Transport Proteins

• The classes of transport proteins include ATP-powered pumps, channel proteins, and carrier proteins.
• ATP-powered pumps move 1-1000 molecules/sec, the slowest of the three.
• Channel proteins move 10^7-10^8 molecules/sec, the fastest of the three.
• Carrier proteins move 10^2-10^4 molecules/sec, a middle pace.

ATP-powered pumps

• Couples ATP hydrolysis to transport a molecule against its concentration gradient.

Channel proteins

• Transport ions down their concentration gradient through a hydrophilic pore in the membrane protein.
• Channel proteins can exist in an open or closed conformation.
• When open, many ions can pass simultaneously.

Carrier proteins

• Bind water-soluble molecules on one side of the membrane and deliver them to the other side.
• This involves a conformational change in the protein and only binds one or a few molecules at a time.

Uniports, Symports, and Antiports

• Channels, pumps, and carriers can be subdivided into uniports, symports, and antiports.
• Uniports work in passive diffusion and are selective for one type of molecule.
• Uniports move molecules down their concentration gradients.
• A symporter and an antiporter use an existing electrochemical gradient rather than direct ATP hydrolysis.
• Symporters and antiporters move one of the molecules against its concentration gradient and a second molecule down its gradient.
• Symporters and antiporters are referred to as secondary active transport.
• Examples include a Na+ symporter that re-uptakes a released neurotransmitter as well as the epithelial Na+-glucose symporter.
• Binding of the two different molecules is cooperative: The conformational change necessary to deliver the two molecules to the other side of the membrane happens only when both are bound to the transporter.

Differences between Uniport and Simple Diffusion

• The rate of substance movement is higher for uniporters versus simple diffusion.
• Partition co-efficient is irrelevant for uniporters because there is no contact with the hydrophobic lipid environment.
• Uniport transport is limited by the number of uniporters in the membrane.
• Transport with a uniporter is specific.
• GLUT1 is the main uniporter for glucose in erythrocytes (blood cells).
• Once glucose enters an erythrocyte, it is rapidly converted to glucose-1-phosphate (and can eventually enter glycolysis).
• This keeps intracellular glucose low and allows for the continuous import of glucose.
• Specificity: Km is a measure of the affinity of an enzyme for its substrate.
• The lower the Km, the tighter the binding between the two.
• GLUT1 has a Km of 1.5 mM for glucose and 30 mM for galactose.

Na+-glucose symporter Effectiveness

• A symporter can generate an internal glucose concentration 30,000 times greater than the external concentration using the Na+ electrochemical gradient (generated by the Na+/K+ pump).
• If the symporter used only 1 mole of Na+, the concentration gradient would be 170 times higher inside compared to outside.

Glucose flux in the gut epithelial cell

• Glucose from digested food arrives in the intestine, where very low glucose concentration compared to the cell.
• Low sodium inside the cell is due to the Na+/K+ pump.
• Glucose symport at the apical domain brings glucose into the cell.
• Passive glucose transport at the basal domain sends glucose into the blood for transport to other tissues.
• Note separation of membrane proteins at basal and apical domains, mediated in part by tight junction proteins that restrict membrane protein movement.

Classes of ATP Pumps

• The classes of ATP pumps are P-class, V-class, F-class, and ABC superfamily.

P-Class

• P-class pumps are heterotetramers with 2 subunits: α and β.
• The beta subunit is phosphorylated during transport.
• Examples of P-class pumps are Na+/K+ ATPase, Ca2+ ATPase, H+/K+ ATPase (acidifies stomach).

V-Class

• V-class pumps have multiple subunits.
• They transport only protons against their gradient.
• V-class pumps acidify lysosomes and vacuoles.

F-Class

• F-class pumps have multiple subunits, related to V-class.
• They is the only member that generates ATP and pumps only protons.
• F-class pumps are located in the inner membrane of mitochondria, thylakoid, and bacterial plasma membrane.

ABC Superfamily

• ABC superfamily pumps have more members than other classes and transport sugars, amino acids, phospholipids, and proteins.
• They are responsible for multidrug resistance by pumping anti-cancer drugs out of cells.

Na+/K+ ATPase: Mechanism

• In its E1 conformation, the Na+/K+ ATPase has three high-affinity Na+-binding sites and two low-affinity K+-binding sites on the cytosolic-facing surface of the protein.
• Due to the high affinity of the E1 conformation to Na+, three Na+ ions bind to the Na+-binding sites despite the low intracellular Na+ concentration.
• Despite the high intracellular K+ concentration, K+ ions are unable to bind to the low-affinity K+-binding sites.
• ATP also binds to its site on the cytoplasmic side.
• The bound ATP is hydrolyzed to ADP, and the liberated phosphate is transferred to a specific aspartate residue in the Na+/K+ ATPase, forming a high-energy acyl phosphate bond, denoted by E1.
• The protein changes its conformation to E2.
• During the E1-E2 transition, the three bound Na+ ions become accessible to the exterior face.
• Transition to the E2 conformation also generates two high-affinity K+ sites and three low-affinity Na+ sites on the extracellular face.
• The Na+ ions then dissociate, and the K+ ions associate with their high affinity sites.
• The aspartyl-phosphate bond in E2 is hydrolyzed, causing E2 to revert to E1.
• During the E2-E1 transition, the two bound K+ ions become accessible to the cytosolic face.
• The K+ sites revert to low-affinity sites, thus releasing the K+ into the cytosol.
• The transporter is now available for another round of ion transport.

Ca2+-ATPase

• The Ca2+-ATPase is another example of a P-class ATPase with high affinity Ca2+ binding sites in the E1 conformation.
• Phosphorylation by ATP causes a conformational change that exposes the Ca2+ to the exterior face, lowering the Ca2+ affinity and causing the ions to be released into the cell exterior.
• Dephosphorylation regenerates the E1 conformation.
• Ca2+-ATPase is important in muscle cells for contraction, nerve cells for neurotransmitter release, fertilized oocytes to start development, and liver cells for glycogen breakdown.