Membrane Protein Pumps

Learning objectives You should be able to understand & discuss: Active transport-Na + /K + ATPase ABC transporters Metabolite transport by lactose permease

Ion pumps: ATP-driven Ion-pumps are energy transducers in that they convert one form of free energy into another. Two types of ATP-driven pumps: (1) P-type ATPases and (2) the ATP-binding cassette (ABC) transporters that undergo conformational changes on ATP binding & hydrolysis and cause a bound ion to be transported across the membrane.

Ion pumps: gradient driven A different mechanism of active transport used the gradient of one ion to drive the active transport of another. An example of such a secondary transporter is the E.coli lactose transporter. Many transporters of this class are present in the membranes of our cells. The expression of these transporters determines which cell metabolites a cell can import from the environment. Transporter expression is therefore a primary means of controlling metabolism.

Expression & metabolic activity e.g. glucose metabolism. Which tissues can make use of glucose is largely governed by the expression of different members of a family of homologous glucose transporters called GLUT1 through GLUT5 in different cell types. GLUT3 binds glucose tightly so these cells have first call on glucose when it is present at low concentrations.

Free energy stored in Concentration gradients Unequal distribution of molecules (concentration gradient) requires an input of energy Passive Active

Free Energy & Transport (a) Free energy in transporting uncharged solute across a membrane (b) Singly charged solute to the side having the same charge. A transport process must be active when  G is positive, whereas it can be passive when  G is negative.

The Free Energy Stored in Concentration Gradient For an uncharged solute molecule:  G = RTln(c 2 /c 1 ) = 2.303RTlog 10 (c 2 /c 1 ) R is the gas constant (8.315x10 -3 kJ/mol) T is temperature in Kelvin Concentration on side 1 of the membrane c 1 Concentration on side 2 of the membrane c 2

The Free Energy Stored in Concentration Gradient For a charged solute molecule:  G = RTln(c 2 /c 1 ) + ZF  V = 2.303RTlog 10 (c 2 /c 1 ) + ZF  V where Z is the charge of the solute  V is the potential across the membrane F is the Faraday constant (96.5 kJ/V/mol)

Two families of membrane proteins use ATP hydrolysis to pump ions and molecules across membranes The extracellular fluid of animal cells has a salt concentration similar to seawater (ca 140 mM). However cells must maintain their intracellular salt concentrations (ca 14 mM). Most animal cells have high K + and low Na + relative to the external medium.

Na + -K + ATPase

These ionic gradients are generated by the Na + -K + ATPase. It transports 3 Na + out and 2 K + into the cell for each ATP hydrolysed. Free energy change = kJ mol -1 Hydrolysis of ATP = -50 kJ mol -1

ATP hydrolysis provides the energy needed to pump Na + out of the cell and K + into the cell generating the gradients. Subsequent purification of other pumps reveals a large family (evolutionarily related) in bacteria, archaea, and eukaryotes including the Ca 2+ ATPase and the H + -K + ATPase. Na + -K + ATPase

Pump action – simple in principle but more complex in detail

Calcium channel Sarcoplasmic Reticulum Calcium-Transporting ATPases (SERCA) (1.5mM) (1.0µM)

Calcium pump structure SR Ca 2+ ATPase, or SERCA P-type ATPase – forms phosphorylaspartate Pumps calcium into the SR of muscle cells (1.5mM in SR compared to 1.0  M in cytoplasm) Important for muscle contraction N – binds nucleotide; P – accepts the phosphoryl group (Asp 351); A is the actuator domain (E1: Ca 2+ bound state) Ca 2+

E2: Calcium free state Calcium free form Ca 2+

Mechanism of P –type ATPases (>70 in the human genome) Eversion

Digitalis inhibits the Na + -K + pump by blocking dephosphorylation of E 2 -P Foxglove (Digitalis purpurea) is the source of digitalis Digitoxigenin is used to treat congestive heart failure. It increases the force of muscle contraction K i = 10 nM

How inhibition of the sodium-potassium pump leads to stronger contraction of the heart Inhibition of the Na + -K + pump by digitalis leads to a higher level of Na + inside the cell. The reduced Na + gradient results in slower extrusion of calcium by the sodium-calcium exchanger. The increase in calcium enhances the ability of the cardiac muscle to contract.

ABC transporters - multidrug resistance The onset of MDR in cultured tumour cells (& presumably tumours in patients) was found to correlate with expression & activity of a membrane protein of 170 kD. This is an ATP- dependant pump that extrudes a wide range of small molecules from the cells that express it. There are ABC transporters containing two transmembrane domains and two ATP-binding domains (cassettes) – (thus termed ABC).

ABC transporters

Vibrio cholerae lipid transporter – an ABC transporter (>150 ABC transporter genes in the human genome) Dimer of 62 kDa chains; N – transmembrane C – ATP binding cassette P-Loop NTPase

Eukaryotic ABC transporters generally transport molecules out of the cell ABC transporter mechanism

Secondary transporters - Lactose permease Archetype secondary transporter The thermodynamically unfavourable flow of one species of ion or molecule up a concentration gradient is driven by the favourable flow of a different species down a concentration gradient.

Secondary transporters cotransporters

Lactose permease This symporter uses the H + gradient across the E.coli membrane (outside H + has higher concentration) generated by the oxidation of fuel molecules, to drive the uptake of lactose and other sugars against a concentration gradient.

Lactose permease Lactose

Glu 269 is the likely proton acceptor Many features similar to ABC transporter (NO ATP!) Lactose permease mechanism [H + ]