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 Polarized 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. Channels are now known to be present in all plant cell types, at the PM and vacuoles and all other membranes as well. 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. 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. 7. Calcium-permeable channels in endomembranes are activated by both voltage and ligands (A)Diagram illustrating channel activities at the guard cell vacuolar membrane during stomatal closure (B)During plasma membrane-based signal transduction 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 1M increasing the activity of the channel. Ca2+ is thought to interact with calmodulin associated with the channel or a channel regulatory protein 8. Plasma membrane anion channels facilitate salt release during turgor adjustment and elicit membrane depolarization after stimulus perception 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 Anion channels function 1. (turgor) 2. Depolarization Ca2+ channel activation 3.Membrane hyperpolarization activation Vm 4.Guard cell cell type ATP, protein Kinase activatione 3.6.9 Vacuolar malate channels participate in malate sequestration 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. 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. Integrated channel activity at the vacuolar and plasma membranes yields sophisticated signaling systems 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 3.7 Water transport through aquaporins 3.7.1 Directionality of water flow is determined by osmotic and hydraulic forces J v = L p ( p - ) p : hydrostatic pressure : osmotic pressure J v : the flux of water across the membrane : Expresses the ability of the osmotically relevant solutes to permeate the membrane relative to water 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. Transcellular osmotic Pressure probe 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. 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. 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). Structure of an aquaporin showing the six transmembrane helices and two conserved NPA (Asn-Pro-Ala) residue 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. 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 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. vacuole 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 Transpiration Requirement for a low-resistance pathway apoplast symplast 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 Requirment for maintenance of cytosolic volume during osmoticstress