生化生物化學(xué)第13章

1、Berg Tymoczko Stryer Biochemistry Sixth EditionChapter 13:Membrane Channels and PumpsOUTLINESThe Transport of Molecules Across a Membrane May Be Active or Passive Two Families of Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Membranes Lactose Permease Is an Archetype of Seco

2、ndary Transporters That Use One Concentration Gradient to Power the Formation of Another Specific Channels Can Rapidly Transport Ions Across Membranes Gap Junctions Allow Ions and Small Molecules to Flow Between Communicating Cells Specific Channels Increase the Permeability of Some Membranes to Wat

3、er Introduction The lipid bilayer is intrinsically impermeable to ions and polar molecules, yet certain such species must be able to cross these membranes for cell function. Permeability is conferred by two classes of membrane proteins, pumps and channels. Pumps use free energy such as ATP hydrolysi

4、s or light absorption to drive the thermodynamically uphill transport of ions or molecules. Channels enable ions to flow rapidly through membranes in a thermodynamically downhill direction. Introduction Pumps are energy transducers in that they convert one form of free energy into another. Two types

5、 of ATP-driven pumps, P-type ATPases and the ATP-binding cassette (ABC) transporters, undergo conformational changes on ATP binding and hydrolysis that cause a bound ion to be transported across the membrane. A different mechanism of active transport utilizes the gradient of one ion to drive the act

6、ive transport of another. Introduction Pumps can establish persistent gradients of particular ions across membranes. Specific ion channels can allow these ions to flow rapidly across membranes down these gradients. These channels are among the most fascinating molecules in biochemistry in their abil

7、ity to allow some ions to flow freely through a membrane while blocking the flow of even closely related species. These gated ion channels are central to the functioning of our nervous systems, acting as elaborately switched wires that allow the rapid flow of current. We conclude with a discussion o

8、f a different class of channel: the cell-to-cell channel, or gap junction, allows the flow of metabolites or ions between cells. For example, gap junctions are responsible for synchronizing muscle-cell contraction in the beating heart.IntroductionIntroduction The flow of ions through a single membra

9、ne channel (channels are shown in red in the illustration at the left) can be detected by the patch-clamp technique, which records current changes as the channel transits between open and closed states. 1 The Transport of Molecules Across a Membrane May Be Active or Passive Lipophilic molecules can

10、pass through cell membranes because they dissolve in the lipid bilayer. The steroid hormones provide a physiological example. These cholesterol derivatives can pass through a membrane in their path of movement, but what determines the direction in which they will move? Such molecules will pass throu

11、gh a membrane down their concentration gradient in a process called simple diffusion. In accord with the Second Law of Thermodynamics, molecules spontaneously move from higher concentration to lower concentration.1 The Transport of Molecules Across a Membrane May Be Active or Passive Ions or polar m

12、olecules are more complicated when they are transported, although they move down their concentration gradients in passive mode. How are they able to do so? Sodium ions pass through specific channels in the hydrophobic barrier formed by membrane proteins. This means of crossing the membrane is called

13、 facilitated diffusion, or passive transport. Channels, like enzymes, display substrate specificity in that they facilitate the transport of some ions, but not other, even closely related ions.1 The Transport of Molecules Across a Membrane May Be Active or Passive How is the sodium gradient establis

14、hed in the first place? In this case, sodium must move, or be pumped, against a concentration gradient. Because moving the ion from a low concentration to a higher concentration results in a decrease in entropy, it requires an input of free energy. The transporters embedded in the membrane are capab

15、le of using an energy source to move the molecule up a concentration gradient. Because an input of energy from another source is required, this means of crossing the membrane is called active transport.1 The Transport of Molecules Across a Membrane May Be Active or Passive An unequal distribution of

16、 molecules is an energy-rich condition because free energy is minimized when all concentrations are equal. Consequently, to attain such an unequal distribution of molecules requires an input of free energy. Can we quantify the amount of energy required to generate a concentration gradient?Quantifica

17、tion of Free Energy1 The Transport of Molecules Across a Membrane May Be Active or Passive Consider an uncharged solute molecule. The free-energy change in transporting this species from side 1, where it is present at a concentration of C1, to side 2, where it is present at concentration C2, is R is

18、 8.315 10-3 kJ mol-1, or 1.987 10-3 kcal mol-1) and T is in kelvins. 1 The Transport of Molecules Across a Membrane May Be Active or Passive For a charged species, the unequal distribution across the membrane generates an electrical potential that also must be considered. The sum of the concentratio

19、n and electrical terms is called the electrochemical potential or membrane potential. The free-energy change is then given by in which Z is the electrical charge of the transported species, V is the potential in volts across the membrane, and F is 96.5 kJ V-1 mol-1 (or 23.1 kcal V-1 mol-1).2 Membran

20、e Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Membranes The extracellular fluid of animal cells has a salt concentration similar to that of seawater. However, cells must control the concentrations of intracellular salt. For instance, most animal cells contain a high concentration o

21、f K+ and a low concentration of Na+ relative to the external medium. These ionic gradients are generated by an enzyme that is called the Na+-K+ pump or the Na+-K+ ATPase. The hydrolysis of ATP by the pump provides the energy needed for the active transport of Na+ out and K+ into the cell, generating

22、 the gradients. The hydrolysis of ATP takes place by this enzyme only when Na+ and K+ are present. This ATPase, like all such enzymes, requires Mg2+.2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Membranes The change in free energy panying the transport of Na+ and K+ can be

23、 calculated. Suppose that Na+ outside and inside the cell are 143 and 14 mM, respectively, and the corresponding K+ are 4 and 157 mM, the membrane potential is -50 m V and a temperature is 37, the free-energy change in transporting 3 mol of Na+ out and 2 mol of K+ into the cell is 3 (5.99) + 2(9.46)

24、 = +36.9 kJ mol-1. The hydrolysis of a single mole of ATP provides about -50 kJ mol-1 under typical cellular conditions, which is sufficient to drive the uphill transport of these ions. One mole of ATP supports 3 Na+ out and 2 K+ in.2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules A

25、cross Membranes The active transport of Na+ and K+ is of great physiological significance. Indeed, more than a third of the ATP consumed by a resting animal is used to pump these ions. The Na+ and K+ gradient in animal cells controls cell volume, renders neurons and muscle cells electrically excitab

26、le, and drives the active transport of sugars and amino acids.2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Membranes P-Type ATPases There are many evolutionarily related ion pumps identified from bacteria, archaea, and all eukaryotes. These pumps are specific for an array

27、 of ions. In addition to the Na+-K+ ATPase, Ca2+ ATPase transports Ca2+ out of the cytoplasm and into the sarcoplasmic reticulum of muscle cells, gastric H+-K+ ATPase is the enzyme responsible for pumping sufficient protons into the stomach to lower the pH below 1.0. These enzymes and the hundreds o

28、f known homologs are referred to as P-type ATPases because they form a key phosphorylated intermediate, a phosphoryl group from ATP is linked to the side chain of a specific conserved aspartate residue in the ATPase to form phosphoryl-aspartate.Figure 13.2 Pump action. A simple scheme for the pumpin

29、g of a molecule across a membrane. The pump interconverts to two conformational states, each with a binding site accessible to a different side of the membrane. 2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Membranes Membrane pumps function by mechanisms that are simple in

30、 principle but often complex in detail. Fundamentally, each pump protein can exist in two principal conformational states, one with ion-binding sites open to one side of the membrane and the other with ion-binding sites open to the other side. To pump ions in a single direction across a membrane, fr

31、ee energy must be provided and coupled to the interconversion between the two conformational states.2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Membranes SR Ca2+ ATPase, or SERCA is the Ca2+ ATPase found in the sarcoplasmic reticulum of muscle cells. This enzyme constitu

32、tes 80% of the protein in the sarcoplasmic reticulum membrane, which plays an important role in muscle contraction, a process triggered by an abrupt rise in the cytoplasmic calcium ion level. Muscle relaxation depends on the rapid removal of Ca2+ from the cytoplasm into the sarcoplasmic reticulum, a

33、 specialized compartment for Ca2+ storage, by SERCA. This pump maintains a Ca2+ concentration of approximately 0.1 mM in the cytoplasm compared with 1.5 mM in the sarcoplasmic reticulum.The structural features of P-type ATPases2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across

34、Membranes Figure 13.3 Calcium-pump structure. The crystal structures of Ca2+ pump have been determined in five different states. The first structure of SERCA to be determined had Ca2+-bound, but no nucleotides present . The two Ca2+ (green) lie in the center of the transmembrane domain. A conserved

35、aspartate residue (Asp 351) that binds a phosphoryl group lies in the P domain. The designation bb refers to backbone carbonyl groups.2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Membranes SERCA is a single 110-kd polypeptide with a transmem-brane domain consisting of 10

36、a helices. The transmembrane domain includes sites for binding two calcium ions. Each calcium ion is coordinated to seven oxygen atoms coming from a combination of side-chain glutamate, aspartate, threonine, and asparagine residues, backbone carbonyl groups, and water molecules. A large cytoplasmic

37、head-piece constitutes nearly half the molecular weight of the protein and consists of three distinct domains. N domain binds the ATP nucleotide, P domain accepts the phosphoryl group on a conserved Asp residue, and A domain serves as an actuator (執(zhí)行器), linking changes in the N and P domains to the

38、transmembrane part of the enzyme.2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Membranes Phosphorylation of P-Type ATPasesFigure 13.4 Conformational changes associated with calcium pumping. This structure (left) was determined in the absence of bound calcium and with a pho

39、sphorylaspartate analog present in the P domain. Notice how different this structure is from the calcium-bound form (right): both the transmembrane part (yellow) and the A. P. and N domains have substantially rearranged. 2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Membra

40、nes Phosphorylation of P-Type ATPases SERCA is remarkably structurally dynamic. In the structure of SERCA without bound Ca2+ and with a phosphoryl-aspartate analog present in the P domain, the N and P domains are closed around the phosphoryl-aspartate analog, and the A domain has rotated substantial

41、ly relative to its position in SERCA with Ca2+ bound and without the phosphoryl analog. Furthermore, the transmembrane part of the enzyme has rearranged substantially and the well-organized Ca2+-binding sites are disrupted. These sites are now accessible from the side of the membrane opposite the N,

42、 P, and A domains. The structural results can be combined with other studies to construct a detailed mechanism for Ca2+ pumping by SERCA. 2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Membranes Transport Mechanism of P-Type ATPases1. The catalytic cycle begins with the enz

43、yme in its unphosphorylated state with two calcium ions bound，the enzyme conformation in this state is referred as E1 with Ca2+ bound, E1-( Ca2+)2. In this conformation, SERCA can exchange calcium ions but only with calcium ions from the cytoplasmic side of the membrane. 2 Membrane Proteins Use ATP

44、Hydrolysis to Pump Ions and Molecules Across Membranes Transport Mechanism of P-Type ATPases2. The enzyme in the same conformation can bind ATP. The domains N, P, and A undergo substantial rearrangement as they close around the bound ATP, but there is no substantial conformational change in the tran

45、smembrane domain. The Ca2+ are now trapped inside the enzyme. 2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Membranes Transport Mechanism of P-Type ATPases3. The phosphoryl group is then transferred from ATP to Asp 351. 2 Membrane Proteins Use ATP Hydrolysis to Pump Ions a

46、nd Molecules Across Membranes Transport Mechanism of P-Type ATPases4. Upon ADP release, the enzyme changes its overall conformation again, including the transmembrane domain this time. This new conformation is referred to as E2 or E2-P in its phosphorylated form. The process of inter-converting the

47、E1 and E2 conformations is sometimes referred to as eversion. In the E2-P conformation, the Ca2+-binding sites e disrupted and the calcium ions are released to the side of the membrane opposite which they entered; ion transport has been achieved. 2 Membrane Proteins Use ATP Hydrolysis to Pump Ions a

48、nd Molecules Across Membranes Transport Mechanism of P-Type ATPases5. The phosphoryl aspartate residue is hydrolyzed to release inorganic phosphate. 2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Membranes Transport Mechanism of P-Type ATPases6. With the release of phosphat

49、e, the interactions stabilizing the conformation E2 are lost, and the enzyme everts back to the El conformation which can bind two calcium ions from the cytoplasmic side.2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Membranes Phosphorylation of P-Type ATPasesFigure 13.5 Pu

50、mping calcium. Ca2+ ATPase transports Ca2+ through the membrane. This mechanism likely applies to other P-type ATPases. For example, Na+-K+ ATPase is an a2b2 tetramer. Its a subunit is homologous to SERCA and includes a key aspartate residue analogous to Asp 351. The b subunit does not directly take

51、 part in ion transport. A mechanism analogous to that stated before applies, with three Na+ ions binding from the inside of the cell to the E1 conformation and two K+ ions binding from outside the cell to the E2 conformation.2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Me

52、mbranes Phosphorylation of P-Type ATPases2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Membranes Digitalis Specifically Inhibits the Na+ -K+ Pump by Blocking Its Dephosphorylation Foxglove (Digitalis purpurea) is the source of digitalis, one of the most Widely used drugs.

53、Certain steroids isolated from plants are potent inhibitors (Ki 10 nM) of the Na+-K+ pump. Digitoxigenin and ouabain are members of this class of inhibitors, which are known as cardiotonic steroids because of their strong effects on the heart. 2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and

54、Molecules Across Membranes Digitalis Specifically Inhibits the Na+ -K+ Pump by Blocking Its Dephosphorylation Figure 13.6 Digitoxigenin. Cardiotonic steroids such as digitoxigenin inhibit the Na+ -K+ pump by blocking the dephosphorylation of E2-P. These compounds inhibit the dephosphorylation of the

55、 E2-P form of the ATPase when applied on the extracellular face of the membrane. Digitalis is a mixture of cardiotonic steroids derived from the dried leaf of the foxglove plant. The compound increases the force of contraction of heart muscle and is consequently a choice drug in the treatment of con

56、gestive heart failure. Inhibition of the Na+-K+ pump by digitalis (毛地黃毒苷) leads to a higher level of Na+ inside the cell. The diminished Na+ gradient results in slower extrusion of Ca2+ by the sodium-calcium exchanger. The subsequent increase in the intracellular level of Ca2+ enhances the ability o

57、f cardiac muscle to contract. 2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Membranes Digitalis Specifically Inhibits the Na+ -K+ Pump by Blocking Its Dephosphorylation It is interesting to note that digitalis was used effectively long before the discovery of the Na+-K+ AT

58、Pase. In 1785, William Withering, a British physician, heard tales of an elderly woman, known as the old woman of Shropshire, who cured people of dropsy (積水) (which today would be recognized as congestive heart failure (充血性心力衰竭) with an extract of foxglove. Withering conducted the first scientific s

59、tudy of the effects of foxglove on congestive heart failure and documented its effectiveness.2 Membrane Proteins Use ATP Hydrolysis to Pump Ions and Molecules Across Membranes Digitalis Specifically Inhibits the Na+ -K+ Pump by Blocking Its Dephosphorylation Yeast genome revealed the presence of 16

60、members of P-type ATPases. 2 proteins transport H+ ions, 2 transport Ca2+, 3 transport Na+, and 2 transport metals such as Cu2+. In addition, there are 5 members, also called flippases, which are involved in the transport of phospholipids with amino acid head groups from the inner to the outer leafl