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

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Presentation on theme: "General properties of ion channels. An action potential as seen in the large internode cells of some algae 1. Ion channels are ubiquitous in plant membranes."— Presentation transcript:

1 General properties of ion channels

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

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 Planar Lipid Bilayers: recording activity of channels at intracellular membranes. Voltage Clamp: classical approach, keeping cell wall and regulatory machinery within cell intact. 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 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 1.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 ─ currents flow across the PM in both directions, into and out of the cytosol Current-voltage relationship

17 K + outward rectifier stabilize V m If hyper-depolarized, outward rectifying channels open cytosol 100 mM K + 1 mM K + E K =-120 mV cytosol E K >-120 mV K+K+ K+K+ K+K+

18 K + inward rectifiers take up K + from the soil and the apoplast cytosol K + < 1mM accumulated by ion coupled carrier K+K+ K+K+ K+K+ 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 +, Ca 2+, 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 Products of multigene family Tissue-specific expression of plant inward rectifiers ─ KAT1 expressed in guard cell K+-selective inward rectifiers ─AKT1 found in roots and hydathodes mature root lateral root hydathodes

22 Radioactive tracer flux analysis 86 Rb + 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 Ca 2+ ─ highly conserved P-domain motif TxGYGD

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

25 Ca 2+ dependence of the KCO1 : KCO1 mediated currents are potentiated by cytosolic free Ca 2+

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 1)Channels : - constitute a major pathway for Na + uptake into plant cells - important implications for salinity tolerance. - partially blocked by external Ca 2+ 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 (A) 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 3.6.5 Monovalent cation channels at the vacuolar membrane are Ca 2+ -sensitive and mediate vacuolar K + mobilization. 1)Vacuoles : - organelles for accumulation and storage of solutes - require massive solute loss in some circumstances 2)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 1)Little selectivity among monovalent cations 2)Inhibited when cytosolic Ca 2+ concentrations exceed 1μM 3)Activated when cytosolic pH increases The vacuolar K+ (VK) channel 1)Highly selective for K + over other monocovalent cations 2)Activated by cytosolic Ca 2+ in the nanomolar to low micromolar concentration range 3)Inhibited by increasing cytosolic pH

31 3.6.6 Calcium-permeable channels in the plasma membrane provide potential routes for entry of Ca 2+ to the cytosol during signal transduction. 1)Ca 2+ : Ca 2+ increase is central to signal transduction. activation of down stream targets, initial signal into the end response. 2) Ca 2+ permeable channels : activation of increase in cytosolic Ca 2+, upstream elements in Ca 2+ - based signal transduction pathway, 3) Voltage-gated Ca 2+_ permeable channels are activated by membrane depolarization.

32 Calcium-based signal transduction in a typical plant cell. 1)Receptor is perceived by signal. 2) Changed the activity of Ca 2+ -ATPases or plasma membrane Ca 2+ channel 3) Change on cytosolic free calcium concentration. 4) release of Ca 2+ from internal stores. 5) 2nd messenger is activation 6) increased free Ca 2+ changes the activity of Ca 2+ - binding proteins 7) final cellular response.

33 External Signals Light (red, blue) Cold stress Heat shock Mechanical stresses (touch, wind & wounding) Pathogens Phytohormones (Auxin, ABA, GA) Gravity [Ca 2+ ] cyt AM, DU, FM Diverse Cellular Responses Ca 2+ -Signal Decoder Calmodulins CDPKs, CBLs Other CBPs Amplitude (AM) Duration (DU) Frequency (FM)

34 Encoding and Decoding Calcium Signaling Stimulus Influx Efflux Ca 2+ spikes Decoders Responses Regulates enzymes Changes gene expression Ca2+ signals are generated by the influx of Ca 2+ through Ca2+ channels. Maintenance of low Ca2+ is archived by Ca 2+ efflux though Ca 2+ active transporters Encoding Decoding

35 Activation of a wheat root plasma membrane Ca 2+ channel by voltage. The channel activates strongly as V m and is fully activated at -100mV.

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

37 (A)Diagram illustrating channel activities at the guard cell vacuolar membrane during stomatal closure (B)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 (A)Current-voltage relationship for rapidly activating(R-type)anion channels. (B)Current-voltage relationship for slowly activating(S-type)channels

41 Anion channels function 1. 팽압 (turgor) 을 조절 기능 2. 특별한 자극에 의해 Depolarization 되어 Ca2+ channel activation 되 게 함 3.Membrane hyperpolarization 으로 activation 되어 Vm 의 조절에 관 여 4.Guard cell 을 포함한 cell type 에서 ATP, protein Kinase 로 부터 activatione 됨

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. 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 3.6.10 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 3.7.1 Directionality of water flow is determined by osmotic and hydraulic forces J v = L p ( ▲ p - σ ▲ п) ▲ p : hydrostatic pressure ▲ п : osmotic pressure J v : the flux of water across the membrane σ: Expresses the ability of the osmotically relevant solutes to permeate the membrane relative to water

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

50 Transcellular osmotic Pressure probe

51 3.7.3. The nonequivalence of P f and P d provides evidence for water channels P f 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 Model for water flow through a single- life, multiple occupancy aquaporin Water movement across biological membranes occurs through both the lipid bilayer and the pores formed by water channels.

53 3.7.4. 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. Extracellular view of eight asymmetrical subunits that form two tetramers. One of the monomers of the central tetramer is colored gold.

56 3.7.5 Aquaporin activity is regulated transcriptionally and posttranslationally H2OH2O Aquaporin TranscriptionPosttranslation Environmental stimuli (blue light, ABA, GA, cold & drought) Phosphorylation by Ca 2+ 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 vacuole 3.7.6 Plasma membrane aquaporins may play a role in facilitating transcellular water flow Plant water channel Plasma membrane Tonoplant intrinsic protein(TIP) Plasma membrane intrinsic protein(PIP) vacuole Plasma membrane

59 Transpiration Requirement for a low-resistance pathway apoplast symplast

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

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