Membrane Transport Xia Qiang, MD & PhD Department of Physiology Room C518, Block C, Research Building, School of Medicine Tel:

Organelles have their own membranes

Cell Membrane (plasma membrane)

Membrane Transport Lipid Bilayer -- primary barrier, selectively permeable

Membrane Transport ◦ Simple Diffusion （单纯扩散） ◦ Facilitated Diffusion （易化扩散） ◦ Active Transport （主动转运） ◦ Endocytosis and Exocytosis （出胞与入胞）

Over time, solute molecules placed in a solvent will evenly distribute themselves. START: Initially higher concentration of molecules randomly move toward lower concentration. Diffusional equilibrium is the result (Part b).

At time B, some glucose has crossed into side 2 as some cross into side 1.

Note: the partition between the two compartments is a membrane that allows this solute to move through it. Net flux accounts for solute movements in both directions.

Simple Diffusion Relative to the concentration gradient movement is DOWN the concentration gradient ONLY (higher concentration to lower concentration) Rate of diffusion depends on The concentration gradient Charge on the molecule Size Lipid solubility

Facilitated Diffusion ◦ Carrier-mediated

The solute acts as a ligand that binds to the transporter protein…. A cartoon model of carrier-mediated transport. … and then a subsequent shape change in the protein releases the solute on the other side of the membrane.

In simple diffusion, flux rate is limited only by the concentration gradient. In carrier- mediated transport, the number of available carriers places an upper limit on the flux rate.

Characteristics of carrier-mediated diffusion net movement always depends on the concentration gradient ◦ Specificity ◦ Saturation ◦ Competition

–Channel-mediated 3 cartoon models of integral membrane proteins that function as ion channels; the regulated opening and closing of these channels is the basis of how neurons function.

A thin shell of positive (outside) and negative (inside) charge provides the electrical gradient that drives ion movement across the membranes of excitable cells.

The opening and closing of ion channels results from conformational changes in integral proteins. Discovering the factors that cause these changes is key to understanding excitable cells.

Characteristics of ion channels ◦ Specificity ◦ Gating （门控）

Three types of passive, non-coupled transport through integral membrane proteins

Outside Inside NH 2 CO 2 H IIIIIIIV Voltage-gated Channel –e.g. Voltage-dependent Na + channel

Na + channel

Balloonfish or fugu

Closed Activated Inactivated Na + channel conformation –Open-state –Closed-state

Ligand-gated Channel –e.g. N 2 -ACh receptor channel

Aquaporin Aquaporins are water channels that exclude ions Aquaporins are found in essentially all organisms, and have major biological and medical importance

The Nobel Prize in Chemistry 2003 "for discoveries concerning channels in cell membranes" Peter AgreRoderick MacKinnon "for the discovery of water channels" "for structural and mechanistic studies of ion channels"

The dividing wall between the cell and the outside world – including other cells – is far from being an impervious shell. On the contrary, it is perforated by various channels. Many of these are specially adapted to one specific ion or molecule and do not permit any other type to pass. Here to the left we see a water channel and to the right an ion channel.

Peter Agre ’ s experiment with cells containing or lacking aquaporin. The aquaporin is necessary for making the cell absorb water and swell

Passage of water molecules through the aquaporin AQP1. Because of the positive charge at the center of the channel, positively charged ions such as H3O+, are deflected. This prevents proton leakage through the channel.

Model for water permeation through aquaporin

In both simple and facilitated diffusion, solutes move in the direction predicted by the concentration gradient. In active transport, solutes move opposite to the direction predicted by the concentration gradient. membrane

Active transport Primary active transport （原发性主动转运） Secondary active transport （继发性主动转运）

Primary Active Transport making direct use of energy derived from ATP to transport the ions across the cell membrane

Concentration gradient of Na + and K + Extracelluar (mmol/L) Intracellular (mmol/L) Na K

Here, in the operation of the Na + -K + -ATPase, also known as the “sodium pump,” each ATP hydrolysis moves three sodium ions out of, and two potassium ions into, the cell.

Na-K Pump

electrogenic pump （生电性泵） Na + -K + pump (Na + pump, Na + -K + ATPase)

Physiological role of Na + -K + pump –Maintaining the Na + and K + gradients across the cell membrane –Partly responsible for establishing a negative electrical potential inside the cell –Controlling cell volume –Providing energy for secondary active transport

Other primary active transport Primary active transport of calcium Primary active transport of hydrogen ions ……

Secondary Active Transport The ion gradients established by primary active transport permits the transport of other substances against their concentration gradients

Secondary active transport uses the energy in an ion gradient to move a second solute.

Cotransport （同向转运） the ion and the second solute cross the membrane in the same direction (e.g. Na + -glucose, Na + -amino acid cotransport) Countertransport （逆向转运） the ion and the second solute move in opposite directions (e.g. Na + -Ca 2+, Na + -H + exchange)

Cotransporters

Exchangers

Ion gradients, channels, and transporters in a typical cell

Here, water is the solvent. The addition of solute lowers the water concentration. Addition of more solute would increase the solute concentration and further reduce the water concentration. Solvent + Solute = Solution Osmosis （渗透）

Begin: The partition between the compartments is permeable to water and to the solute. After diffusional equilibrium has occurred: Movement of water and solutes has equalized solute and water concentrations on both sides of the partition.

Begin: The partition between the compartments is permeable to water only. After diffusional equilibrium has occurred: Movement of water only has equalized solute concentration.

Role of Na-K pump in maintaining cell volume

Response to cell shrinking

Response to cell swelling

Endocytosis and Exocytosis

Alternative functions of endocytosis: 1.Transcellular transport 2.Endosomal processing 3.Recycling the membrane 4.Destroying engulfed materials

Endocytosis

Exocytosis

Two pathways of exocytosis Constitutive exocytosis pathway -- Many soluble proteins are continually secreted from the cell by the constitutive secretory pathway Regulated exocytosis pathway -- Selected proteins in the trans Golgi network are diverted into secretory vesicles, where the proteins are concentrated and stored until an extracellular signal stimulates their secretion

Steps to exocytosis Vesicle trafficking: In this first step, the vesicle containing the waste product or chemical transmitter is transported through the cytoplasm towards the part of the cell from which it will be eliminated Vesicle tethering: As the vesicle approaches the cell membrane, it is secured and pulled towards the part of the cell from which it will be eliminated Vesicle docking: In this step, the vesicle comes in contact with the cell membrane, where it begins to chemical and physically merge with the proteins in the cell membrane Vesicle priming: In those cells where chemical transmitters are being released, this step involves the chemical preparations for the last step of exocytosis Vesicle fusion: In this last step, the proteins forming the walls of the vesicle merge with the cell membrane and breach, pushing the vesicle contents (waste products or chemical transmitters) out of the cell. This step is the primary mechanism for the increase in size of the cell's plasma membrane

Epithelial Transport （上皮转运）

Measurement of voltage in an epithelium

Models of epithelial solute transport

Mechanisms of intestinal glucose absorption

Models of “isotonic” water transport in a leaky epithelium

Glands

Case Hereditary Spherocytosis ( 遗传性球形红细胞增多症 ) A 20-year-old woman suffers from anemia and occasional jaundice. A thorough review of her medical records reveals that over the past l0 years she has had episodes of more severe anemia, usually after periods of febrile illness. The patient has a markedly enlarged spleen. Microscopic examination of the patient's blood showed a large number of microspherocytes (red blood cells [RBCs] that are round and somewhat smaller than erythrocytes). The osmotic fragility (measured by putting RBCs in hypotonic solutions) was much greater than that of RBCs from healthy individuals. When the patient's erythrocytes were incubated in a buffer solution at 37  C under sterile conditions, the fraction of the RBCs that were hemolyzed was much larger than the hemolyzed fraction from a healthy individual. This "autohemolysis" could be greatly diminished by including glucose and adenosine triphosphate (ATP) in the RBC incubation solution. RBCs from fresh blood had a normal content of Na+ and K+. The permeabilities of the patient's erythrocyte membranes to Na+ and K+ were found to be about three times normal. The level of Na+, K+-ATPase in the patient's RBC membranes was also about three times the level in RBCs from healthy individuals. The average life span of the patient's erythrocytes was well below the normal life span. When an aliquot of the patient's RBCs was labeled and injected intravenously into a healthy individual, the patient's RBCs had a markedly reduced survival time compared with normal RBCs. When labeled RBCs from a healthy individual were infused into the patient, the survival time of the normal RBCs was comparable with their survival time in the donor. The patient's spleen was removed, and after the splenectomy, the patient's anemia was largely ameliorated.

Questions l. Why should the patient's erythrocytes have a greater osmotic fragility than RBCs from healthy individuals? 2. Why might the patient's RBCs "autohemolyze" more rapidly than normal erythrocytes when they are incubated at 37  C under sterile conditions? 3. Why should including glucose and ATP in the incubation mixture diminish the extent of autohemolysis? 4. Why should the patient's RBCs have a reduced life span? What might the spleen have to do with this? 5. What is proved by the observations that the patient's RBCs have a reduced life span in the circulation of a healthy individual and that the RBCs of a healthy individual have a normal life span in the patient's circulation? 6. Why might the patient have more severe episodes of anemia following febrile illnesses? 7. Why should splenectomy largely correct the patient's anemia?

Answers l. The spherical shape of the patient's red blood cells (RBCs) would be expected to lead to greater osmotic fragility. When a normal RBC hemolyzes, it first swells to an approximately spherical shape. Any attempt to increase its volume beyond this point causes hemolysis because membranes cannot stretch appreciably (although they can bend). In hemolysis focal microscopic "breaks" in the membrane allow the internal contents of the RBC to equilibrate with the extracellular fluid. The patient's RBCs are already spherical, so they cannot first swell to become spherical when placed in a hypotonic solution. The spherocytes thus hemolyze more readily when put in hypotonic solutions. 2. A major way in which cells prevent osmotic lysis is by pumping Na+ out of the cell by the Na+, K+- ATPase. The Na+ gradient helps to counterbalance the osmotic gradient in the other direction because of intracellular impermeant substances and permeant ions that are in equilibrium across the membrane. When RBCs are incubated at 37  C, in the absence of substrates for producing energy, ATP levels in the cell decrease. Eventually the pumping fates of Na+ and K+ diminish. Na+ leaking into the cell down its electrochemical gradient transfers osmotic equivalent to the RBC interior. This process leads to swelling, and ultimately to lysis, of the RBC. Because the patient's RBCs are three times more permeable to Na+ than normal, the swelling and osmotic lysis occur more rapidly. Moreover, the hemolysis of the spherical RBCs occurs more readily than hemolysis of normal RBCs for the reasons discussed in the answer to question l. 3. The patient's RBCs are aided in resisting swelling and hemolysis by having an elevated level of Na+,K+-ATPase in their plasma membranes. Thus when Na+ leaks into the cells at an elevated rate (compared with normal cells), it can be pumped back out of the cell at a similarly elevated rate by the greater number of Na+, K+-ATPase molecules. When the ATP level falls during incubation, the Na+, K+-ATPase is no longer able to keep up with Na+ influx. Cell swelling and lysis result. Providing the RBCs with ATP and with glucose (from which the cells can make ATP) allows the Na+, K+- ATPase molecules to keep pumping Na+ out of the cell at elevated rates that compensate for the elevated rate of Na+ leak into the cell. In this way glucose and ATP help to prolong the time of incubation that the patient's cells can undergo before the onset of appreciable, hemolysis. The ability of glucose and ATP to prevent an elevated rate of autohemolysis is one of the best diagnostic criteria of the disease known as hereditary spherocytosis. This criterion helps to distinguish hereditary spherocytosis from other microcytic anemias. 4. Flexibility of erythrocytes is required for them to deform sufficiently to pass through the narrow slit in the basement membrane that separates the splenic cords from the venous sinuses of the spleen. The patient's spherical RBCs are less deformable than normal discoidal RBCs. The patient's RBCs are thus retained in the splenic cords to a greater extent than normal. Response to this engorgement of splenic cords is believed to contribute to the splenomegaly observed in patients with hereditary spherocytosis. While the patient's RBCs are delayed in the splenic cords, they tend to deplete their glucose and then their ATP levels, which results in osmotic hemolysis by the mechanisms described earlier. Moreover, splenic macrophages engulf and destroy RBCs retained in the splenic cords. 5. These observations prove that there is no defect in the patient's spleen but rather that the defect is in the patient's RBCs. 6. The patient's decreased RBC life span, and the resultant anemia, are partially compensated by an increased rate of erythropoiesis. In a febrile illness, the rate of erythropoiesis is decreased, leading to transiently increased anemia. 7. A major contributor to the shortened life span of the patient's RBCs is the increased rate of destruction of RBCs in the patient's spleen, for the reasons discussed in the answer to question 4. Removing the patient's spleen dramatically prolongs the life of the average microspherocyte and thus leads to a marked improvement in the patient's anemia.

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