Structural Biochemistry/Membrane Proteins/Active Transport

Transport of Molecules Across Membrane
Transport of molecules across membrane is the movement of a molecule from inside the membrane to outside or vice versa. There are two factors to determine if a molecule will cross a membrane:

- The permeability of the molecule in a lipid bilayer: Molecules move spontaneously from high concentration to low concentration due to the second law of thermodynamics. However, molecules with high polarity, such as sodium, are not able to freely enter the cell membrane because the charged ion cannot pass through the hydrophobic core of the membrane. This transport of molecule across membrane can be either passive (where movement is driven by a gradient) or active (where movement is against a gradient and requires energy).

- Availability of an energy source:  Energy is minimized when all concentrations are equal, so an uneven distribution of molecules is a form of potential energy, which can be used to drive other processes. One process is the transport of molecules from one side of a membrane to the other. The equation that describes the amount of energy required for this process is :

$$ G= RTln(side2/side1)$$,

where R = 8.315*10-3 KJ/mol, the molar gas constant, and T = temperature.

When the molecules involved are charged, an electrical potential can build up. The cumulative effect of the electrical potential and the uneven distribution of concentrations gives us a modified free energy equation:

$$ G= RTln(side2/side1) + ZFV $$,

where F is Faraday’s constant and Z is electrical charge of transported species and V is potential difference across the membrane.

Active Transport
It requires work to pump a molecule across a membrane against its gradient. Moving ions from low concentration to high concentration leads to decrease in entropy, which requires an input of free energy. Therefore, this type of membrane traffic is called active transport. The transport proteins that move solutes against a concentration gradient are called carrier proteins. On the other hand, channel proteins are involved in passive transport.

Types of Active Transport
Sodium-Potassium Pumps are an example of active transport. It is known that cells contain high concentrations of potassium ions but low concentrations of sodium ions. Therefore, it was deduced that a protein existed on the plasma membrane which actively pumped the two ions against their biological gradients. This protein was discovered in the 1950s by Jens Christian Skou and for his discovery, was awarded the Nobel Prize in 1997. This pump works by binding sodium ions which stimulates phosphorylation by the addition of a phosphate group from ATP. This phosphorylation causes a change in the 3D shape of the protein, making it open up to the extracellular world, and decreases the protein's affinity for sodium ions. In turn, the new shape has a high affinity for potassium ions which bind and force a 3D conformational change while triggering the release of the phosphate group. This causes the protein to open up the intercellular world and the loss of the phosphate causes the pump to have a lower affinity for potassium and a higher affinity for sodium. The cycle repeats.

In this sodium/potassium pump, sodium is transferred out of the plasma membrane and potassium is pumped inside the plasma membrane. Since active transport requires energy, it uses ATP or it couples to molecules moving down the concentration gradient. In the sodium/potassium pump, sodium and phosphate (the phosphate from the breakdown of ATP: ATP → ADP + P) are coupled to the pump, which takes both of them out of the cell and brings the potassium inside. For this process to take place, both the potassium and sodium pumping must occur at the same time because if the ability to pump one of them is lost, then the ability to pump the other ion will be lost as well. The sodium/potassium pump is active transport because there is coupled transport where one molecule's transfer is dependent on the other molecule's transfer. This example of active transport is antiport because molecules are being moved in opposite directions.

Cotransport Pumps, or coupled transport, is a type of active transport in which the transport of a specific solute indirectly facilitates the active transport of another solute. The general mechanism is that, through the use of ATP, a specific solute is driven up its concentration gradient, analogous to moving water up a hill. In the second step, the specific solute runs back down its concentration gradient while forcing the other solute up its own concentration gradient, analogous to coupling water running downhill to force the work of another machine.

Endocytosis is another type of active transport. In the previous examples, active transport was used on small molecules. In endocytosis, energy is used to take in biological molecules and large particles by the formation of new vesicles. There are multiple types of endocytosis, with the major categories being phagocytosis, pinocytosis and receptor-mediated endocytosis.

Exocytosis is also another type of active transport, utilizing energy to do the opposite of endocytosis. In exocytosis, the vesicle fuses with the plasma membrane thereby releasing all the contents and waste outside of the cell. This type of active transport is mainly used by secretory cells where they secrete insulin or neurotransmitters.

Efflux Pumps Active Efflux is a type of active transport and is the mechanism largely responsible for the extrusion of drugs such as antibiotics, toxic substances and other xenobiotics. Bacteria efflux pumps are separated into five families.

1. Major facilitator superfamily (MFS) 2. ATP binding cassette (ABC) 3. Small multidrug resistance family (SMR) 4. Resistance-nodulation-cell division superfamily (RND) 5. Multi-antimicrobial extrusion protein (MATE)

These efflux pumps are largely responsible for antibiotic drug resistance due to the presence of the efflux pumps that export toxins out of the cell and inhibit the drug's effects. Gram-negative bacteria have a greater resistance to antiseptics and antibiotics. The RND family of efflux pumps is exclusive to gram-negative bacteria and is very effective in generating resistance against antibiotics.

In the case of E. Coli, two homologues, AcrB and AcrB complex together with the outer membrane protein channel TolC and utilizing the proton-motive force, this complex can effectively export a variety of drugs across the periplasmic place and out of through the outer membrane. This is possible due to AcrB's ability to assume an asymmetric structure in which each subunit exhibits different conformations to attach to the attach to the substrate and move them out of the transporter.