Sinh

Ha Noi national university
Scientific natural university
Faculty of biology
Teacher guide : Professor- Vu Van Vu;
Student : Vu Thi Luyen;
Class : K8-honor BA Biology.
Transport Across Membranes
Table of contents
Aquaporins;
Facilitated diffusion of ions and molecules;
Active transport:
Direct active transport;
Indirect active transport;
Endocytosis and exocytosis;
The patch clamp technique;
Hypotonic solutions;
Isotonic solutions;
Hypertonic solutions.



Something distinguish active transport from passive diffussion.
Active transport can move substances either with or against a concentration gradient.
Active transport requires the expenditure of energy which come from the high-energy molecule ATP .
Passive diffusion can’t move substances against a concentration gradient.
Passive diffusion don’t require the expenditure of energy which come from the high-energy molecule ATP .
Aquaporins
Aquaporins are made up of six transmembrane α-helices arranged in a right-handed bundle, with the amino and the carboxyl termini located on the cytoplasmic surface of the membrane.
Aquaporins form tetramers in the cell membrane, and facilitate the transport of water and, in some cases, other small uncharged solutes, such as glycerol, CO2, ammonia and urea across the membrane depending on the size of the pore.
Aquaporins
Aquaporins
Aquaporins in plants
The presence of aquaporins in the cell membranes seems to serve to facilitate the transcellular symplastic pathway for water transport.
Aquaporins in plants are separated into four main groups :
Plasma membrane Intrinsic Protein (PIP) ;
Tonoplast Intrinsic Protein (TIP) ;
Nodulin-26 like Intrinsic Protein (NIP) ;
Small basic Intrinsic Protein (SIP) .
Gating of Plant Aquaporins
The gating of aquaporins is carried out to stop the flow of water through the pore of the protein.
The gating of an aquaporin is carried out by an interaction between a gating mechanism and the aquaporin which causes a change in the protein so that it blocks the pore and thus disallows the flow of water through the pore.
In plants it has been seen that there are at least two forms of aquaporin gating.
These are gating by the dephosphorylation of certain serine residues, which has been linked as a response to drought, and the protonation of specific histidine residues in response to flooding. The phosphorylation of an aquaporin has also been linked to the opening and closing of a plant in response to temperature.
X-ray structure of the open & closed forms of the gated spinach aquaporin .
Eight water molecules (red spheres) are seen to line up along the water transport channel & the grey dotted tube represents the diameter of the water-channel. In the open conformation (right) there is space for water molecules to move through the entire length of the pore, whereas in the closed conformation (left) the aquaporin is blocked from the inside of the cell (bottom).
Under normal conditions the aquaporin is phosphorylated (indicated by P) & the water channel is open.
During drought stress these aquaporins close in response to the removal of phosphate groups from two conserved serine residues.
During flooding the aquaporins close in response to the protonation of a conserved histidine (indicated by H+).
Facilitated diffusion
Some substances which require specific carriers to take them through the membrane .The carriers bind to the molecule to be transported and take them through in the direction determined by concentrations on either site of the membrane .The movement will be from high-concentration to low-concentration.
The Potassium Channel consists of four major parts, the outer helix, the inner helix, the pore helix and the selectivity filter. The structures of the Potassium Channel work together to help diffuse K+ ions into the cell.
The Potassium Channel`s Selectivity Filter

The red sticks pointing at the K+ ions are oxygen atoms from the backbone of the protein. They help stabilize the K+ ion when it loses its water molecules and enters the selectivity filter.

Valinomycin is a carrier for K+.
It is a circular molecule, made up of 3 repeats of the sequence shown above.
The Potassium Channel`s Selectivity Filter

Two of the K+ ions are inside the channel marking the beginning and the end of the selectivity filter. Although it appears as if three ions are inside the channel, the bottom two magenta spheres are two seperate postitions for the second K+ ion (only one K+ ion in either position).
The Potassium Channel`s Selectivity Filter

The two K+ ions inside the selectivity filter repel each other. The repulsion of the K+ ions helps overcome the interaction of the K+ ion with the backbone oxygens and allows the K+ ion to proceed down the Potassium Channel.
A comparison of K+ ion in the selectivity filer and Na+ in the selectivity filter. K+ ion easily makes contact with the oxygen`s and stabilizes. The Na+ ion, however, cannot reach all of the oxygen atoms and does not remain stable in the selectivity filter.
The Potassium Channel`s Selectivity Filter

Val and Tyr point away from the pore and form the structural support for the Potassium Channel`s Selectivity Filter .
Reactivity K ~ Rb > Cs > Na > Li
Radius(A) 1.33 1.48 1.69 0.95 0.60
Ligand-gated ion channels
(external ligands)

Internal ligands
Model of a voltage-gated ion channel
Model of a voltage-gated ion channel
Patch Clamping
The technique of patch clamping is used to study ion channel activity.
A narrow bore micropipet may be pushed up against a cell or vesicle, and then pulled back, capturing a fragment of membrane across the pipet tip.


Patch Clamping
A voltage is imposed between an electrode inside the patch pipet and a reference electrode in contact with surrounding solution. Current is carried by ions flowing through the membrane.
If a membrane patch contains a single channel with 2 conformational states, the current will fluctuate between 2 levels as the channel opens and closes.
The increment in current between open and closed states reflects the rate of ion flux through one channel.
Overview of the recording set-up: microscope, patch clamp amplifier, computer.
A close-up view of the recording scope. The recording electrode is attached to the blue headstage.
A close-up view of the microscope stage. The cells are plated on a glass coverslip that is adhered to the bottom of the plastic dish. The electrode holder does not have a pipette on it in this picture.
Tobias and Kamran are using the computer to run the patch clamp amplifier and collect data. 
Active transport
The movement of substances from a low concentration to a high concentration requires the expenditure of energy and will need specific protein carriers for the transport to take place.
The Ca2+- ATPase of skeletal muscle
Three classes of ATPases
P-type transport Na+, K+ and Ca2+ through the eukaryotic plasma membrane. When translocating, they are phosphorylated.
F-type run in reverse to convert H+ gradients into ATP in mitochondria and bacteria (ATP synthase).
V-type use ATP to form H+ gradients across plant tonoplasts.
The pumping of protons across a membrane by an energy-expending uniport generates a proton motive force, which can be exploited by an ATPase running `in reverse` to generate ATP. This is how bacteriorhodopsin, oxidative phosphorylation and photosynthesis all work:
ABC-transporters
Typical ABC-transporters will contain two transmembrane domains (TMs), each of which consists of α-helices which cross the phospholipid bilayer multiple times. These helices form between six to eleven (usually six) membrane-spanning regions. These transmembrane domains provide the specificity for the substrate, and prevent unwanted molecules from using the transporter. In between the TMs is a ligand binding-domain, which is on the extracellular side of the protein for importers and on the cytoplasmic side for exporters.

All ABC proteins also contain either one or two ATP-binding domain(s), (nucleotide-binding folds (NBFs)) and are located on the cytoplasm side of the membrane. These folds are divided into parts or motifs, called Walker A and Walker B, which are separated by approximately 90-120 amino acids. In addition, there is a third short and highly conserved motif (called LSGGQ motif, C motif, or "signature" motif) located after the Walker B motif. Unlike the Walker A and Walker B motifs, which are found in other proteins which hydrolyze ATP, the signature motif is unique to ABC transporters. These folds form the “cassettes” which the protein family is named after. The transmembrane domains and nucleotide-binding folds are often arranged in the order NH3+-TM-NBF-TM-NBF-COO-.
ABC-transporters
ABC transporters may be classified as either half transporters or full transporters. Full transporters consist of the typical two TMs and NBFs. Half transporters consist of only one TM and one NBF and must combine with another half transporter to gain functionability. Half transporters can thus form homodimers if two identical ABC transporters join, and heterodimers if two unlike ABC transporters join.
ABC-transporters
When an ATP molecule binds to each cassette of an ABC-transporter, it induces a conformational change in which the NBFs and TMs shift to reveal a cavity called the translocation pathway. When the translocation pathway opens, molecules which were previously bound to the ligand binding-domain are free to travel through the pathway. This action is called the power stroke.
ABC-transporters
Function’s ABC-transporters to plant.
Encompasses polar auxin transport ;
Lipid catabolism ;
Xenobiotic detoxification ;
Disease resistance ;
Stomatal function .
Uniport. Transports one species. Also known as a pump ;
Symport. Transports two species in the same direction, often one against its gradient, one with it;
Antiport. Transports two species in opposite directions, often one against its gradient, one with it.
Symport pumps
Symport pumps
The glucose/Na+ symport uses the energy stored in the Na+ gradient (produced by the Na+/K+ ATPase) to transport glucose against its concentration gradient.
Antiport pumps
Na+/K+-ATPase
Endocytosis is one way in which large particles that normally would not pass through the membrane can be taken into the cell.
Phagocytosis is the movement into the cell of other organisms (bacteria) or fragments of organic matter.
Pinocytosis is the movement of liquid into the cell.
Receptor-mediated endocytosis requires specific receptors on the cell surface to detect molecules and to allow the fast movement of molecules into the cell, e.g. hormones.
Exocytosis is the movement of large particles out of the cell via secretory granules originating from the Golgi apparatus, and can include hormones, neurotransmitters, digestive enzymes etc.
If the solution is isotonic relative to the cell, then the solute concentrations are the same on both sides of the membrane and water moves equally in both directions
Animal cells
Plant cells
A hypertonic solution has increased solute, and a net movement of water outside causing the cell to shrink.
Animal cells
Plant cells
A hypotonic solution has decreased solute concentration, and a net movement of water inside the cell, causing swelling or breakage.
Thank you very much