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]
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1
UNITED IN DIVERSITY: MECHANOSENSITIVE ION CHANNELS IN PLANTS
2 3
Eric S. Hamilton, Angela M. Schlegel & Elizabeth S. Haswell*
4 5
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
16 17
1. Introduction
18
2. Mechanosensitive Ion Channels: Transducing Force into Current
19
2.1 Models for MS Channel Gating
20
3. Physiological Roles for MS Channels
21
3.1 Animal MS Channels
22
3.2 Plant MS Channels
23
4. Approaches Used to Study Plant MS Ion Channels
24
4.1 Pharmacological Inhibition and Activation
25
4.2 Electrophysiology
26
4.3 Genetics
27
5. Criteria for Assignment as a Mechanosensitive Ion Channel
28
6. MSL Channels
29
6.1 A Diverse Family of MS Channels
30
6.2 Evidence that MSLs are MS Ion Channels
31
6.3 MSLs Serve as Osmotic Conduits in the Plastid Envelope
32
6.4 MSLs at the Plant Plasma Membrane
33
6.5 Beyond the Paradigm of Emergency Release Valves
34
7. MCA Channels
35
7.1 MCA Proteins Modulate Ca2+ Influx in Response to Mechanical Stimuli
36
7.2 Structural Features of MCAs
2
37
8. TPK Channels
38
9. Other Activities Yet to be Identified
3
39
KEYWORDS
40
Mechanotransduction, MscS, MSL, MCA, TPK1
4
41
ABSTRACT
42 43
Mechanosensitive (MS) ion channels are a commonly used mechanism for the
44
perception and response to mechanical force. This class of mechanoreceptors is
45
capable of transducing membrane tension directly into ion flux. In plant systems, MS ion
46
channels have been proposed to play a wide array of roles, from the perception of touch
47
and gravity to osmotic homeostasis of intracellular organelles. Three families of plant
48
MS channels have been identified: the MscS-Like (MSL), Mid1-Complementing Activity
49
(MCA), and Two-Pore Potassium (TPK) families. Channels from these three families
50
vary widely in terms of structure and function, localize to multiple cellular compartments,
51
and conduct chloride, calcium, and/or potassium ions. However, they are still likely to
52
represent only a fraction of the MS ion channel diversity present in plant systems.
53
5
54
1. INTRODUCTION
55 56
How cells sense mechanical force is a long-standing question in biology. Mechanical
57
signals such as touch, gravity, and osmotic pressure are critical to proper development,
58
environmental stress response, and overall cellular health in a wide variety of
59
prokaryotic and eukaryotic organisms and cell types. The perception of force can be
60
mediated
61
reorganization, or nuclear deformation (reviewed in (32)).
through
the
actions
of
integrins
or
focal
adhesions,
cytoskeletal
62 63
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
66
ions across the lipid bilayer, are responsible for a wide range of functions across all of
67
life, including the production of action potentials in nerve cells (102), maintaining the
68
ionic conditions required for metabolism in plants (132) and Ca2+ signaling in all cells
69
(19). Interested readers are referred to two recent reviews on plant ion channel function
70
(48, 121).
71 72
The flux of ions through a channel can be regulated by a variety of stimuli, including
73
transmembrane voltage (10), ligand binding (57), light (23), and mechanical force (78). It
74
is the latter stimulus that defines a diverse group of channels known as
75
mechanosensitive (MS) ion channels, also referred to as stretch-activated or force-
6
76
gated channels. MS ion channels are found in all three domains of life (5, 58, 61),
77
pointing to the fundamental requirement for mechanosensation in all cells.
78 79
Several recent reviews describe the wide variety of mechanical stimuli that land plants
80
must sense and respond to during their lifespan (21, 85, 114). In addition, as many as
81
18 distinct MS ion channel activities have been identified in plant membranes by patch-
82
clamp electrophysiology (these are described in detail below), implying that
83
mechanically gated ion channels play an important role in plant systems. In this review
84
we provide a general introduction to MS ion channel structure and function, outline the
85
approaches used to study plant MS ion channels, and summarize what is currently
86
known about three families of plant MS ion channels, emphasizing their diverse
87
structure, evolutionary history, and physiological roles.
88 89
2. MECHANOSENSITIVE ION CHANNELS: TRANSDUCING FORCE INTO CURRENT
90 91
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
95
possible the first identification of MS ion channel activities in animal skeletal cells (41,
96
43). Shortly thereafter, MS ion channel activities were detected in tobacco, broad bean
97
and giant Escherichia coli protoplasts (33, 80, 106). The structures of two of the MS
7
98
channels identified in those E. coli protoplasts have been solved at atomic resolution,
99
providing the foundation for many elegant experimental and theoretical investigations
100
into the molecular mechanism of MS channel activity (reviewed in (111)).
101 102
2.1 Models for MS Channel Gating
103
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
107
gradient in either direction across the membrane through the channel pore (49).
108 109
For some classes of MS ion channels, increased membrane tension leads directly to
110
gating. This behavior has been described by a number of biophysical models that
111
address the energetic interactions at the membrane-protein interface (reviewed in (47)).
112
One proposed mechanism is the lipid disordering model illustrated in Figure 1A. An ion
113
channel increases the free energy of the membrane in which it is embedded, as the
114
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
116
tension, a conformational change in the channel that reduces the local deformation
117
imposed on the membrane, while opening the channel pore, is favored (79, 115).
118
8
119
Another driving force for MS channel gating may be the thinning of the lipid bilayer
120
under increased membrane tension (Figure 1B). According to this model, membrane
121
thinning results in a mismatch between the height of the channel’s hydrophobic
122
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
124
conformational change in the channel that maintains energetically favorable interactions
125
between the TM domain and the lipid bilayer (such as rotating a TM helix within the
126
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
128
mutually exclusive, and that there are likely many other mechanisms capable of driving
129
intrinsic mechanosensitivity in ion channels (96, 98).
130 131
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
134
Caenorhabditis elegans—are likely to be gated indirectly by tethering to other cellular
135
components (29). Tension applied to a physical link between a channel and the
136
extracellular matrix or the intracellular cytoskeletal system could directly stretch open
137
the channel, reorient the channel in the lipid bilayer, or lead to lipid raft reorganization
138
(3, 13, 47). A unifying theme in all of these models, however, is that the responsiveness
139
of a MS ion channel to force depends on highly dynamic interactions with the lipid
140
bilayer.
9
141 142
3. PHYSIOLOGICAL ROLES FOR MS CHANNELS
143
There are many ways in which an organism might employ a mechanosensor capable of
144
transducing force into ion flux; many MS channels from animals have been studied in
145
detail and shown to improve fitness during development or in a changeable
146
environment. Here we briefly summarize what is known about the physiological roles of
147
MS channels in C. elegans, D. melanogaster, and other metazoans—as these studies
148
inform our understanding of MS channels in plants, whether or not the channels are
149
evolutionarily related—and then address their potential functions in plants.
150 151
3.1 MS Ion Channels in Animals
152
Several distinct families of MS channels are thought to underlie the senses of touch,
153
pain, hearing, proprioception, and gravity sensation in animal systems. For example,
154
response to light touch is mediated by the Degenerin/Epithelial Sodium Channel
155
(Deg/ENaC) family in C. elegans. The Transient Receptor Potential (TRP) family
156
mediates nose touch and proprioception in C. elegans and hearing, nociception, and
157
bristle touch in Drosophila melanogaster (reviewed in (5)). Two-Pore Potassium (TPK)
158
MS ion channels are required for pressure-responsive vasodilation and also appear to
159
regulate the pain threshold for cold and heat in mice (reviewed in (50)). The recently
160
identified Piezo family mediates diverse mechanosensory events in animals, from touch
161
and pain in sensory neurons to intercellular communication and osmotic control
162
(reviewed in (119), see sidebar: Piezo).
10
163 164
3.2 MS Ion Channels in Plants
165
Plants sense and respond to many of the same mechanical stimuli as animals, including
166
touch, gravity, and osmotic stress (16, 85, 114). They also respond to unique signals
167
associated with developmental processes such as lateral root emergence, pollen tube
168
growth, cell wall damage, and plant-pathogen interactions (4, 52, 72). In many of these
169
cases, applying a mechanical stimulus leads to a rapid burst of ion flux, and it has long
170
been speculated that this correlation may be attributed to the action of MS ion channels
171
in the stimulated cells, in part because of the speed of the response (reviewed in (34,
172
51, 85)).
173 174
The flux of Ca2+ in particular has been implicated in various mechanosensory pathways.
175
For example, gravity stimulation (introduced by rotating a root or shoot 90 degrees) is
176
associated with membrane depolarization, the rapid influx of Ca2+ ions and subsequent
177
alkalinization of the cells in the root cap (reviewed in (114)). Ca2+ influx is also
178
associated with touch stimulus (reviewed in (34)), osmotic stress (108), and bending
179
(84), consistent with the action of a mechanically gated calcium channel in these
180
processes. After influx, Ca2+ could serve as a second messenger in a large number of
181
downstream events—some of which, such as the activation of calmodulin and
182
calmodulin-like proteins, are also implicated in mechanotransduction (16).
183 184
4. APPROACHES USED TO STUDY MS ION CHANNELS IN PLANTS
11
185 186
4.1 Pharmacological Inhibition or Activation
187
Evidence that MS ion channels are an integral part of a particular mechanosensory
188
process can be obtained by pharmacological treatments with known inhibitors or
189
activators of MS ion channels. The Ca2+ influx associated with mechanical stimulation
190
can be inhibited by lanthanides (26, 83, 89), ruthenium red (67) or cytoskeletal inhibitors
191
(25). However, these treatments are often non-specific. For example, the commonly
192
used lanthanide Gd3+ blocks a wide variety of channels, not only Ca2+-selective or
193
mechanosensitive (69). Furthermore, Gd3+ can indirectly inhibit the action of non-
194
selective MS ion channels by reducing overall membrane fluidity (31, 74). On the other
195
hand, the chemical trinitrophenol (TNP), which increases curvature and tension when
196
applied to membranes (79), behaves as a MS channel activator and can induce Ca2+
197
flux or lower the threshold for mechanical stimulation (37, 89, 103). While sensitivity to
198
one of these pharmacological agents can provide evidence that MS ion channels are
199
involved in a particular response, confirmation will likely require knowing the molecular
200
identity of the channels involved and characterization of their channel properties through
201
the methods described below.
202 203
4.2 Electrophysiology
204
The gold standard technique for the analysis of MS ion channels is patch-clamp
205
electrophysiology. Patch-clamping involves the production of a high resistance seal
206
between the glass of a micropipette tip and a small patch of membrane containing the
12
207
channels of interest (105). The pipette can either remain attached to the cell with an
208
intact patch (cell-attached), remain attached to the cell with a ruptured patch (whole-
209
cell), or be completely removed from the cell along with the patch (excised). In all of
210
these configurations, membrane tension is increased by introducing positive or negative
211
pressure through the patch pipette, and the resulting current across the membrane is
212
recorded over time. This technique has been used to identify and characterize plant MS
213
channels in their native membranes or heterologously expressed in Xenopus oocytes,
214
as described in more detail below. While patch clamping allows the identification of
215
individual MS ion channels—and in the excised patch configuration, control over the
216
ionic conditions on both sides of the membrane—a drawback especially relevant to
217
plant systems is that it requires isolation of cells from the tissue and the removal of the
218
cell wall.
219 220
4.3 Genetics
221
Molecular genetic approaches in Arabidopsis thaliana and other model plant systems
222
have added another dimension to the study of MS channels in recent years. Although a
223
forward genetic screen has not yet successfully been used to identify a MS channel,
224
reverse genetics motivated by either phylogenetics or a functional assay has identified
225
several candidates (45, 73, 89). Once a candidate gene is identified, the protein can be
226
heterologously expressed and tested for MS channel activity in cell survival or
227
electrophysiological assays. Genetic ablation or overexpression of candidate MS
13
228
channel genes in planta can be powerful tools for characterizing channels in their native
229
systems.
230 231
5. CRITERIA FOR ASSIGNMENT AS A MECHANOSENSITIVE ION CHANNEL
232 233
Establishing that a particular gene encodes the primary force-transducer in a MS
234
response, as opposed to an accessory or downstream component of the MS response,
235
is a challenging endeavor. The following criteria have previously been established: 1)
236
proper expression and localization for the observed MS response; 2) the channel is
237
required for the response, but not for the normal development of the cell or tissue in
238
which the response occurs (unless the MS response being measured is the
239
development of the tissue itself); 3) evidence of MS gating in isolation or in heterologous
240
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
242
categorize MS ion channels can be difficult, especially if MS ion channels function as
243
heteromultimers or with other cellular structures.
244 245
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
247
evidence that does exist in favor of this interpretation in each case. Table 1 summarizes
248
relevant information about the three families of plant MS channels that have so far been
249
identified: the MscS-Like (MSL), Mid1-Complementing Activity (MCA), and Two-Pore
14
250
Potassium (TPK) channels. As summarized below, channels from these three families
251
vary widely in terms of structure and function, localize to multiple cellular compartments,
252
and conduct diverse subsets of ions.
253 254
6. MSCS-LIKE CHANNELS
255 256
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-
258
established model system for the study of MS ion channels ((12, 82), see sidebar:
259
MscS). MscS serves as an “emergency release valve” under conditions of hypoosmotic
260
shock in E. coli (12, 68). Genes predicted to encode homologs of MscS are found
261
throughout bacterial and archaeal genomes, in some protist genomes—including
262
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.
264 265
The predicted evolutionary relationship among representative members of the MscS
266
superfamily is presented in Figure 2A, using the ~100 amino acid domain conserved
267
among the MscS superfamily (99). This sequence maps to the pore-lining helix and the
268
upper part of the cytoplasmic vestibule of MscS and is marked in cyan in Figure 2B (8).
269
A number of highly conserved motifs within this domain are important for function in
270
bacterial and plant channels (7, 22, 53). Land plant MscS homologs fall into three
15
271
phylogenetic groups, I-III, which also correspond to three different subcellular
272
localizations (see below).
273 274
6.1 A Diverse Family of MS Channels
275
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,
279
providing further evidence that they serve a diverse set of functions within the cell
280
(Figure 2B).
281 282
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
284
predicted to contain five TM helices, the last helix corresponding to the pore-lining
285
domain of MscS, and a C-terminus that is located in the stroma or matrix. Group III MSL
286
proteins are predicted or shown to localize to the plasma membrane (44, 46) and to
287
contain six TM helices, again with the most C-terminal TM segment corresponding to
288
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.
291
16
292
The A. thaliana genome encodes ten MSL genes (Table 1), and they show a range of
293
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.
295
MSL2 and MSL3 are 50% identical at the amino acid level; MSL4 and MSL5 are 68%
296
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
298
genome may indicate functional redundancy (as with MSL2 and MSL3, see below), but
299
may also have permitted the evolution of unique characteristics (as with MSL9 and
300
MSL10, see below).
301 302
6.2 Evidence that MSLs are MS Ion Channels
303
It is currently accepted that MS ion channel activity has been retained among most
304
members of the MscS superfamily (however, for one exception see (15)). All six MscS
305
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
307
MSY1 from fission yeast (92).
308 309
Several lines of evidence support the hypothesis that MSLs form functional MS ion
310
channels in A. thaliana. Plastid-localized MSL3 was able to partially rescue the
311
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
313
plasma membrane-localized channels MSL9 and MSL10, which are genetically required
17
314
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
316
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
319
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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913
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914 915
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916
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918
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919 920
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922
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923
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924
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925
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926
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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
