Reginald H. Garrett Charles M. Grisham Chapter 9 Membranes and Membrane Transport

Transport Energy input drives active transport. Primary active transport is driven by ATP. Some transport processes are driven by light energy. Secondary active transport is driven by ion gradients.

9.8 How Does Energy Input Drive Active Transport Processes? Energy input drives transport In active transport, solutes flow against their thermodynamic potential (against a concentration and/or charge gradient). Energy input drives such transport. Energy source and transport machinery are "coupled". Energy source may be ATP, light or a concentration gradient.

The Sodium Pump aka Na + /K + -ATPase This is a large protein with 120 kD α-subunits and 35 kD β-subunits. It is an α 2 β 2 tetramer. Maintains intracellular Na + low and K + high. Crucial for all organs, but especially for neural tissue and the brain. ATP hydrolysis drives Na + out and K + in. Alpha subunit has ten transmembrane helices with large cytoplasmic domain.

Na +,K + -ATPase Uses ATP Energy to Drive Sodium and Potassium Transport Figure 9.48 Schematic (a) and structure (b) of Na +,K + -ATPase. inside outside

Na +,K + -ATPase Uses ATP Energy to Drive Sodium and Potassium Transport Figure 9.49 A mechanism for Na +,K + -ATPase. The model assumes two principal conformations, E 1 and E 2. Binding of Na + ions to E 1 is followed by phosphorylation and release of ADP. ATP hydrolysis occurs via an E-P intermediate. Mechanism involves two enzyme conformations known as E 1 and E 2. Cardiac glycosides inhibit by binding to outside. inside outside

Na +,K + -ATPase

The Sodium Pump Steps in transport by the Na + /K + -ATPase E1 is open to the inside, has a high affinity for Na + (K M = 0.2 mM) and poor binding of K +. So, 2K + are released and 3 Na + are bound on the inside. E1 also has a high affinity for ATP which binds. Phosphorylation of Asp 369 occurs only in presence of Na + and ATP (needs Mg ++ ). After phosphorylation the 3 Na + are tightly bound, ADP leaves and the E1~P3 Na + complex changes conformational to E2~P3 Na +.

The Sodium Pump Steps in transport by the Na + /K + -ATPase E2 is open to the outside, has a high affinity for K + (K M = 0.05 mM) and poor binding of Na +. 3 Na + are released to the outside and E2 ~P binds 2 K + forming E2 ~P2 K +. Hydrolysis of aspartyl-P occurs only in presence of K +. Dephosphorylation then occurs giving E22 K + and Pi. The loss of Pi results in a conformational change back to E12 K +. 2 K + is released to the inside and the cycle starts again.

Free Energy of the Na +,K + -ATPase The Na + /K + pump: 3 Na + in 3 Na + out 2 K + out 2 K + in Approx conc.: Na + out = 145 mM Na + in = 15 mM K + out = 5 mM K + in = 150 mM  = 70 mV The potential inside = (-) and outside = (+).

Free Energy of the Na +,K + -ATPase For Na + moving in to out at 37 o C:  G = RT ln C o /C i + ZF  = 8.314(310) ln 145/15 + 1(96480)(0.070) Note: Na + is moving from a region of (-) charge to a region of (+) charge which is energetically unfavorable and this term will contribute to a (+)  G so the membrane potential is (+).  G = = J/mol or 12.6 kJ/mol  G is (+) so this energy must be provided to move 1 mol Na +, and for 3 mol of Na + = 37.8 kJ.

Free Energy of the Na +,K + -ATPase For K + at 37 o C:  G = RT ln C i /C o + ZF  = 8.314(310) ln 150/5 + 1(96480)(-0.070) Note: K + is moving from a region of (+) charge to a region of (-) charge which is energetically favorable and this term will contribute (-) to  G so the membrane potential is (-).  G = = 2011 J/mol or 2.01 kJ/mol  G is (+) so this energy must be provided to move 1 mol K +, and for 2 mol of K + = 4.02 kJ

Free Energy of the Na +,K + -ATPase Total energy required for transport:  G = = kJ This occurs concurrent with hydrolysis of 1 mol ATP. At normal physiological concentrations the  G for ATP hydrolysis is ~ -51 kJ/mol. Therefore, one concludes that sufficient energy is available to run this pump.

Calcium Transport Is Accomplished in the Sarcoplasmic Reticulum by Ca 2+ -ATPase A process similar to Na +,K + transport Calcium levels in resting muscle cytoplasm are maintained low by Ca 2+ -ATPase (a Ca 2+ pump). Calcium is pumped into the sarcoplasmic reticulum (SR) by a 110 kD protein that is very similar to the alpha subunit of Na,K-ATPase. Aspartyl phosphate E-P intermediate is at Asp 351 and Ca 2+ -pump also fits the E 1 -E 2 model.

Calcium Transport Is Accomplished in the Sarcoplasmic Reticulum by Ca 2+ -ATPase Figure 9.51 The transport cycle of the sarcoplasmic reticulum Ca 2+ -ATPase involves at least five different conformations of the protein, represented by the blue- shaded boxes here. Sarcoplasmic reticulum Cytoplasm

The Gastric H +,K + -ATPase The enzyme that keeps the stomach at pH 0.8 The parietal cells of the gastric mucosa (lining of the stomach) have an internal pH of 7.4. H +,K + -ATPase pumps protons from these cells into the stomach (using energy of ATP) to maintain a pH difference across a single plasma membrane of 6.6 ! This is the largest known transmembrane gradient in eukaryotic cells.

The Gastric H +,K + -ATPase Maintains the Low pH of the Stomach Figure 9.52 The H +,K + -ATPase and a K + /Cl - cotransport system work together to achieve net transport of H + and Cl -.

The Gastric H +,K + -ATPase H +,K + -ATPase is similar in many respects to Na +,K + -ATPase and Ca 2+ -ATPase. All three enzymes form covalent E-P intermediates (P-type pumps). All three have similar sequences for the large (α) subunit. All three are involved in active transport.

ABC Transporters Use ATP to Drive Import and Export Functions and Multidrug Resistance Cells “clean house” with membrane transporters known as multidrug resistance (MDR) pumps. MDR pumps are designed to recognize foreign organic molecules in cells and pump them out. Among these are the ABC transporters, some export therapeutic drugs from cancer cells, others import nutrients. In bacteria, these pumps are used to import nutrients into the cell. ABC transporters use the hydrolytic energy of ATP do not phosphorylate the enzyme.

ABC Transporters Use ATP to Drive Import and Export Functions and Multidrug Resistance All ABC transporters consist of two transmembrane domains (TMDs) which form the pore and two cytosolic nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. ABC transporters contain p-loops in the NBDs that interact with the phosphates of ATP. Bacterial ABC transporters are multimeric (importers tend to be tetrameric and exporters dimeric). Eukaryotic ABC pumps are monomeric.

ABC Transporters Use ATP to Drive Import and Export Functions and Multidrug Resistance Figure 9.54 Influx pumps in the inner membrane of Gram- negative bacteria bring nutrients into the cell; efflux pumps export cellular waste products.

ABC Transporters Use ATP to Drive Import and Export Functions and Multidrug Resistance Figure 9.56 Several ABC transporters are shown in different stages of their transport cycles. MBP = multidrug binding protein.

9.9 How Are Certain Transport Processes Driven by Light Energy? Bacteriorhodopsin is a light-driven proton pump Protein opsin and retinal chromophore Retinal is bound to opsin via a Schiff base linkage. The Schiff base (at Lys 216 ) can be protonated, and this site is one of the sites that participate in H + transport. The carboxyl groups of Asp 85 and Asp 96 also serve as proton binding sites during transport. These Asp residues lie in hydrophobic environments. Their carboxyl pK a values are near 11.

9.9 How Are Certain Transport Processes Driven by Light Energy? Figure 9.57 The Schiff base linkage between the retinal chromophore and Lys 216.

9.9 How Are Certain Transport Processes Driven by Light Energy? Lys 216 is buried in the middle of the 7-TMS structure of bR, and retinal lies mostly parallel to the membrane and between the helices. Light absorption converts retinal from all-trans to 13-cis configuration, triggering conformation changes that induce pK a changes. This facilitates proton transfers from Asp 96 to the Lys Schiff base to Asp 85 and net proton transport across the membrane. The transmembrane proton hopping causes cis- retinal to convert back to the trans form.

9.10 How Is Secondary Active Transport Driven by Ion Gradients? The gradients of H +, Na + and other cations and anions established by ATPases can be used for secondary active transport of various substrates. Many amino acids and sugars are accumulated by cells in transport processes driven by Na + and H + gradients. Many of these are symports, with the ion and the transported amino acid or sugar moving in the same direction. In antiport processes, the ion and the transported species move in opposite directions.

AcrB is a Secondary Transport System AcrB is the major MDR transporter in E. coli. It is responsible for pumping a variety of molecules. AcrB is part of a tripartite complex that bridges the E. coli inner and outer membranes and spans the entire periplasmic space. AcrB works with AcrA and TolC to transport drugs and other toxins from the cytoplasm across the entire cell envelope and into the extracellular medium using energy from a proton gradient.

AcrB is a Secondary Transport System Figure 9.59 A tripartite complex of proteins comprises the large structure in E. coli that exports waste and toxin molecules. The transport pump is AcrB, embedded in the inner membrane.

AcrB is a Secondary Transport System AcrB is a secondary active transport system and a H + -drug antiporter. As protons flow spontaneously inward through AcrB in the E. coli inner membrane, drug molecules are driven outward. Remarkably, the three identical subunits of AcrB adopt slightly different conformations, denoted loose (L), tight (T), and open (O). These three conformations are three consecutive states of a transport cycle. As each monomer cycles through L, T, and O states, drugs enter tunnel, are bound and then exported.

AcrB is a Secondary Transport System Figure 9.60 In the AcrB trimer, the three identical subunits adopt three different subunits. Possible transport paths of drugs through the tunnels are shown in green.

AcrB is a Secondary Transport System Figure 9.61 A model for drug transport by AcrB involves three different conformations.

Ionophores Ionophores are carriers or channel formers that transport ions. Carrier:Valinomycin – a cyclic depsipeptide. It has 12 residues and the bonding arrangement alternates -ester-peptide-ester-peptide- (-L-Val-D-hydroxyisoVal-D-Val-L-Lactate-) 3 Carries K + in the center of the cyclic structure. Transports K + at a rate of 10 4 ions/sec.

Valinomycin

Ionophores Channel former: Gramicydin – a helical peptide. It has 15 residues that alternate in stereochemistry except for one Gly. Formyl-V-G-A-L-A-V-V-V-W-L-W-L-W-L-W-ethanolamine L L D L D L D L D L D L D L Has mostly non-polar sidechains. It dimerizes N-term to N-term to span the membrane and K + ions flow through the core of the helix. Transports K + at a rate of 10 7 ions/sec.

Gramicydin The dimer has adjacent N-terminal residues. Not an α-helix. It is more like a cylinder of parallel beta sheet. H- bonds are like those in parallel beta sheet. K + ions flow through the hollow core.

End Chapter 9 Membranes and Membrane Transport