Passages through membrane Membrane Transport 1. Passive transport • Direct diffusion across membrane • Through channel • Facilitated diffusion through carrier 2. Energy-dependent transport • Active transport Primary active transport - pump Secondary active transport - carrier • Group translocation 1

Four criteria to classify transport process: 1. Does transport occur across the bilayer or is it protein-mediated?

Nicholls and Ferguson. 2002.

2

Four criteria to classify transport process: 2. Is transport passive or directly coupled to metabolism?

Nicholls and Ferguson. 2002.

3

Four criteria to classify transport process: 3. Does the transport process involve a single ion or metabolite, or are fluxes of two or more species directly coupled?

movement of one molecule at a time

Nicholls and Ferguson. 2002.

tightly coupled transport of two different molecules in the same direction

tightly coupled transport of two different molecules in the opposite directions 4

Four criteria to classify transport process: 4. Does the transport process involve net charge transfer across the membrane?

no net transfer of charge

creating a charge separation across the membrane

Electrogenic/ Electrophoretic Nicholls and Ferguson. 2002.

5

endorsed by the International Union for Biochemistry and Molecular Biology

(carrier or porters)

Luckey, M. 2008. Membrane Structural Biology.

6

Passive transport driving force The Electrochemical Potential Difference solute moving across a membrane is subject to its own gradients of both concentration and electrical potential

∆

µ~ i F

= z ∆E M i

i RT  Cinside + ln i F  Coutside

   

Volt

Active transport driving force Proton motive force (pmf) is a measure of the free energy

stored in a transmembrane electrochemical potential difference for H+ (gradients of proton)

2.3RT ∆p = ∆EM − ∆pH in −out F ∆p = ∆EM − 59∆pH in −out

mV 7

Proteins involved in transport carry out either active or passive transport. Active transporters enable a cell to accumulate solutes against electrical and/or concentration gradients by making use of energy sources to “pump” them thermodynamically “uphill”. Passive transporters allow the “downhill” flow of solutes across the membrane until their electrical and/or concentration gradients are dissipated.

Luckey, M. 2008. Membrane Structural Biology.

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Class I: Channels and pores Proteins (peptides) that allow the relatively free flow of solute through the membrane. Five subclasses:  α-helical protein channels  β-barrel porins  channel-forming toxins, including colicins, diphtheria toxin, etc.  nonribosomally synthesized channels (e.g. gramicidin and alamethicin)  holins (function in export of enzymes that digest cell walls in an early step of cell lysis)

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Ion Channels: large, membrane-spanning proteins Ionic selectivity • Cation channel-- K+, Ca2+ • Anion channel --ClIon channels can be very specific, showing a high degree of selectivity with regard to the solutes transported. But not all channels exhibit such high selectivity. The imperfection of ion channel transport may be very important, and may well be a key to the entry of many ‘undesirable’ ions into plants. Anion channels appear to discriminate poorly between NO-3 and Cl-.

Tester. 1996.

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All ion channels have an ion-conduction pathway and a gate. Features of the ion-conduction pathway determine the specificity and the rate of ion conduction, whereas the gate functions as a switch, opening and closing the pore at the desired time. Channels can change conformation rapidly between catalytic (‘open’) and non-transporting (‘closed’) states. The movement is called ‘gating’: • • • •

Voltage-gated Ligand-gated Stretch-activated Light-activated

A cell can not allow rapid, dissipative fluxes over long periods of time, and most channels are not transporting for most of the time.

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I. Voltage-gated ion channels A voltage-gated ion channel can be defined as a membrane-spanning protein, is made up of 3 basic parts: a) the voltage sensor, b) the pore/conducting pathway and c) the gate. Basic of K+ channel

conducting pathway/ selectivity filter K+ H2 O

voltage sensor

subunit 1 Chung et al. 2007. Biological Membrane Ion Channels.

subunit 1

gate

subunit 2

Voltage-gated channels can be traced back at least 2.5 billion years. 12

Chung et al. 2007. Biological Membrane Ion Channels.

I. Voltage-gated ion channels

The functional channels are made up of four subunits (K+ channels) or one protein with four homologous domains (Na+ and Ca2+ channels). Each subunit has six transmembrane segments and a pore loop. The fifth and sixth transmembrane segments (S5 and S6) and the pore loop were responsible for ion conduction. The S4 segment contains several basic residues, arginines or lysines and was voltage sensor.

13

Chung et al. 2007. Biological Membrane Ion Channels.

Example 1 Plasma membrane potassium channels Potassium channels are responsible for shaping the electrical behavior of cell membranes. K+ channel currents set the resting membrane potential, control action potential duration, control the rate of action potential firing, control the spread of excitation and Ca2+ influx and provide active opposition to excitation. There are a large number of K+ channel types, with a great deal of phenotypic diversity. Ann Plant Reviews. V15. 2004

The family of potassium channels is found in bacteria, archaea, and eukaryotes. They are both selective and fast, their selectivity for K+ over Na+ is over 1000-fold, the conduction rates of about 108 ions s-1 are close to diffusion limited. The diversity of potassium channels surpasses that of any other channel family.

Luckey, M. 2008. Membrane Structural Biology.

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In the plasma membrane of plant cells, three distinct types of voltage-gated potassium channels were identified, which are active in different voltage ranges: 1. hyperpolarisation-activated K+ channel—Kin 2. depolarisation-activated K+ channels– Kout 3. weakly rectifying K+ channels

Ann Plant Reviews. V15. 2004

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a. Inward K+ channel (Shaker K+-channel family) Hyperpolarisation-activated K+ channels (Kin) open at membrane voltages more negative than around -100 mV, whereas at more positive voltages they are closed.

Database of membrane proteins with known three-dimensional structure, see blanco.biomol.uci.edu/Membrane_Proteins

Shaker Kv1.2

16

Kuo, A. et al. 2003. Science 300:1922-1926

17

Crichton. 2008. Biological Inorganic Chemistry.

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b. Outward rectifying K+ channel KCO1, the only plant outward-rectifying K+ channel identified, has four transmembrane domains. Animal 6-transmembrane Shaker channels are all outward-rectifying.

KCO1 Depolarisation-activated K+ channels, Kout, open at membrane voltages more positive than the equilibrium potential for potassium, EK. At more negative voltages they are closed.

19 Buchanan et al. 2000

c. Weakly rectifying K+ channels—Kweak channels Kweak channels belong to the Shaker K+ channel family. These channels appear to be active in the entire physiological voltage range and thus mediate both, potassium inward and potassium outward fluxes. A characteristic fingerprint of weakly rectifying K+ channels is the block by extracellular protons and Ca2+. As is the case for AKT2 from Arabidopsis, Kweak channels can act in two different gating modes: while in mode 1, AKT2 activates like a hyperpolarisation-activated K+ channel, in mode 2 it is open in the entire physiological voltage range. It was proposed that the setting of the gating mode is determined by the phosphorylation status of the protein. Thus AKT2 can be switched from an inward-rectifying channel to a leak-like K+ channel by phosphorylation events. At the present time, Kweak channels are supposed to provide a background conductance that stabilizes the membrane voltage, e.g. in phloem processes. 20

Ann. Plant Reviews. 2004. V15.

Example 3 Plasma membrane Ca2+ channel Calcium concentrations in the cytoplasm of cells are maintained at a low level. Ca2+ channels activate quickly such that the opening of ion channels can rapidly change the cytoplasmic environment. Ca2+ inside the cell acts as a 'secondary messenger' prompting responses by binding to a variety of Ca2+ sensitive proteins. Ca2+ channels play role in stimulating muscle contraction, in neurotransmitter secretion, gene regulation, activating other ion channels. Ca2+ channels are distinguished as either voltage-activated or responding to the binding of calcium or other agonists that release calcium from intracellular stores. Ca2+ channels have diverse biological, pharmacological, and physiological properties.

21

Chung et al. 2007. Biological Membrane Ion Channels.

Ca2+ channel possess a poreforming α1 subunit in four repeats of a domain with six transmembrane-spanning segments that include the voltage sensing S4 segment and the pore forming (P) region. The α1 subunit is large (190250 kDa) and incorporates the majority of the known sites regulated by secondary messengers, toxin, and drugs. This subunit is usually complexed with at least three auxiliary subunits, α2, δ, β and δ, with the α2 and δ subunits always linked by a disulfide bond. L-type Ca2+ channels are a member of the HVA or high voltage activation type because the channels are activated by strong depolarizations typically to 0 or +10 mV and are long-lasting (slow to inactivate). They are blocked by the lipid-soluble 1,422 dihydropyridines. Chung et al. 2007. Biological Membrane Ion Channels.

Example 4 Anion channels The anion transport system is one of facilitated diffusion system of the erythrocyte membrane. Cl- and HCO3- ions are exchanged across the red cell membrane by a 95 kD transmembrane protein.

Diagram of anion channel of red blood cells. Cl- and HCO3- may move in either directions, according to concentration gradients across the membrane. The counter flow of the ions prevents any electrical potential developing across the membrane.

Diagram to illustrate attachment of anion channel protein and glycophorin to the cytoskeleton.

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II. Ligand-gated ion channels (LGICs) LGICs are fast-responding channels in which the receptor, which binds the activating molecule (the ligand), and the ion channel are part of the same nanomolecular protein complex. The gating of ligand-gated ion channels occurs only after binding of specific compound to form a complex.

24 Ann. Plant Reviews. V15. 2004.

Why do cells need ion channels that are regulated through binding of agonists rather than via changes in membrane voltage? Ligand-gated channels are excellent molecular switches that can fulfill complex tasks in information processing. In many biological processes, information has to be rapidly converted from one type of signal into another. Where signals from inter- or intracellular messengers have to be transduced into electrical signals, ligand-gated ion channels form the obvious mechanism. Ligand-gated ion channels may also be crucial when ion fluxes and transmembrane signals need to be controlled at intracellular membranes where large deviations in membrane potential are either unlikely to occur or where control of the membrane potential may be difficult. ER membrane, Golgi membrane or nuclear envelopes may not experience large swings in membrane potential, providing little scope for regulating ionic fluxes through voltages-gated channels. Most ligand-gated ion channels appeared relatively late in evolution (around 800 million years ago), after eukaryotic life was established, and most of these transporters are found only in multicellular organisms where they play specific roles in inter- and intracellular communication. 25 Ann. Plant Reviews. V15. 2004.

Schematic longitudinal and cross-sectional diagrams of a typical LGIC.

LGIC database: http://www.ebi.ac.uk/ compneur-srv/LGICdb/LGICdb.php)

A. Two of the five subunits and the pathway for ions entering the exterior end of the channel and moving into the cell interior via lateral portals at the cytoplasmic end of the channel. B. A cross-sectional view showing the four transmembrane segments of each of the five subunits. The five M2 segments line the pore region of the channel within 26 the membrane lipid bilayer.

Ca2+ permeable channels in endomembrane are activated by both voltage and ligands.

Two types are activated by ligands--inositol 1,4,5-triphosphate (IP3) and cyclic ADP-ribose (cADPR). Slow activating vacuolar (SV) channel response slowly to membrane depolarization and is strongly activated by Ca2+-calmodulin. The Ca2+-induced Ca2+ release (CICR) phenomenon is generated through the activity of one of the two voltage-activated Ca2+ channel. 27

Buchanan et al. 2000

Current flows through ion channel

Current–voltage relationships (Part 1)

(∆I/∆V)

28

Current–voltage relationships (Part 2)

29

Current–voltage relationships (Part 3) outward K+ channel

inward K+ channel

30

Channel for non-ionic molecules Over 350 different AQPs have been identified in all forms of life. Mammals have 11 isoforms, designated AQP0 to AQP10, that fall into 2 classes: AQPs and glyceroaquaporins (GlpF). The high-sequence homology among AQPs suggests that they are a common architecture to accomplish selective water transport.

Water channels are typically bidirectional, allowing influx and efflux of water molecules in response to changing osmotic conditions. They are extremely fast: water flows through a single AQP1 molecules at a rate of 3 billion molecules per second. They are very selective, allowing the passage of water molecules without protons or other ions. AQPs transport only water, while

GlpF conduct small organic molecules like glycerol, urea, glyceraldehyde and glycine, in addition to water. Luckey, M. 2008. Membrane Structural Biology.

DL-

31

Tetramers of AQP1

GlpF channel

Luckey, M. 2008. Membrane Structural Biology.

32

Blocking of proton permeation of AQP1 Partial charges from the helix dipoles restrict the orientation of the water molecules passing through the constriction of the pore. Hydrogen bonding of a water molecule with Asn 76 and/or Asn 192, which extend their amido groups into the constriction of the pore. (N=Asparagin, polar and neutral)

34 Luckey, M. 2008. Membrane Structural Biology.

Role of aquaporins in cell osmoregulation and water uptake The plasma membrane and the tonoplast have strikingly different solute and water transport properties. In tobacco, the tonoplast had high permeability values to water (Pf~ 690 µm s-1) and urea (Purea~75×10-3 µm s-1) as compared to low values in the plasma membrane (Pf~ 6 µm s-1; Purea~1×10-3 µm s-1). These striking differences can be accounted for by a higher level of aquaporins activity in the tonoplast. ... Vacuoles isolated from various plant materials had remarkably high water permeabilities (200-1000 µm s-1). ...by not limiting water and solute transport across the tonoplast, plant cells may be able to use the vacuole space for efficient osmoregulation of the cytosol. A consistent set of data gathered from various plant species has accumulated over the years suggesting that aquaporins in the membrane contribute a large part (50-90%) to root hydraulic conductivity. This means that parallel water transport across cell wall (apoplastic path) or cytoplasmic continuities (symplasmic path) may not be as important as was initially hypothesized.

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Ann. Plant Reviews. 2004. V15.

Gas Channel

The AmtB ammonium channel of E. coli. Midway through the membrane, the channel narrows over a 20 Å span. Two pore-lining residues, His168 and His318, stabilize three NH3 molecules (Am2, Am3 and Am4) through hydrogen bonding. The molecules return to equilibrium as NH4+ in the inner vestibule. 36 Khademi et al.. 2004.

Taiz and Zeiger. 2002.

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