1. General properties of ion channels

2. 1. Ion channels are ubiquitous in plant membranes
An action potential as seen in the large internode cells of some algae

3. Minosa pudica leaf evokes an action potential that cause the pulvinus to lose turgor.
Some insectivorous plants also use action potentials to couple the sensing of prey.

4. Channels are now known to be present in all plant cell types,
: at the PM and vacuoles and all other membranes as well.

5. 2. Ion channels are studied with electrophysiological techniques
Path Clamp: detecting tiny electrical currents, resolving activity of single protein molecules.
Planar Lipid Bilayers: recording activity of channels at intracellular membranes.
Voltage Clamp: classical approach, keeping cell wall and regulatory machinery within cell intact.

6. Path Clamp: detecting tiny electrical currents, resolving activity of single protein molecules.

8. Planar Lipid Bilayers: recording activity of channels at intracellular membranes.
Voltage Clamp: classical approach, keeping cell wall and regulatory machinery within cell intact.

9. Ion flow through channels is passive.
3.5.3 Ionic fluxes through channels are driven solely by electrochemical potential differences
Ion flow through channels is passive.

10. Current-voltage (I-V) curve for a single channel.

11. 4. Ion channels exhibit ionic selectivity
Cation channels
monovalent: K+, H+
divalent: Ca2+,
Anion channels
Cl-, No3-, organic acids
The fact that channels are selective implies that binding sites capable
of distinguishing specific ions must be located within the channel pores.

12. 5. Ion channels are gated, often by voltages or ligands,
through changes in open state probability.

13. The open state probability

14. Ion channels in action

15. Voltage dependent K+ channels at the PM stabilize Vm and allow
controlled K+ uptake and loss
Outward rectifying K+ channels: decrease K+ in cytosol
Inward rectifying K+ channels: increase K+ in cytosol

16. Whole cell currents in the plant cell
Current-voltage relationship
Whole cell currents in the plant cell
currents flow across the PM in both directions, into and out of the cytosol

17. K+ outward rectifier stabilize Vm
cytosol
100 mM K+
1 mM K+
EK=-120mV
If hyper-depolarized, outward rectifying channels open
cytosol
EK >-120mV K+

18. K+ inward rectifiers take up K+ from the soil and the apoplast
cytosol
K+ < 1mM
accumulated
by ion coupled carrier K+ K+
In guard cell,
Outward rectifiers are activated by the small increase in cytosolic pH (elicited by ABA)
Inward rectifiers are subject to modulation by G-protein and are inhibited by increased free cytosolic Ca+ and by PP2B

19. ABA : activate outward rectifying K+ channels
inactivate inward rectifying K+ channels

20. 2. Plant cell inward rectifiers are members of
the Shaker family of votage-gated channels
Shaker family
Superfamily of voltage-gated K+, Ca2+, Na+
Plant inward rectifiers
S4 domain : positively charged residues every third residue
voltage sensor that open the channel in response to a permissive voltage

21. K+-selective inward rectifiers
Products of multigene family
Tissue-specific expression of plant inward rectifiers
KAT1 expressed in guard cell
AKT1 found in roots and hydathodes
mature root lateral root hydathodes

22. Radioactive tracer flux analysis 86Rb+ uptake in WT and akt1-1 roots
akt1-1 shows the reduced K+ uptake and less growth in media with low K+ concentrations

23. The outward rectifier Outward rectifying K+ channel, KCO1
Member of the two-pore K+ channel family
Four transmembrane spans in each subunit
Two-EF hands reside toward the C-terminus
: sensitive to the concentrations of cytosolic Ca2+
highly conserved P-domain motif TxGYGD

24. Hyperpolarizing voltage conformational change : opening the gate
screw out of the membrane
conformational change
: opening the gate

25. Ca2+ dependence of the KCO1
: KCO1 mediated currents are potentiated by cytosolic free Ca2+

26. 4. Voltage – insensitive cation channels may be a major pathway for Na+ uptake across the plasma membrane and for salt release to the xylem
Channels :
- constitute a major pathway for Na+ uptake into plant cells
- important implications for salinity tolerance.
- partially blocked by external Ca2+
2) Less selective channels :
- observed in the plasma membrane of xylem
- almost as permeable to anions as to cations.
- perhaps this channel could provide a pathway for release of salts from the symplasm into the xylem.

27. Na+-dependent currents across the plasma membrane of protoplasts.

28. (B) I-V relationship for the currents in A showing substantial influx of Na+

29. Monovalent cation channels at the vacuolar membrane are Ca2+-sensitive and mediate vacuolar K+ mobilization.
Vacuoles : - organelles for accumulation and storage of solutes
- require massive solute loss in some circumstances
Hypo-osmotic stress, during stomatal closure,
: mobilization of ions from vacuoles to restore normal cell turgor
3) Guard cell has two different classes of K+-permeable channels in releasing K+ from the vacuole.
- the fast vacuolar (FV) channel
- the vacuolar K+ (VK) channel

30. The fast vacuolar (FV) channel
Little selectivity among monovalent cations
Inhibited when cytosolic Ca2+ concentrations exceed 1μM
Activated when cytosolic pH increases
The vacuolar K+ (VK) channel
Highly selective for K+ over other monocovalent cations
Activated by cytosolic Ca2+ in the nanomolar to low micromolar concentration range
Inhibited by increasing cytosolic pH

31. Calcium-permeable channels in the plasma membrane provide potential routes for entry of Ca2+ to the cytosol during signal transduction.
Ca2+ : Ca2+ increase is central to signal transduction.
activation of down stream targets, initial signal into the end response.
2) Ca2+ permeable channels : activation of increase in cytosolic Ca2+,
upstream elements in Ca2+ - based signal transduction pathway,
3) Voltage-gated Ca2+_ permeable channels are activated by membrane depolarization.

32. Calcium-based signal transduction in a typical plant cell.
Receptor is perceived by signal.
2) Changed the activity of Ca2+ -ATPases or plasma membrane Ca2+ channel
3) Change on cytosolic free calcium concentration.
4) release of Ca2+ from internal stores.
5) 2nd messenger is activation
6) increased free Ca2+ changes the activity of Ca2+ - binding proteins
7) final cellular response.

33. External Signals Diverse Cellular Responses [Ca2+]cyt Amplitude (AM)
Light (red, blue)
Cold stress
Heat shock
Mechanical stresses
(touch, wind &
wounding)
Pathogens
Phytohormones
(Auxin, ABA, GA)
Gravity
Ca2+-Signal
Decoder
Diverse
Cellular
Responses
[Ca2+]cyt
AM, DU, FM
Calmodulins
CDPKs, CBLs
Other CBPs
Amplitude (AM)
Let me remind you about calcium signaling in plant. Many kinds of external signals such as light, cold, heat, mechanical stresses, pathogens, phytohormones, and gravity can increase many different forms of cytosolic Ca2+ concentration. Different Ca2+ spike with different amplitude, duration and frequency can be formed dependent on the external signals. Different Ca2+ spike are recognized by Ca2+ decoders such as CaMs, CDPKs, CBLs and other CBPs. Ca2+ decoders modulate downstream enzyme and gene expression and results in appropriate cellular responses.
Duration (DU)
Frequency (FM) 33

34. Encoding and Decoding Calcium Signaling
Influx
Encoding
Ca2+ spikes
Stimulus
Efflux
Decoding
Decoders
Regulates enzymes
Changes gene expression
Responses
Ca2+ signals are generated by the influx of Ca2+ through Ca2+ channels.
Maintenance of low Ca2+ is archived by Ca2+ efflux though Ca2+ active transporters 34

35. Activation of a wheat root plasma membrane Ca2+ channel by voltage.
The channel activates strongly as Vm and is fully activated at -100mV.

36. 7. Calcium-permeable channels in endomembranes are activated by both voltage and ligands

37. Diagram illustrating channel activities at the guard cell vacuolar membrane during stomatal closure
During plasma membrane-based signal transduction

38. Activity of the SV channel increases with increasing cytosolic concentration of Ca2+
(A) Slow activation of the SV channel in barley aleurone vacuoles in response to positive voltages
(B) Ca2+-dependence of whole-vacuole channel activity. Increasing free calcium above approximately 1μM increasing the activity of the channel. Ca2+ is thought to interact with calmodulin associated with the channel or a channel regulatory protein

39. 8. Plasma membrane anion channels facilitate salt release during turgor adjustment and elicit membrane depolarization after stimulus perception

40. Anion channels in guard cell
Current-voltage relationship for rapidly activating(R-type)anion channels.
Current-voltage relationship for slowly activating(S-type)channels

41. Anion channels function
팽압(turgor)을 조절 기능
특별한 자극에 의해 Depolarization되어 Ca2+ channel activation되게 함
Membrane hyperpolarization으로 activation되어 Vm의 조절에 관여
Guard cell을 포함한 cell type에서 ATP, protein Kinase로 부터 activatione됨

42. 3.6.9 Vacuolar malate channels participate in malate sequestration

43. Current-voltage relationship for vacuolar uptake of malate through time-dependent anion channel in the tonoplast.Malate uptake by anion channel is strongly promoted by negative membrane otentials and increases with cytosolic malate concentration. In this figure, cytosolic malate concentration were 10mM(filled squares), 20mM(open squares), 50mM(open circles), and 100mM(filled triangles)- all with a vacuolar malate concentration of 10mM. Malate uptake with equal concentration of malate(50mM) presente on both side of the membrane is indicated by stars.

44. Accumulation of malate in the root of CAM plants
Accumulation of malate in the root of CAM plants. Malate2- is thought to enter the vacuole though malate-selective channels.These channel are strongly inward rectifying and do not allow substantial malate2- efflux. Once inside the vacuole,malate2- is protonated to H.malate and H2.malate0. This maintains the effective concentration difference for malate2- across the membrane.

45. Integrated channel activity at the vacuolar and plasma membranes yields sophisticated signaling systems

46. Ca2+ signaling coordinates the activities of multiple ion channels and H+-pumps during stomatal closure. In this model,perception of ABA by a receptor(R) results in an increase in cytosolic free Ca2+ through Ca2+influx or Ca2+ release from internal stores. Increased cytosolic Ca2+ promotes opening of plasma membrane anion and K+out channels and inhibits opening of K+in channels. As more ions leave the cell than enter it, water follows, turgor is lost, and the stomatal pore is closed

47. 3.7 Water transport through aquaporins

48. Jv = Lp(▲p- σ▲п) ▲p: hydrostatic pressure ▲п : osmotic pressure
3.7.1 Directionality of water flow is determined by osmotic and hydraulic forces
Jv = Lp(▲p- σ▲п)
▲p: hydrostatic pressure
▲п : osmotic pressure
Jv: the flux of water across the membrane
σ: Expresses the ability of the osmotically relevant solutes to permeate the membrane relative to water

49. Membrane permeability to water can be measure in two ways:
3.7.2 Membrane permeability to water can be defined with either an osmotic coefficient (Pf) or a diffusional coefficient (Pd)
Membrane permeability to water can be measure in two ways:
+ The imposition of an osmotic or a hydrostatic pressure (Pf) difference across the membrane can be used to generate a water flow.
Pf = Lp RT/Vw
+ The assessing water permeability relies on measuring the diffusional permeability (Pd) with isotopic water.

50. Transcellular osmotic
Pressure probe
Transcellular osmotic

51. 3.7.3. The nonequivalence of Pf and Pd provides evidence for water channels
Pf involves net flow of water. Each water molecule entering the channel form the left will knock out one molecule on the right.
In the diffusion flow case, a molecule of labeled water entering the channel from the left can diffuse back into the solution on the left.

52. Water movement across biological membranes occurs through both the lipid bilayer and the pores formed by water channels.
Model for water flow through a single-life, multiple occupancy aquaporin

53. Aquaporins are members of the major intrinsic protein family, which can form water channels when expressed in heterologous systems
Water channels or aquaporins are highly expressed in plant membrane. Member of a family of transmembrane channels known as the major intrinsic protein MIP family and these proteins are about 25 to 30 kDa, probably span the bilayer six times and contain an internal repeat sequence indicative of origins from a gene duplication and fusion.
Aquaporins are also characterized by the highly conserved NPA (Asn-Pro-Ala) residue in the N and C terminal.
In plant the aquaporins have been identified in both vacuolar (known as tonoplant intrinsic protein TIP) and plasma membrane (plasma membrane intrinsic protein PIP).

54. Structure of an aquaporin showing the six transmembrane helices and two conserved NPA (Asn-Pro-Ala) residue

55. Three-dementional structure of aquaporin-1 from human erythrocytes
Three-dementional structure of aquaporin-1 from human erythrocytes. Extracellular view of eight asymmetrical subunits that form two tetramers. One of the monomers of the central tetramer is colored gold.

56. Environmental stimuli (blue light, ABA, GA, cold & drought)
3.7.5 Aquaporin activity is regulated transcriptionally and posttranslationally
H2O
Aquaporin
Transcription
Posttranslation
Environmental stimuli (blue light, ABA, GA, cold & drought)
Phosphorylation by Ca2+ dependent protein kinase

57. Figure . Schematic representation of putative mechanisms involved in plant aquaporin egulation. (a) Control of transcription and protein abundance. Drought and salinity, as other environmental stimuli, are known to act on aquaporin gene transcription and possibly interfere with aquaporin translation and degradation, thereby determining protein abundance. The
formation of PIP hetero-tetramers was demonstrated in Xenopus oocytes (Fetter et al. 2004) and is still hypothetical in plant cells. This mechanism might favour transfer of PIP1 homologues to the plasma membrane (PM). (b) Sub-cellular relocalization. The redistribution of a TIP aquaporin, from the tonoplast (TP) to small intracellular vesicles, was demonstrated in Mesembryanthemum
crystallinum suspension cells exposed to a hyperosmotic treatment (Vera-Estrella et al. 2004). The occurrence of a similar relocalization mechanism for PIP aquaporins is shown but remains hypothetical.

58. 3.7.6 Plasma membrane aquaporins may play a role in facilitating transcellular
water flow
vacuole
Tonoplant intrinsic protein(TIP)
Plant water
channel
Plasma membrane
Plasma membrane intrinsic protein(PIP)
vacuole
Plasma membrane

59. Transpiration apoplast symplast
Requirement for a low-resistance pathway

60. 3.7.7Differential water permeabilities of the vacuolar and plasma membranes
can prevent large changes in cytoplasmic volume during water stress
The water permeability of the vacuolar membrane
The water permeability of the plasma membrane

61. Requirment for maintenance of cytosolic volume during osmoticstress