1. Chapter 13: Membrane Channels and Pumps Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition

2. Types of Transport

3. Simple Diffusion Simple diffusion moves molecules across a membrane with a concentration gradient. The rate of diffusion varies linearly with concentration and the diffusion constant of the given molecule. Diffusion Constants Water5 x 10 -3 Urea3 x 10 -6 Glycerol3 x 10 -6 Glucose5 x 10 -8 Cl - 7 x 10 -10 K + 5 x 10 -12 Na + 1 x 10 -12

4. Passive Mediated This method also transports with a gradient but requires a protein to mediate transport across the membrane and differs from simple diffusion by: rate of diffusion and specificity exhibits saturation behavior susceptable to inhibition susceptable to inactivation Although ionophores are different they behave in this manner also.

5. Active Transport This method moves molecules against a gradient and requires a protein to mediate transport across the membrane as well as an energy source which may be ATP. The protein may effect movement in one of three ways: Uniport moves one molecule in either direction. Symport moves two molecules in the same direction. Antiport moves two molecules in opposite direction.

6. Types of Transport Proteins These are gated systems for active transport

7. Ionophores Carriers or channel formers that transport ion. 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.

8. Valinomycin

9. 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 end to end to span the membrane and K + ions flow through the core of the helix. Transports K + at a rate of 10 7 ions/sec.

10. Gramicydin The dimer has adjacent N-terminal residues. H-bonds are like parallel beta sheet. K + ions flow through the hollow core.

11. Membrane Transport Site Requires sat’d Gradient energy (1) Simple diffusion No withNo (2) Channels and pores Less withNo (3) Passive transporters Yes withNo (4) Active transporters Yes againstYes (2) requires an integral peptide or carrier, is limited by rate of diffusion and molecular size. (3) & (4) involve integral proteins.

12. Free Energy of Transport  G transport =  G chem. +  G elect.  G for movement from inside a membrane to outside due to a concentration gradient (chem.): [C out ]  G transport = 2.3 RT log ------ [C in ]  G for movement from inside a membrane to outside due to a potential gradient (charge):  G transport = zF  The potential inside = (-) and outside = (+).

13. Thermodynamics, cont. When both a concentration gradient and a charge gradient are involved the equation is: [C out ]  G transport = 2.3 RT log ------- + zF  [C in ] , the membrane potential, is the charge difference across the membrane in volts, z is the charge on the species being moved and F is Faraday’s constant, 96500 joules/volt.

14.  G Calculation Concentration gradient only: Assume that “A” is 0.05 mM extracellular and that the intracellular concentration is maintained at 15 mM at 37 o C. For movement outside to inside:  G = RT ln Ci /C o = 8.314(310) ln 15 x 10 -3 /0.05 x 10 -3 = 2577 (ln 300) = 2577 (5.7) = 14700 J/mol or 14.7 kJ/mol (  G is (+) so this energy must be provided in order to move 1 mol of “A”)

15.  G Calculation Assume that ATP is available to provide energy for this transport.  G o' = -30.5 kJ/mol Available concentrations: ATP = 2.5 mM; ADP = 1.5 mM and Pi = 0.5 mM  G =  G o' + RT ln ([ADP][Pi]/[ATP])  G = -30500 + 2577ln ([1.5x10 -3 ][0.5x10 -3 ]/[2.5x10 -3 ])  G = -30500 + 2577 ln (3 x 10 -4 )  G = -30500 + 2577 (-8.111) = -30500 - 20900  G = -51.4 kJ/mol There is sufficient energy from 1 mol of ATP hydrolysis to move 3 mols of “A”.

16.  G Calculation Membrane potential: What membrane potential would be needed to move 1 mol of “A” ?  G = zF  14700 = 1(96480)   = 14700/96480 = 0.152 V = 152 mV

17.  G Calculation Normal membrane potentials range from ~ 60 mV to 100 mV. Assume a membrane potential of 60 mV. What intracellular concentration of “A” could be reached if driven by this potential ? zF  = RT ln C i /C o 1(96480)(0.06) = 2577 (ln C i /0.05 x 10 -3 ) 5789 / 2577 = 2.246 = ln C i /0.05 x 10 -3 9.454 = C i /0.05 x 10 -3 and C i = 4.72 x 10 -4 Therefore, a potential difference of 60 mV could not maintain the desired 15 mM conc.

18.  G transport vs C 2 /C 1

19.  G transport vs (  2  1 )

20. Na + /K + Pump  G Calculation 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 = (+).

21. Na + /K + Pump  G Calculation For Na + moving in to out at 37 o C:  G = RT ln C o /Ci + 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 = 5846 + 6754 = 12600 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.

22. Na + /K + Pump  G Calculation 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 = 8765 - 6754 = 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

23. Na + /K + Pump  G Calculation Total energy required for transport:  G = 37.8 + 4.02 = 41.82 kJ This occurs concurrent with hydrolysis of 1 ATP. At normal physiological concentrations the  G for ATP hydrolysis is ~ -51 kJ/mol, so sufficient energy is available.

24. Typical Pumping Scheme

25. Types of Pumps Primary Pumps (direct energy source, e.g. ATP): P-type ATPases (use energy from ATP and involve phosphoryation of the transport protein): Na + /K + ATPase, Ca ++ ATPase, H + /K + ATPase ABC ATPases (use energy from ATP but no phosphorylation): Small molecule pump Secondary pumps (no ATP, e.g. ion gradient): H + /lactose - lactose permease in E.coli Na + /glucose – plasma membrane ATP/ADP transporter - mitochondria

26. Ca ++ ATPase The Ca ++ pump in the sarcoplasmic reticulum (SR) is a primary transport (75%). Muscle resting state: Ca ++ SR = 0.1 μM Muscle contraction, Ca ++ released to cytosol Ca ++ cyt = 15 mM Ca ++ levels must be reduced to relax muscle Reaction: 2 Ca ++ cytosol + ATP 2 Ca ++ SR + ADP + Pi

27. Ca ++ ATPase The transporter is a monomeric 110 kD protein and binds 2 Ca ++. It cycles between two major conformations, E 1 and E 2, has 10 transmembrane helices and 4 domains. Transmembrane domainbinds 2 Ca ++ N domainbinds ATP (nucleotide) P domainaccepts P at Asp 351 A domainactuator The pump is regulated by calmodulin which activates the pump at high cytosolic Ca ++ levels. K M of the ATPase goes from 20 μM to 0.5 μM. Calmodulin binds 4 Ca ++, two in each domain.

28. Ca ++ ATPase, events E 1 is open to the cytosol. 2 Ca ++ are bound from the cytosol. E 1  2 Ca ++ then ATP binds which traps the Ca ++. Phosphorylation of Asp 351 occurs using ATP (needs Mg ++ ) and E 1 ~P  2 Ca ++  ADP is formed. ADP is then released triggering a conformational change to E 2 ~P  2 Ca ++.

29. Ca ++ ATPase, events E 2 is open to the SR. E 2 ~P  2 Ca ++ does not bind Ca ++ well and the 2 Ca ++ are released to the SR giving E 2 ~P. Dephosphorylation of E 2 ~P occurs giving E 2 and Pi. This results in a conformational change back to E 1 and the cycle starts again.

30. Ca ++ Pump 0.1  M in cytoplasm 1.5 mM in sarcoplasmic ret.

31. Sarcoplasmic reticulum Ca ++ ATPase Noncovalent binding of Ca ++ (green) (SERCA)

33. Na + /K + ATPase, events The Na + /K + pump in the plasma membrane is an antiporter and is a primary transport system: 3 Na + in + 2 K + out + ATP 3 Na + out + 2 K + in + ADP + Pi The transporter has 2 types of subunits (α = ~120 kD and β = ~35 kD), is an α 2 β 2 tetramer and cycles between two major conformations, E 1 and E 2. It has 10 transmembrane helices. The mechanism is reported to be ordered sequential.

34. Na + /K + ATPase, events E 1 is open to the inside, has a high affinity for Na + (K M = 0.2 mM) and poor binding of K +. So, 3 Na + are bound from inside the cell. E 1 also has a high affinity for ATP and it binds. Phosphorylation of Asp 369 occurs only in presence of Na + and ATP (needs Mg ++ ). After phosphorylation the 3 Na + are tightly bound and ADP leaves. The E 1 ~P  3 Na + complex then causes a conformational change to produce E 2 ~P  3 Na +.

35. Na + /K + ATPase, events E 2 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 the cell and E 2 ~P binds 2 K + from outside forming E 2 ~P  2 K +. Hydrolysis of Asp-P occurs only in presence of K +. Dephosphorylation occurs giving E 2  2 K + and Pi A conformational change resuts giving to E 1  2 K + 2 K + is then released to the inside and the cycle starts again.

36. ABC ATPase ATP binding casette (ABC) transporters move small molecules across a membrane and vary in specificity for substrate. They bind ATP and use the energy of ATP hydrolysis for transport but do not phosphorylate the transporter. Most are found in the plasma membrane and are pumps but some have been shown to be ion channels. They are members of a superfamily of transporters that represents the oldest line of transporters known.

37. ABC ATPase The ABC transporters typically contain two membrane spanning domains and two ATP binding casettes (nucleotide binding domains - NBDs) where binding and hydrolysis of ATP occurs. Some are single polypeptide chains, others are dimers each containing a membrane spanning domain of about six transmembrane helices and an NBD. These proteins contain p-loops as do the NMP kinases. The amino acid residues in the P-loop interact with the phosphate groups of the bound nucleotide.

38. ABC ATPase E 1 is open to the inside of the cell and binds substrate E 1  S. E 1  S changes conformation, retains substrate but allows 2 ATPs to bind: E 1  S  2 ATP. Binding of ATP induces a conformational change to E 2  S  2 ATP. E 2 is open to the outside of the cell and S exits outside, leaving E 2  2 ATP. Hydrolysis of ATP then occurs and both ADP and Pi leave. E 2 reverts to E 1 and the cycle begins again.

40. ABC ATPase

41. H + /Lactose Transporter Lactose permease in E. Coli is a symporter and is a secondary transport system. It is another transporter that exhibits two states. E 1 has high lactose affinity and is open to the outside of the cell. E 2 has low lactose affinity and is open to the inside of the cell. Lactose permease has two membrane spanning domains each containing six transmembrane helices. There is some evidence to indicate that this is a random multisubstrate process.

42. H + /Lactose Transporter E 1 is open to the outside and binds a proton (Glu 269 ?) giving E 1 ~H. Then lactose binds from the outside to form E 1 ~H  Lactose and the E 1 sites are full. A conformational change occurs to give E 2 ~H  Lactose. E 2 is open to the inside, so, E 2 ~H  Lactose loses lactose giving E 2 ~H. A proton is then lost to the inside and the E 2 sites are empty. A conformational change occurs back to E 1 and the cycle starts again.

43. Lactose Permease

44. End of Chapter 13 Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition