injected) ooctyes in

495

response to negative pressure. These data support the model derived from other

496

studies that MCAs form mechanically gated Ca2+-permeable ion channels, but still falls

497

short of establishing this unequivocally by introducing a mutation that alters channel

498

behavior. As the single channel activities attributed to MCA1 appear rare (detected in 16

499

and 5 patches out of 71, respectively, (36)), it is possible that association with a plant-

500

specific component is required for full activity.

501 502

8. TWO-PORE POTASSIUM CHANNELS

503 504

A third group of plant MS channels includes channels related to those in the mammalian

505

TPK family (also designated K2P; see Table 1 for a summary of their relevant

506

properties). As is evident from their name, TPKs possess two pore domains and are K+-

507

selective. TPK activity is pH sensitive, voltage-independent and can often be activated

508

by increased [Ca2+]cyt (30). Membrane tension has been shown to modulate the open

509

probability of several mammalian TPKs (9, 17) and they are proposed to play a variety

26

510

of mechanosensory roles in cardiomyocytes, the smooth muscle of the stomach and

511

intestines, and in pain perception (50).

512 513

In plants, a subset of TPK channels is localized to the vacuolar membrane (summarized

514

in (118)). AtTPK1, a vacuolar-membrane localized TPK from A. thaliana, is required for

515

normal stomatal closure kinetics, K+ homeostasis in multiple tissue types, and efficient

516

seed germination (39). The cytoplasmic domains of many plant TPKs harbor predicted

517

14-3-3 protein binding domains and Ca2+-binding EF hand motifs; accordingly their

518

activity is activated by co-expression of 14-3-3 proteins and elevated cytosolic Ca2+

519

levels. Recently, TPKs from A. thaliana (AtTPK1), barley (HvTPK1), and rice (OsTPKa)

520

were expressed in A. thaliana mesophyll cell protoplasts isolated from plants lacking the

521

two major vacuolar K+ channels, TPK1 and TPC1. In the vacuolar membrane from these

522

protoplasts, increased current in response to membrane tension, osmotic shock and

523

TNP treatment was observed (73). While TPK channels from both plants and animals

524

gate more readily in the presence of membrane tension, they still exhibit basal activity in

525

the absence of tension, and are often referred to as “spontaneous” or “leaky” (e.g. (30,

526

39)).

527 528

9. OTHER MS CHANNEL ACTIVITIES IN PLANT MEMBRANES

529 530

While the MSLs, MCAs and TPKs are certain to play important roles in plant biology,

531

and provide both cation- and anion-permeable MS channels, they are unlikely to

27

532

account for all of the endogenous MS ion channel activities that have been identified in

533

plant membranes (Figure 3). Many unidentified MS ion channel activities have been

534

observed in plant plasma membranes, including Cl--permeable channels in A. thaliana

535

mesophyll cells and stem-derived suspension cultures of N. tabacum (33, 103); Ca2+-

536

permeable channels in onion epidermal cells (25); and MS ion channel activities of

537

unknown permeability in Zostera muelleri (38), A. thaliana hypocotyl cells (69), and the

538

vacuolar membranes of onion parenchyma (6). MS ion channels permeable to both K+

539

and Cl- have been identified in the plasma membrane of A. thaliana mesophyll cells

540

(110) and in the vacuolar membranes of Beta vulgaris (2). Particularly interesting from a

541

physiological standpoint may be the MS ion channel activities that have been detected

542

in pollen grains and pollen tubes (27), in guard cells (20, 37, 71, 106, 130), and in the

543

leaf-moving organ (pulvinus) of Samanea saman (86).

28

544

SUMMARY POINTS LIST

545 546 547

1. MS ion channels transduce mechanical force into biochemical signals for a wide range of physiological purposes.

548

2. Plants sense and respond to diverse mechanical forces including touch, gravity,

549

osmotic pressure and developmental events. MS ion channels are likely

550

participants in some or all of these processes.

551

3. MS ion channel activities are well represented among different plant species, cell

552

types, and cellular compartments, but only three families have yet been

553

characterized in plants; other MS ion channels known to be present have not yet

554

been identified at the molecular level.

555

4. A broad assortment of techniques exist to study plant MS ion channels, but

556

additional approaches are needed to preserve the cell-and tissue-specific context

557

in which MS channel function in planta.

558

5. A subset of MSL channels localize to mitochondrial and plastidic envelopes, and

559

serve to relieve hypoosmotic stress in plastids during normal growth and

560

development.

561

6. Another class of MSLs localize to the plasma membrane and ER where they are

562

required for the predominant MS channel activity in root protoplasts. The

563

physiological function(s) of these channels have been elusive, though at least

564

one has been implicated in stress-induced cell death signaling.

29

565

7. MCA proteins were identified in a functional screen in yeast. MCAs in multiple

566

plant species are required for Ca2+ influx in response to mechanical events and

567

mediate Ca2+ homeostasis.

568 569

8. Plant TPK channels reside in vacuolar membranes and exhibit ion channel activity that is modulated by membrane tension.

30

570

FUTURE ISSUES LIST

571 572

1. No plant MSL, MCA, or TPK channel has fully satisfied the four criteria for

573

confident assignment as a MS channel. Establishing a physiological stimulus for

574

MSL10 and demonstrating a change in MS channel properties in response to

575

mutations in MCA1, MSL10, or TPK1 will be first steps towards this goal.

576

2. Establishing the physiological functions of plasma membrane-localized MSLs and

577

the relevance of the structural diversity within the MSL family will require creative

578

functional assays and new structural studies.

579

3. Atomic structures will be needed if we are to make significant progress in

580

understanding the gating mechanism, regulation, and other functional aspects of

581

plant MS channels. A structure will be particularly revealing for MCAs, where no

582

information is available from homologs in other systems.

583

4. Current techniques for studying MS ion channels are limited in that they often

584

require removal or damage of the cell wall and analyzing membranes outside of

585

their natural context. New tools are needed to bypass these limitations.

586

5. An exciting, if ambitious, goal for the future will be to match each of the activities

587

that have been detected in plant membranes with a known gene and

588

corresponding channel structure. New functional screens as well as forward

589

genetics and phylogenetics may play an important role in this endeavor.

590 591

31

592

ACKNOWLEDGEMENTS

593 594

We thank our many colleagues in the plant biology, mechanobiology, and signal

595

transduction communities, and apologize to those whose work we were unable to

596

include due to size constraints. We are grateful to the current members of the Haswell

597

lab for insightful comments on this manuscript while it was in preparation. Our current

598

work on MS ion channels in plants is supported by NSF (MCB-1253103), NASA

599

(NNX13AM55G) and NIH (2R01GM084211).

600

32

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890

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912

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913

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914 915

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916

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917

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918

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919 920

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47

921

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922

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923

Arabidopsis. Plant Physiol. 152(3):1284–96

924

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925

calcium-permeable channels in Vicia faba guard cells are regulated by actin

926

dynamics. Plant Physiol. 143:1140–51

927

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928

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929

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930 931

of

an

anion-selective

mechanosensitive

channel

of

small

132. Zhu J-K. 2003. Regulation of ion homeostasis under salt stress. Curr. Opin. Plant Biol. 6(5):441–45

932

933

48

934

GLOSSARY

935 936 937 938 939 940 941 942 943

1. Ion channel = A gated macromolecular pore in a cell membrane that, once opened, permits ions to flow down their electrochemical gradient. 2. Gating = Conformational change undergone by an ion channel in response to stimuli that creates an ion-permeable pore through the membrane 3. Conductance = A measurement of the ease with which current flows through an ion channel at a given voltage, measured in Siemens (S). 4. Open probability = the ratio of channels that are open to those that are closed in a particular population

944

5. Patch-clamp electrophysiology = A technique for measuring current across an

945

isolated patch of membrane under conditions that maintain a particular

946

transmembrane voltage

947

6. MS = mechanosensitive

948

7. Protoplast=a bacterial, fungal, or plant cell from which the cell wall has been

949 950 951

removed 8. Hypoosmotic shock = Rapidly decreasing the osmolarity of the media for a membrane-bound cell or organelle; results in water influx into the cell

952

9. MSL = MscS-Like; MS channels that protect cells/organelles from osmotic stress

953

in bacteria, archaea, fungi, and plants; may have additional physiological

954

functions

49

955

10. MCA = Mid1-Complementing Activity; plasma membrane-localized MS calcium

956

channels that mediate osmotic stress response and calcium homeostasis in

957

plants

958

11. TPK = Two-pore Potassium (K+): mechanically modulated, potassium-selective

959

channels that localize to the plant vacuole and participate in ion homeostasis.

960

12. Plastid = A plant-specific endosymbiotic organelle in which photosynthesis,

961

biosynthesis and/or storage of cell metabolites take place

962

13. Chloroplast = A plastid specialized for photosynthesis

963

14. TNP = Trinitrophenol or picric acid; a negatively charged amphipath that inserts

964 965 966 967 968

into the outer bilayer of a membrane and induces curvature 15. CHO cells = Chinese Hamster Ovary cells, a commonly used cell line for protein expression and tissue culture work 16. Isoxaben = Herbicide that prevents the incorporation of glucose into cell walls by inhibiting cellulose synthase subunits

969

17. DEG/ENaC = Degenerin/Epithelial sodium (Na) Channels; cation-selective ion

970

channels that mediate touch response in animals; especially well-characterized in

971

C. elegans

972

18. TRP = Transient Receptor Potential channels; a family of cation-selective

973

candidate mechanosensitive ion channels potentially mediating multiple sensory

974

pathways in animals

975 976

19. FtsZ = Filamentous temperature sensitive Z; a GTPase that forms filaments required for fission in bacteria and plastids

50

977 978

20. Pulvinus = Organ consisting of central vascular tissue surrounded by two groups of cortical cells whose alternate swelling/shrinking produces leaf movement

51

979

Sidebar1: Piezo channels (line 162)

980

Piezo channels, named for the Greek word for pressure, are believed to mediate the

981

perception of mechanical stimuli in animal systems (reviewed in (94, 119, 128)).

982

mPiezo1 and mPiezo2 were identified in a tour de force RNA silencing screen for the

983

gene underlying mechanosensitivity in a mouse tissue culture cell line, and genes

984

encoding Piezo homologs were identified throughout the animal kingdom, in protists,

985

amoebae, and, surprisingly, in plants. Piezos are exceptionally large proteins,

986

comprising 2000-4000 amino acids and 20-40 predicted transmembrane helices.

987

Expressed in both sensory and non-sensory tissues, Piezos are required for response

988

to gentle touch and for vascular development in Zebrafish and mouse. They are further

989

implicated in noxious touch response, cellular extrusion, and red blood cell volume

990

regulation in flies, fish, mouse, rat, and humans. While heterologous expression of

991

Piezo channels can confer mechanosensitivity on an insensitive cell, it is not yet known

992

if they require other cellular components or a specialized lipid environment for

993

mechanosensitivity. Mutations in human Piezo genes are associated with a number of

994

diseases.

995 996

Sidebar2: Escherichia coli MscS (line 259)

997

The Mechanosensitive channel of Small conductance of E. coli was among the first

998

mechanosensitive channels to be identified and is now one of the best understood in

999

any system (reviewed in (12, 47, 61)). MscS is a weakly anion-preferring channel with a

1000

conductance of ~1 nS, and contributes to cellular survival of hypoosmotic shock ranging

52

1001

from 500-1000 mOsm. MscS activity can be reconstituted with only recombinant protein

1002

and lipids, indicating that it is gated directly through membrane tension. The C-terminal

1003

domain also undergoes a structural rearrangement upon gating and has been proposed

1004

to help regulate the osmolytes that are available to pass through the channel pore. Five

1005

crystal structures of prokaryotic MscS homologs in conducting and non-conducting

1006

conformations, molecular dynamic simulations and a slew of structure-function studies

1007

support several models for the MscS gating mechanism. Taken together, these studies

1008

form a solid foundation for future investigations into the structure, biophysical

1009

mechanism, and physiological function of MscS homologs in plants.

53

1010

FIGURE CAPTIONS

1011 1012

Figure 1. Models for Mechanosensitive Ion Channel Gating. In the lipid reordering

1013

model (a) MS ion channels force the membrane to distort to establish favorable

1014

interactions with the channel (top). Lateral membrane tension (horizontal arrows)

1015

increases bilayer energy as the membrane structure is further altered. The open

1016

conformation of the channel is then favored as it reduces lipid disordering through a

1017

lower energy interface with the membrane (bottom). The level of lipid disordering is

1018

indicated by yellow shading and the conformational changes of channel relative to the

1019

membrane emphasized by dashed lines. In the hydrophobic mismatch model (b),

1020

membrane bilayers create favorable interactions between the polar lipid heads and

1021

polar residues of an embedded protein (top). Lateral membrane tension (horizontal

1022

arrows) results in a thinner bilayer, disrupting some of these favorable interactions

1023

(middle). The open conformation of the channel, which has a shorter channel profile

1024

within the membrane, restores these interactions (bottom). Increasing hydrophobicity of

1025

regions is shown as a gradient from very hydrophilic (red) to very hydrophobic (blue).

1026 1027

Figure

2

Phylogenetic

Relationships

and

Subcellular

Localization,

and

1028

Topologies of MscS-Like Channels. (a). The inferred phylogeny of 44 members of the

1029

MscS superfamily is presented as an unrooted radial tree. Sequences were identified by

1030

Phytozome BLAST analysis (http://www.phytozome.net/) or inclusion in previous

1031

analyses (15, 44, 65, 92, 93, 100, 117, 131). The MscS-like region of each protein was

54

1032

identified by InterProScan (55) and aligned using ClustalW (113) with a gap-opening

1033

penalty of 3.0 and a gap extension penalty of 1.8. The evolutionary history was inferred

1034

using the Neighbor-Joining (104) method with a JTT distance matrix (54) using MEGA6

1035

software (112). The reliability of the tree was determined via bootstrapping (n = 1,000

1036

replicates) (35) and branches with bootstrap values of less than 50% were collapsed.

1037

Scale bar, 4.0 amino acid substitutions per site. The phylogenetic origin or cluster is

1038

indicated in the colored boxes. The sequences used in this analysis and their UniProt

1039

accession numbers, TAIR accession numbers, or Phytozome (cite) accession numbers

1040

are: E. coli MscS (P0C0S1), YbdG (P0AAT4); Synechocystis sp. PCC6803 bCNGa

1041

(M1ME31); H. pylori MscS (E1Q2W1); C. glutamicum MscCG (P42531); T.

1042

tengcongensis MscS (Q8R6L9); T. gondii (B6KM08); P. falciparum (Q8IIS3); D.

1043

discoideum (Q54ZV3); S. pombe MSY1 (O74839), MSY2 (O14050); C. reinhardtii

1044

MSC1 (A3KE12), MSC2 (A8HM43), MSC3 (A8HM47); A. thaliana MSL1 (At4g00290),

1045

MSL2 (At5g10490), MSL3 (At1g58200), MSL8 (At2g17010), MSL9 (At5g19520), MSL10

1046

(At5g12080);

1047

(GRMZM2G125494, GRMZM2G028914, GRMZM2G005013); O. sativa (Os02g45690,

1048

Os04g48940,

Os06g10410,

1049

Bradi5g19160,

Bradi3g51250);

1050

Vv00002410001); P. patens (Pp1s79_156, Pp1s314_12, Pp1s2_4320); C. papaya

1051

(supercontig_55.26, _22.80,_126.38_20.126). (b) Predicted topology and subcellular

1052

localization of representative MSLs from A. thaliana. Topologies were drawn according

1053

to predictions on Aramemnon (http://aramemnon.botanik.uni-koeln.de/index.ep). The

P.

trichocarpa

(Pt002G105900,

Os02g44770); V.

vinifera

B.

Pt004G178900);

distachyon

(Vv00015105001,

Z.

mays

(Bradi1g15920, Vv00026926001,

55

1054

region of highest homology to E. coli MscS is cyan, the Group I-specific C-terminal

1055

extension is green, and the Group III-specific N-terminal region is highlighted in orange.

1056 1057

Figure 3. Molecularly Uncharacterized MS Ion Channel Activities Identified in

1058

Plant Membranes. (a) Plasma membrane- and (b) vacuolar-localized MS ion channels

1059

identified through patch-clamp electrophysiology and activated through increased

1060

membrane tension are presented and categorized by established ion permeability.

1061

Relevant citations are indicated beneath each channel. Arrows indicate the ion

1062

permeability but do not specify the direction of ion flux in or out of the cell or vacuole.

1063

56

Table 1. Plant Mechanosensitive Ion Channels

Family

Mid-1 Complementing Activity

MscS-Like

Two-Pore K+

a

predicted

Organism

Mutant/Silenced Phenotype

Associated MS Ion Activity in Heterologous Systems Channel Characteristics

Gene

MCA1

At4g35920

the roots of mca1 mutants are less efficient at penetrating hard agar, do not induce lignin deposition Arabidopsis nor alter carbohydrate gene experssion patterns in thaliana response to isoxaben; mca1 mca2 double mutants are hypersensitive to MgCl2 and show developmental delays

MCA2

At2g17780

mca2 mutants show a reduction in Ca2+ uptake; mca1 Arabidopsis mca2 double mutants are hypersensitive to MgCl2 and thaliana show developmental delay

NtMCA1

AB622811

Nicotiana tabacum

NtMCA2

AB622812

Nicotiana tabacum

OsMCA1

Os03g0157300

MSL1

At4g00290

Arabidopsis thaliana

mitochondriaa

MSL2

At5g10490

msl2 null mutants show defective leaf shape; msl2 msl3 Arabidopsis double mutants have enlarged chloroplasts and thaliana enlarged, round non-green plastids; msl2 msl3 double mutant chloroplasts exhibit multiple division rings

plastid envelope, poles

MSL3

At1g58200

msl2 msl3 double mutants have enlarged chloroplasts Arabidopsis and enlarged, round non-green plastids; msl2 msl3 thaliana double mutant chloroplasts exhibit multiple division rings

plastid envelope, poles

MSL4

At1g53470

Arabidopsis msl4 msl5 msl6 msl9 msl10 quintuple mutants lack MS thaliana channel activity in root protoplasts

plasma membranea

Haswell, Peyronnet et al., 2008

MSL5

At3g14810

Arabidopsis msl4 msl5 msl6 msl9 msl10 quintuple mutants lack MS thaliana channel activity in root protoplasts

plasma membranea

Haswell, Peyronnet et al., 2008

MSL6

At1g78610

Arabidopsis msl4 msl5 msl6 msl9 msl10 quintuple mutants lack MS thaliana channel activity in root protoplasts

plasma membranea

Haswell, Peyronnet et al., 2008

MSL9

At5g19520

msl9 mutants lack a ~45 pS MS channel activity in root Arabidopsis protoplasts; msl9 msl10 mutants lack a ~50 pS MS thaliana channel activity in root protoplasts

plasma membrane and ER

Haswell, Peyronnet et al., 2008

MSL10

At5g12080

msl10 mutants lack a ~140 pS MS channel activity in Arabidopsis cell death, increased H2O2 accumulation, induction of root protoplasts; msl9 msl10 mutants lack a ~50 pS MS thaliana SAG12, OSM34, DOX1, PERX34, KTI1 channel activity in root protoplasts

expression is associated with a plasma membrane ~100 pS conductance in and ER Xenopus oocytes with a moderate preference for anions

Haswell, Peyronnet et al., 2008; Maksaev and Haswell, 2012; Veley et al., 2014

TPK1

At5g55630

tpk1 mutants lack an instantaneous tonoplast K+ current in all shoot cell types, show modest sensitivity to high Arabidopsis resistant to high and low K+ in the media, fast guard cell vacuolar membrane and low K+ in the media, slow guard cell closing kinetics closing kinetics in response to ABA, fast germination thaliana in response to ABA, and slow germination especially in the presence of ABA

OsTPK1a

Os03g541002

Oryza sativa

expression is associated with an instantaneous K+-selective vacuolar membrane channel activity that is increased with membrane tension.

HvTPK1

EU926490

Hordeum vulgare

expression is associated with an instantaneous K+-selective vacuolar membrane channel activity that is increased with membrane tension.

OsMCA1-silenced lines exhibit slower growth, reduced Oryza sativa aequorin luminescnece in response to hypoosmotic shock or to the membrane distorting agent TNP

Overexpression phenotype

Subcellular Localization

Protein

Key References

survival and Ca2+ uptake in response to challenge with mating pheremone in S. cerevisiae; stretch-activtated Ca2+ influx in CHO cells

Nakagawa et al., 2007; Yamanaka et al., 2010; Denness et al., 2011; Wormit et al., 2012; Furuichi et al., 2012;

plasma membrane

survival and Ca2+ uptake in response to challenge with mating pheremone in S. cerevisiae

Yamanaka et al., 2010

increased Ca2+ uptake in cultured tobacco cells, higher expression of NtERF4

plasma membrane, punctate signal

survival and Ca2+ uptake in response to challenge with mating pheremone, Ca2+ Kusuru et al., 2011 uptake in response to hypoosmotic shock in S. cerevisiae

increased Ca2+ uptake in cultured tobacco cells, higher expression of NtERF4

plasma membrane, punctate signal

survival and Ca2+ uptake in response to challenge with mating pheremone, Ca2+ Kusuru et al., 2011 uptake in response to hypoosmotic shock in S. cerevisiae

increased Ca2+ uptake in cultured rice cells

plasma membrane

Kurusu et al., 2012

increased Ca2+ uptake in seedling roots, increased Ca2+ influx (measured as aequorin signal) in response to hypoosmotic shock and TNP treatment, high level expression of TCH3

plasma membrane

expression is associated with ~15 or ~35 pS conductance mechanically gated channel in Xenopus ooctyes

survival of hypo-osmotic shock in E. coli

Haswell, 2007 Haswell & Meyerowitz, 2006; Wilson et al., 2011;Jensen & Haswell, 2011; Veley et al., 2012;

survival of hypo-osmotic shock in E. coli

expression is associated with an instantaneous K+-selective channel activity that is increased with membrane tension.

Haswell & Meyerowitz, 2006; Wilson et al., 2011; Veley et al., 2012

Gobert et al., 2007; complements a K+ uptake-deficient E. coli Matthuis 2011; Isayenkov mutant et al., 2013

Isayenkov, 2011; Matthuis 2011

Boscari, 2009; Matthuis 2011

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