3.23: Diffusion, Active Transport and Membrane Channels - Biology

3.23: Diffusion, Active Transport and Membrane Channels - Biology

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Transport Across Cell Membranes

All cells acquire the molecules and ions they need from their surrounding extracellular fluid (ECF). There is an unceasing traffic of molecules and ions in and out of the cell through its plasma membrane (Examples: glucose, (Na^+), (Ca^{2+})). In eukaryotic cells, there is also transport in and out of membrane-bounded intracellular compartments such as the nucleus, endoplasmic reticulum, and mitochondria (Examples: proteins, mRNA, (Ca^{2+}), and ATP).

The following problems can occur during transport:

1. Relative concentrations

Molecules and ions move spontaneously down their concentration gradient (i.e., from a region of higher to a region of lower concentration) by diffusion. Molecules and ions can be moved against their concentration gradient, but this process, called active transport, requires the expenditure of energy (usually from ATP).

2. Lipid bilayers are impermeable to most essential molecules and ions.

The lipid bilayer is permeable to water molecules and a few other small, uncharged, molecules like oxygen (O2) and carbon dioxide (CO2). These diffuse freely in and out of the cell. The diffusion of water through the plasma membrane is of such importance to the cell that it is given a special name - osmosis. Lipid bilayers are not permeable to ions such as K+, Na+, Ca2+ (called cations because when subjected to an electric field they migrate toward the cathode [the negatively-charged electrode]) and Cl-, HCO3- (called anions because they migrate toward the anode [the positively-charged electrode]). They are also not permeable to small hydrophilic molecules like glucose and macromolecules like proteins and RNA. The cells solve the problem of transporting ions and small molecules across their membranes with the help of the following two mechanisms:

  • Facilitated diffusion: Transmembrane proteins create a water-filled pore through which ions and some small hydrophilic molecules can pass by diffusion. The channels can be opened (or closed) according to the needs of the cell.
  • Active transport: Transmembrane proteins, called transporters, use the energy of ATP to force ions or small molecules through the membrane against their concentration gradient.

Facilitated Diffusion of Ions

Facilitated diffusion of ions takes place through proteins, or assemblies of proteins, embedded in the plasma membrane. These transmembrane proteins form a water-filled channel through which the ion can pass down its concentration gradient. The transmembrane channels that permit facilitated diffusion can be opened or closed. They are said to be "gated"; some types of gated ion channels:

  • ligand-gated
  • mechanically-gated
  • voltage-gated
  • light-gated

Ligand-gated ion channels

Many ion channels open or close in response to binding a small signaling molecule or "ligand". Some ion channels are gated by extracellular ligands; some by intracellular ligands. In both cases, the ligand is not the substance that is transported when the channel opens.

External ligands

External ligands (shown here in green) bind to a site on the extracellular side of the channel.


  • Acetylcholine (ACh). The binding of the neurotransmitter acetylcholine at certain synapses opens channels that admit Na+ and initiate a nerve impulse or muscle contraction.
  • Gamma amino butyric acid (GABA). Binding of GABA at certain synapses — designated GABAA — in the central nervous system admits Cl- ions into the cell and inhibits the creation of a nerve impulse

Internal ligands

Internal ligands bind to a site on the channel protein exposed to the cytosol. Examples:

  • "Second messengers", like cyclic AMP (cAMP) and cyclic GMP (cGMP), regulate channels involved in the initiation of impulses in neurons responding to odors and light respectively.
  • ATP is needed to open the channel that allows chloride (Cl-) and bicarbonate (HCO3-) ions out of the cell. This channel is defective in patients with cystic fibrosis. Although the energy liberated by the hydrolysis of ATP is needed to open the channel, this is not an example of active transport; the ions diffuse through the open channel following their concentration gradient.

Mechanically-gated ion channels

Sound waves bending the cilia-like projections on the hair cells of the inner ear open up ion channels leading to the creation of nerve impulses that the brain interprets as sound. Mechanical deformation of the cells of stretch receptors opens ion channels leading to the creation of nerve impulses.

Voltage-gated ion channels

In so-called "excitable" cells like neurons and muscle cells, some channels open or close in response to changes in the charge (measured in volts) across the plasma membrane. For example, as an impulse passes down a neuron, the reduction in the voltage opens sodium channels in the adjacent portion of the membrane. This allows the influx of (Na^+) into the neuron and thus the continuation of the nerve impulse. Some 7000 sodium ions pass through each channel during the brief period (about 1 millisecond) that it remains open. This was learned by use of the patch clamp technique.

The Patch Clamp Technique

The properties of ion channels can be studied by means of the patch clamp technique. A very fine pipette (with an opening of about 0.5 µm) is pressed against the plasma membrane of either an intact cell or the plasma membrane can be pulled away from the cell and the preparation placed in a test solution of desired composition. Current flow through a single ion channel can then be measured.

Such measurements reveal that each channel is either fully open or fully closed; that is, facilitated diffusion through a single channel is "all-or-none". This technique has provided so much valuable information about ion channels that its inventors, Erwin Neher and Bert Sakmann, were awarded a Nobel Prize in 1991.

Facilitated Diffusion of Molecules

Some small, hydrophilic organic molecules, like sugars, can pass through cell membranes by facilitated diffusion. Once again, the process requires transmembrane proteins. In some cases, these — like ion channels — form water-filled pores that enable the molecule to pass in (or out) of the membrane following its concentration gradient.


Maltoporin. This homotrimer in the outer membrane of E. coli forms pores that allow the disaccharide maltose and a few related molecules to diffuse into the cell.

Example: The plasma membrane of human red blood cells contain transmembrane proteins that permit the diffusion of glucose from the blood into the cell.

Note that in all cases of facilitated diffusion through channels, the channels are selective; that is, the structure of the protein admits only certain types of molecules through. Whether all cases of facilitated diffusion of small molecules use channels is yet to be proven. Perhaps some molecules are passed through the membrane by a conformational change in the shape of the transmembrane protein when it binds the molecule to be transported.

In either case, the interaction between the molecule being transported and its transporter resembles in many ways the interaction between an enzyme and its substrate.

Active Transport

Active transport is the pumping of molecules or ions through a membrane against their concentration gradient. It requires a transmembrane protein (usually a complex of them) called a transporter and energy. The source of this energy is ATP.

The energy of ATP may be used directly or indirectly.

  • Direct Active Transport. Some transporters bind ATP directly and use the energy of its hydrolysis to drive active transport.
  • Indirect Active Transport. Other transporters use the energy already stored in the gradient of a directly-pumped ion. Direct active transport of the ion establishes a concentration gradient. When this is relieved by facilitated diffusion, the energy released can be harnessed to the pumping of some other ion or molecule.

Direct Active Transport

The Na+/K+ ATPase

The cytosol of animal cells contains a concentration of potassium ions (K+) as much as 20 times higher than that in the extracellular fluid. Conversely, the extracellular fluid contains a concentration of sodium ions (Na+) as much as 10 times greater than that within the cell. These concentration gradients are established by the active transport of both ions. And, in fact, the same transporter, called the Na+/K+ ATPase, does both jobs. It uses the energy from the hydrolysis of ATP to

  • actively transport 3 Na+ ions out of the cell
  • for each 2 K+ ions pumped into the cell.

This accomplishes several vital functions:

  • It helps establish a net charge across the plasma membrane with the interior of the cell being negatively charged with respect to the exterior. This resting potential prepares nerve and muscle cells for the propagation of action potentials leading to nerve impulses and muscle contraction.
  • The accumulation of sodium ions outside of the cell draws water out of the cell and thus enables it to maintain osmotic balance (otherwise it would swell and burst from the inward diffusion of water).
  • The gradient of sodium ions is harnessed to provide the energy to run several types of indirect pumps.

The crucial roles of the Na+/K+ ATPase are reflected in the fact that almost one-third of all the energy generated by the mitochondria in animal cells is used just to run this pump.

The H+/K+ ATPase

The parietal cells of your stomach use this pump to secrete gastric juice. These cells transport protons (H+) from a concentration of about 4 x 10-8 M within the cell to a concentration of about 0.15 M in the gastric juice (giving it a pH close to 1). Small wonder that parietal cells are stuffed with mitochondria and uses huge amounts of ATP as they carry out this three-million fold concentration of protons.

The Ca2+ ATPases

A Ca2+ ATPase is located in the plasma membrane of all eukaryotic cells. It uses the energy provided by one molecule of ATP to pump one Ca2+ ion out of the cell. The activity of these pumps helps to maintain the ~20,000-fold concentration gradient of Ca2+ between the cytosol (~ 100 nM) and the ECF (~ 20 mM). In resting skeletal muscle, there is a much higher concentration of calcium ions (Ca2+) in the sarcoplasmic reticulum than in the cytosol. Activation of the muscle fiber allows some of this Ca2+ to pass by facilitated diffusion into the cytosol where it triggers contraction.

After contraction, this Ca2+ is pumped back into the sarcoplasmic reticulum. This is done by another Ca2+ ATPase that uses the energy from each molecule of ATP to pump 2 Ca2+ ions.

Pumps 1. - 3. are designated P-type ion transporters because they use the same basic mechanism: a conformational change in the proteins as they are reversibly phosphorylated by ATP. And all three pumps can be made to run backward. That is, if the pumped ions are allowed to diffuse back through the membrane complex, ATP can be synthesized from ADP and inorganic phosphate.

ABC Transporters

ABC ("ATP-Binding Cassette") transporters are transmembrane proteins that

  • expose a ligand-binding domain at one surface and a
  • ATP-binding domain at the other surface.

The ligand-binding domain is usually restricted to a single type of molecule.

The ATP bound to its domain provides the energy to pump the ligand across the membrane.

The human genome contains 48 genes for ABC transporters. Some examples:

  • CFTR — the cystic fibrosis transmembrane conductance regulator
  • TAP, the transporter associated with antigen processing
  • The transporter that liver cells use to pump the salts of bile acids out into the bile.
  • ABC transporters that pump chemotherapeutic drugs out of cancer cells thus reducing their effectiveness.

ABC transporters must have evolved early in the history of life. The ATP-binding domains in archaea, eubacteria, and eukaryotes all share a homologous structure, the ATP-binding "cassette".

Indirect Active Transport

Indirect active transport uses the downhill flow of an ion to pump some other molecule or ion against its gradient. The driving ion is usually sodium (Na+) with its gradient established by the Na+/K+ ATPase.

Symport Pumps

In this type of indirect active transport, the driving ion (Na+) and the pumped molecule pass through the membrane pump in the same direction. Examples:

  • The Na+/glucose transporter. This transmembrane protein allows sodium ions and glucose to enter the cell together. The sodium ions flow down their concentration gradient while the glucose molecules are pumped up theirs. Later the sodium is pumped back out of the cell by the Na+/K+ ATPase. The Na+/glucose transporter is used to actively transport glucose out of the intestine and also out of the kidney tubules and back into the blood.
  • All the amino acids can be actively transported, for example out of the kidney tubules and into the blood, by sodium-driven symport pumps.
  • Sodium-driven symport pumps also return neurotransmitters to the presynaptic neuron.
  • The Na+/iodide transporter. This symporter pumps iodide ions into the cells of the thyroid gland (for the manufacture of thyroxine) and also into the cells of the mammary gland (to supply the baby's need for iodide).
  • The permease encoded by the lac operon of E. coli that transports lactose into the cell.

Antiport Pumps

In antiport pumps, the driving ion (again, usually sodium) diffuses through the pump in one direction providing the energy for the active transport of some other molecule or ion in the opposite direction. Example:

Ca2+ ions are pumped out of cells by sodium-driven antiport pumps. Antiport pumps in the vacuole of some plants harness the outward facilitated diffusion of protons (themselves pumped into the vacuole by a H+ ATPase) to the active inward transport of sodium ions. This sodium/proton antiport pump enables the plant to sequester sodium ions in its vacuole. Transgenic tomato plants that overexpress this sodium/proton antiport pump are able to thrive in saline soils too salty for conventional tomatoes. Antiport pumps to the active inward transport of nitrate ions (NO3)

Some inherited ion-channel diseases

A growing number of human diseases have been discovered to be caused by inherited mutations in genes encoding channels.


  • Chloride-channel diseases
    • cystic fibrosis
    • inherited tendency to kidney stones (caused by a different kind of chloride channel than the one involved in cystic fibrosis)
  • Potassium-channel diseases
    • the majority of cases of long QT syndrome, an inherited disorder of the heartbeat
    • a rare, inherited tendency to epileptic seizures in the newborn
    • several types of inherited deafness
  • Sodium-channel diseases
    • inherited tendency to certain types of muscle spasms
    • Liddle's syndrome. Inadequate sodium transport out of the kidneys, because of a mutant sodium channel, leads to elevated osmotic pressure of the blood and resulting hypertension (high blood pressure)


Osmosis is a special term used for the diffusion of water through cell membranes. Although water is a polar molecule, it is able to pass through the lipid bilayer of the plasma membrane. Aquaporins — transmembrane proteins that form hydrophilic channels — greatly accelerate the process, but even without these, water is still able to get through. Water passes by diffusion from a region of higher to a region of lower concentration. Note that this refers to the concentration of water, NOT the concentration of any solutes present in the water. Water is never transported actively; that is, it never moves against its concentration gradient. However, the concentration of water can be altered by the active transport of solutes and in this way the movement of water in and out of the cell can be controlled. Example: the reabsorption of water from the kidney tubules back into the blood depends on the water following behind the active transport of (Na^+).

  • Hypotonic solutions: If the concentration of water in the medium surrounding a cell is greater than that of the cytosol, the medium is said to be hypotonic. Water enters the cell by osmosis. A red blood cell placed in a hypotonic solution (e.g., pure water) bursts immediately ("hemolysis") from the influx of water. Plant cells and bacterial cells avoid bursting in hypotonic surroundings by their strong cell walls. These allow the buildup of turgor within the cell. When the turgor pressure equals the osmotic pressure, osmosis ceases.
  • Isotonic solutions: When red blood cells are placed in a 0.9% salt solution, they neither gain nor lose water by osmosis. Such a solution is said to be isotonic. The extracellular fluid (ECF) of mammalian cells is isotonic to their cytoplasm. This balance must be actively maintained because of the large number of organic molecules dissolved in the cytosol but not present in the ECF. These organic molecules exert an osmotic effect that, if not compensated for, would cause the cell to take in so much water that it would swell and might even burst. This fate is avoided by pumping sodium ions out of the cell with the Na+/K+ ATPase.
  • Hypertonic solutions: If red cells are placed in sea water (about 3% salt), they lose water by osmosis and the cells shrivel up. Sea water is hypertonic to their cytosol. Similarly, if a plant tissue is placed in sea water, the cell contents shrink away from the rigid cell wall. This is called plasmolysis. Sea water is also hypertonic to the ECF of most marine vertebrates. To avoid fatal dehydration, these animals (e.g., bony fishes like the cod) must continuously drink sea water and then desalt it by pumping ions out of their gills by active transport.

Marine birds, which may pass long periods of time away from fresh water, and sea turtles use a similar device. They, too, drink salt water to take care of their water needs and use metabolic energy to desalt it. In the herring gull, shown here, the salt is extracted by two glands in the head and released (in a very concentrated solution — it is saltier than the blood) to the outside through the nostrils. Marine snakes use a similar desalting mechanism.

Biochemistry. 5th edition.


The flow of ions through a single membrane 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 the open and closed states. [(Left) (more. )

The lipid bilayer of biological membranes, as discussed in Chapter 12, is intrinsically impermeable to ions and polar molecules. Permeability is conferred by two classes of membrane proteins, pumps and channels. Pumps use a source of free energy such as ATP or light to drive the thermodynamically uphill transport of ions or molecules. Pump action is an example of active transport. Channels, in contrast, enable ions to flow rapidly through membranes in a downhill direction. Channel action illustrates passive transport, or facilitated diffusion.

Pumps are energy transducers in that they convert one form of free energy into another. Two types of ATP-driven pumps, P-type ATPases and the ATP-binding cassette pumps, undergo conformational changes on ATP binding and hydrolysis that cause a bound ion to be transported across the membrane. Phosphorylation and dephosphorylation of both the Ca 2+ -ATPase and the Na + -K + -ATPase pumps, which are representative of P-type ATPase, are coupled to changes in orientation and affinity of their ion-binding sites.

A different mechanism of active transport, one that utilizes the gradient of one ion to drive the active transport of another, will be illustrated by the sodium�lcium exchanger. This pump plays an important role in extruding Ca 2+ from cells.

We begin our examination of channels with the acetylcholine receptor, a channel that mediates the transmission of nerve signals across synapses, the functional junctions between neurons. The acetylcholine receptor is a ligand-gated channel in that the channel opens in response to the binding of acetylcholine (Figure 13.1). In contrast, the sodium and potassium channels, which mediate action potentials in neuron axon membranes, are opened by membrane depolarization rather than by the binding of an allosteric effector. These channels are voltage-gated. These channels are also of interest because they swiftly and deftly distinguish between quite similar ions (e.g., Na + and K + ). The flow of ions through a single channel in a membrane can readily be detected by using the patch-clamp technique.

Figure 13.1

Acetylcholine Receptors. An electron micrograph shows the densely packed acetylcholine receptors embedded in a postsynaptic membrane. [Courtesy of Dr. John Heuser and Dr. Shelly Salpeter.]

The chapter concludes with a view of a different kind of channel—the cell-to-cell channel, or gap junction. These channels allow the transport of ions and metabolites between cells.

  • 13.1. The Transport of Molecules Across a Membrane May Be Active or Passive
  • 13.2. A Family of Membrane Proteins Uses ATP Hydrolysis to Pump Ions Across Membranes
  • 13.3. Multidrug Resistance and Cystic Fibrosis Highlight a Family of Membrane Proteins with ATP-Binding Cassette Domains
  • 13.4. Secondary Transporters Use One Concentration Gradient to Power the Formation of Another
  • 13.5. Specific Channels Can Rapidly Transport Ions Across Membranes
  • 13.6. Gap Junctions Allow Ions and Small Molecules to Flow between Communicating Cells
  • Summary
  • Problems
  • Selected Readings

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

What is Passive Diffusion

Passive diffusion refers to the movement of ions or molecules across the cell membrane through a concentration gradient without utilizing the cellular energy. Therefore, passive diffusion uses the natural entropy of the molecules to pass through the cell membrane. The movement of molecules occurs until their concentration becomes equal on both sides. The four main types of passive diffusion are osmosis, simple diffusion, facilitated diffusion, and filtration.

Simple Diffusion

The simple movement of molecules across a permeable membrane is called simple diffusion. Small, non-polar molecules use simple diffusion. The diffusion distance should be less in order to maintain a better flow. Simple diffusion is shown in figure 3.

Figure 3: Simple Diffusion

Facilitated Diffusion

Polar molecules and large molecules pass through the cell membrane by facilitated diffusion. The three types of transport proteins involved in the facilitated diffusion are channel proteins, aquaporins, and carrier proteins. Channel proteins make hydrophobic tunnels across the membrane, allowing the selected hydrophobic molecules to pass through the membrane. Some channel proteins are opened at all times, and some are gated like ion channel proteins. Aquaporins allow water to cross the membrane quickly. Carrier proteins change their shape, transporting target molecules across the membrane. Facilitated diffusion is shown in figure 4.

Figure 4: Facilitated Diffusion


Filtration is the movement of solutes along with water due to the hydrostatic pressure generated by the cardiovascular system. It occurs in Bowman’s capsule in the kidney. Filtration is shown in figure 5.

Figure 5: Filtration


Osmosis is the movement of water across a selectively permeable membrane. It occurs from a high water potential to a low water potential. The effect of the osmotic pressure on red blood cells is shown in figure 6. Red blood cells in a hypertonic solution may lose water from cells. Hypertonic solutions contain a higher concentration of solutes than the cytoplasm of red blood cells. Isotonic solutions contain a similar concentration of solutes as in the cytoplasm. So, the net movement of water in and out of the cell is zero. Hypotonic solutions contain low solute concentrations than the cytoplasm. Red blood cells receive water from hypotonic solutions.

Figure 6: Osmotic Pressure on Red Blood Cells

The lipid soluble molecules passively pass through the phospholipid bilayer. Water soluble molecules pass through the cell membrane by means of transmembrane proteins.


Biological membranes are permeable to water but not to ions or small polar organic molecules. Due to this difference in permeability, water moves across a membrane from a region of low solute concentration to a region of high solute concentration. This passive transport of water is called osmosis. Imagine two chambers that contain different concentrations of a solute, and that are separated by a membrane permeable to water but not to the solute. There will be a net flow of water from the compartment with the lower concentration of solute to the compartment with the higher concentration until equilibrium is achieved and the two compartments contain equal concentrations of solute. The compartment containing the higher concentration of solute is the hypertonic solution, and the compartment containing the lower concentration of solute is the hypotonic solution. When the compartments contain solutions of equal concentration, they are isotonic solutions.

What is Diffusion

Diffusion is the passive movement of molecules along a concentration gradient of a higher concentration to a lower concentration. Three major diffusion methods can be identified: simple diffusion, facilitated diffusion, and osmosis.

Simple Diffusion

Simple diffusion is an unassisted type of diffusion in which a particle moves from a higher to a lower concentration. Once, the molecules are evenly distributed by simple diffusion, the molecules on the either sides of the cell membrane achieve an equilibrium where no net movement of molecules is observed. Small, nonpolar molecules like oxygen, carbon dioxide, and ethanol move across the cell membrane by simple diffusion.

Facilitated Diffusion

Facilitated diffusion is the transport of substances across a biological membrane through a concentration gradient by means of a carrier molecule. Large ions and polar molecules which are dissolved in water are transported by specific transmembrane proteins in the cell membrane. Polar ions diffuse through transmembrane channel proteins and large molecules diffuse through transmembrane carrier proteins. Aquaporins are the other type of transmembrane proteins, which transport water through the cell membrane quickly.

Figure 1: Facilitated Diffusion through Carrier Proteins


Osmosis refers the free diffusion of water molecules across the cell membrane through an osmotic pressure.

Part 2: Alternating Access of the Glucose Transporter

00:00:09.01 Hi. I'm Nieng Yan.
00:00:10.05 I'm a professor in the School of Medicine,
00:00:13.28 Tsinghua University, Beijing, China.
00:00:15.02 Welcome to iBiology seminar series.
00:00:17.25 In part 2,
00:00:19.13 I'd like to share with you
00:00:20.29 one major research interest in my lab.
00:00:24.03 That is, the structure elucidation
00:00:25.29 of one very fundamental physiological process
00:00:29.16 , the cellular uptake of glucose.
00:00:33.25 We all know glucose is the primary energy source
00:00:37.10 to most of the lives on Earth.
00:00:39.28 From the textbook of biochemistry
00:00:42.23 or cellular biology,
00:00:44.10 you all learned how glucose is burned
00:00:48.08 to release energy to support life.
00:00:50.14 We know through glycolysis,
00:00:52.08 one glucose molecule is split to
00:00:55.29 two pyruvate molecules,
00:00:57.06 and during this process
00:00:59.01 two ATP molecules are generated.
00:01:01.23 And in the aerobic conditions,
00:01:05.13 the pyruvate molecules
00:01:08.06 are further burned through the TCA cycle,
00:01:10.24 or the citric acid cycle,
00:01:13.02 and the electron transport chain,
00:01:15.17 to generate carbon dioxide.
00:01:20.03 And during this process.
00:01:21.20 I mean, if it's complete metabolism,
00:01:24.05 then one glucose can be used to
00:01:26.26 produce over 30 ATP molecules.
00:01:30.00 That is the energy currency for all life.
00:01:34.13 However, before the metabolism of glucose,
00:01:38.12 there is also one critical step
00:01:40.17 -- that is to take the glucose into the cell.
00:01:44.15 From part 1,
00:01:46.07 I already told you that glucose
00:01:47.28 is highly hydrophilic,
00:01:51.08 that means, they are water soluble.
00:01:52.08 However, the cell is surrounded by
00:01:56.22 the hydrophobic lipid bilayer.
00:01:57.26 So, glucose cannot enter the cell
00:02:00.02 through free diffusion.
00:02:01.13 There must be different proteins
00:02:04.24 to mediate this process.
00:02:06.18 These proteins are called glucose transporters.
00:02:10.14 So, as we see here,
00:02:14.19 glucose transporter is important,
00:02:16.16 is essential for cellular uptake of glucose.
00:02:20.07 And, throughout the years,
00:02:23.14 we have identified different types of glucose transporters,
00:02:27.01 and more glucose transporters are being identified,
00:02:31.06 but among all of those,
00:02:33.03 the most rigorously characterized ones
00:02:37.13 are called GLUTs, as shown here,
00:02:38.25 G-L-U-T,
00:02:40.26 glucose transporters.
00:02:43.18 So, in human bodies,
00:02:47.22 there is a huge family called
00:02:49.15 major facilitator superfamily,
00:02:51.07 and the GLUTs belong to this family.
00:02:52.16 Even within the GLUT family,
00:02:54.23 there are 14 different isoforms
00:02:57.01 that exhibit tissue specificity
00:02:59.28 and substrate specificity.
00:03:02.05 As summarized here, for example,
00:03:05.04 GLUT1 functions in brain and red blood cells,
00:03:08.28 and GLUT2 is for liver.
00:03:11.28 GLUT3 is also called neuronal glucose transporter,
00:03:14.27 indicating that it functions in neurons.
00:03:17.10 And GLUT4 is very famous
00:03:19.14 -- it take glucose into adipocytes and muscle cells.
00:03:24.27 So, these are the four most famous GLUTs
00:03:28.19 -- GLUT 1, 2, 3, 4.
00:03:30.13 And for the other 10 different isoforms,
00:03:32.28 unfortunately, for some of them,
00:03:35.07 their substrates remain uncharacterized.
00:03:38.24 Oh, besides, for these glucose transporters,
00:03:41.07 despite their sequence similarity with each other,
00:03:44.00 they actually may have
00:03:47.11 different binding affinities for glucose
00:03:51.06 and for other similar sugars,
00:03:54.19 and they have different turnover rates.
00:03:56.24 For example, GLUT1 can take
00:04:00.25 up to 1200 glucose per second,
00:04:04.09 but that's. yeah, that's very fast.
00:04:06.13 however, GLUT3.
00:04:08.19 for GLUT3, the number is 6000.
00:04:10.09 it's five times faster than GLUT1,
00:04:13.15 and this is amazing.
00:04:15.05 Because of their fundamental
00:04:18.02 significance in physiology,
00:04:19.28 you can imagine,
00:04:21.10 malfunction or misregulation of these proteins
00:04:24.29 are associated with various diseases.
00:04:28.17 For example, GLUT1 deficiency syndrome
00:04:31.20 is actually a rare genetic disease
00:04:34.13 manifested by early onset seizure
00:04:37.24 or retarded development.
00:04:40.10 And GLUT2, because it's associated with the liver.
00:04:43.15 so, mutations of GLUT2
00:04:46.11 are associated with
00:04:50.12 a type of disease called Fanconi-Bickel syndrome.
00:04:52.24 And more and more evidence shows that
00:04:57.02 GLUT1 and GLUT3 are overexpressed in cancer cells,
00:05:01.19 especially solid tumor cells,
00:05:04.11 because of the so-called Warburg effect.
00:05:06.01 I just told you,
00:05:08.26 without oxygen, one glucose can be
00:05:11.23 converted to pyruvate.
00:05:13.01 During this process, two ATP molecules are generated.
00:05:15.17 However, in the presence of oxygen,
00:05:18.23 that is, aromatic.
00:05:20.18 or, sorry, aerobic conditions,
00:05:22.28 about. I mean, over 30 ATP molecules
00:05:25.02 can be generated.
00:05:26.10 For solid tumors,
00:05:27.26 it's usually under hypoxic conditions.
00:05:30.23 That's. you know, that means it can only.
00:05:34.22 one glucose can only generate two ATPs.
00:05:37.10 Consequently, more glucose transporters
00:05:39.25 have to be expressed
00:05:43.14 to take more sugar to
00:05:46.08 compensate for this amounts.
00:05:47.23 And for GLUT4,
00:05:49.12 it's very famous because of
00:05:51.10 its association with type 2 diabetes mellitus
00:05:54.17 and obesity.
00:05:56.29 So, as I just mentioned,
00:05:59.03 glucose transporters belong to
00:06:01.12 the so-called major facilitator superfamily.
00:06:04.25 As a matter of fact,
00:06:06.27 they are the prototypes of this
00:06:09.10 largest secondary active transporter family.
00:06:12.22 And for members in this family,
00:06:16.00 actually, they are widespread across species,
00:06:19.04 from bacteria to human beings.
00:06:20.17 And members in this family
00:06:22.07 have a very broad spectrum of substrates,
00:06:25.22 from ions, sugars,
00:06:28.07 amino acids, or even peptides.
00:06:31.05 And in terms of transport mechanisms,
00:06:35.26 if you watched part 1 already,
00:06:39.08 actually, members in this family can be
00:06:42.02 uniporters, symporters, or antiporters.
00:06:44.15 That's in terms of the orientations of the transport.
00:06:47.00 And, as I also told you,
00:06:49.20 a general alternating access
00:06:53.18 model or mechanism
00:06:55.08 has been proposed to account
00:06:56.29 for all the secondary transporters.
00:06:58.23 Especially for MFS members,
00:07:01.05 this works very well.
00:07:02.27 And we thought that because
00:07:04.24 GLUTs are the prototypes in the understanding of this family,
00:07:07.17 so structural and biochemical characterization of GLUTs
00:07:12.17 may also shed light on the understanding
00:07:15.04 of other members of this largest family.
00:07:18.07 Okay, why is it a prototype,
00:07:20.16 especially GLUT1?
00:07:22.08 Because it was one of the
00:07:24.29 first transporters to be cloned
00:07:26.26 and characterized.
00:07:29.06 So, let me bring you to the history.
00:07:31.05 Actually, the characterization of glucose uptake
00:07:35.15 into our blood cells
00:07:38.01 can be dated back to about a century ago.
00:07:40.15 And at that time it was already discovered that
00:07:45.22 the uptake rate or the "diffusion".
00:07:47.15 at that time people didn't know it's active transport,
00:07:50.03 so they still called it diffusion.
00:07:52.25 but one PhD student found that
00:07:56.03 the diffusion coefficient is actually concentration dependent,
00:07:59.09 suggesting it was not free diffusion.
00:08:02.00 In 1948, LeFevre, in one paper,
00:08:05.24 speculated on the active transport component,
00:08:10.03 although he didn't specify
00:08:11.22 whether it was protein or something else,
00:08:13.15 but he just speculated there would be
00:08:16.15 an active transport mechanism.
00:08:19.04 And in the 1950s, Widdas,
00:08:21.00 in his very famous paper,
00:08:23.12 proposed a so-called mobile solute carrier mechanism.
00:08:26.21 As a matter of fact,
00:08:29.02 this mechanism was so famous
00:08:30.19 that all the secondary transporters in humans
00:08:35.15 are named after SLC.
00:08:38.24 So, for example, GLUT1 is actually.
00:08:41.08 the gene name for GLUT1 is SLC2A1,
00:08:45.06 but don't call it slack,
00:08:47.02 because scientists don't like that name,
00:08:49.11 so it's SLC.
00:08:51.17 And then.
00:08:53.04 and so far it's all about these components.
00:08:55.04 And in 1977, these scientists
00:08:58.18 actually were able to purify
00:09:01.14 the protein component from red blood cells
00:09:04.01 and reconstitute them into a liposome,
00:09:07.01 and they reconstituted the uptake of glucose.
00:09:10.13 So, they named this protein component
00:09:12.22 GLUT1.
00:09:14.17 And then, in 1985,
00:09:16.12 Harvey Lodish's lab cloned GLUT1,
00:09:19.20 and when the sequence was available
00:09:22.20 it was clear that this protein contains
00:09:25.16 12 transmembrane helices.
00:09:27.24 And in the 1990s,
00:09:30.26 the study efforts were shifted
00:09:33.16 to the pathophysiological investigations,
00:09:35.25 as well as structural characterizations,
00:09:38.10 because we would like to understand their structure,
00:09:41.07 to see their structure,
00:09:42.17 so as to understand
00:09:44.18 its functional mechanism and disease mechanism.
00:09:47.13 However, 30 years.
00:09:49.24 almost 30 years passed.
00:09:52.11 so what we learned from the textbook
00:09:54.01 about the structure of GLUT1 was still this,
00:09:56.26 the one published by Harvey Lodish in 1985.
00:10:00.19 This is the topological structure.
00:10:03.06 Alright, umm.
00:10:05.12 so, I started my lab in 2007
00:10:07.17 and we were very interested in the structure of GLUTs
00:10:10.14 because we thought it could help
00:10:13.00 address a lot of interesting questions,
00:10:14.25 as listed here.
00:10:16.05 Of course, the first thing is
00:10:19.01 you try to see the architecture of GLUTs,
00:10:21.18 that's the most direct, but superficial, purpose.
00:10:25.04 And with the structure
00:10:27.14 we might be able to reveal
00:10:29.11 the molecular basis underlying the substrate selectivity,
00:10:32.14 why it selects glucose,
00:10:35.22 but not, for example, maltose.
00:10:38.19 And we.
00:10:40.14 because we understand that these transporters
00:10:43.09 follow this alternating access cycle,
00:10:46.07 so we'd like to reveal the conformational changes
00:10:48.26 during the transport cycle,
00:10:50.12 to understand their functional mechanism.
00:10:54.00 And we also hope to
00:10:57.12 provide a molecular interpretation
00:10:59.10 for all these disease-related mutations.
00:11:03.26 And, for my own research,
00:11:06.27 I'm also very interested in the difference,
00:11:08.21 the mechanistic difference,
00:11:10.05 between symporters,
00:11:11.20 particularly proton symporters,
00:11:13.16 and facilitators,
00:11:15.01 but I may not have time to go into the details of this part.
00:11:17.27 And finally, because membrane proteins
00:11:21.18 are embedded in the lipid bilayer,
00:11:24.05 we would really like to understand
00:11:27.00 how they are modulated by lipids,
00:11:28.28 and there are more and more questions.
00:11:30.15 they just emerge during your research.
00:11:32.26 So, to address these questions,
00:11:34.27 we started not with a glucose transporter,
00:11:37.26 but with their relatives,
00:11:40.21 their relatives from E. coli,
00:11:42.21 which are technically easier than the human protein.
00:11:46.24 So we determined the two structures of
00:11:50.09 E. coli proton-sugar symporters,
00:11:53.02 FucP and XylE.
00:11:55.21 So, as the name indicated,
00:11:58.02 they are proton symporters,
00:11:59.12 meaning they exploit this transmembrane proton gradient
00:12:02.18 to drive the uptake of the substrates,
00:12:05.08 either L-fucose or D-xylose,
00:12:07.21 from a low concentration environment
00:12:10.20 to the high concentration interior of the cell.
00:12:13.13 In the past three years, we were very lucky.
00:12:16.27 We were finally able to determine
00:12:18.21 the crystal structures of GLUT1,
00:12:21.08 and its closely related GLUT3,
00:12:24.25 in three different conformations.
00:12:27.02 That means they adopt different states
00:12:30.04 during a transport cycle,
00:12:31.21 as shown here.
00:12:32.25 So, all the way from
00:12:35.22 outward-open, occluded, and inward-open.
00:12:37.18 When I say outward or inward,
00:12:40.08 that refers to the substrate binding site,
00:12:42.21 that is. remember, for the alternating access,
00:12:47.01 that is. the substrate binding site
00:12:48.28 can never be exposed
00:12:50.29 to both sides of the membrane,
00:12:54.09 so it's always open to one side,
00:12:55.27 the substrate comes,
00:12:57.07 and this protein undergoes conformational change
00:13:00.04 to expose the substrate
00:13:02.07 to the other side.
00:13:03.20 This is called alternating access.
00:13:05.13 So, with these three structures,
00:13:07.00 we have a relatively better understanding
00:13:08.23 of this transport cycle of GLUTs.
00:13:11.16 Alright, first thing.
00:13:13.06 To address the question of the architecture.
00:13:15.19 but, before that, I know
00:13:19.18 many people are interested in the crystallization of membrane proteins
00:13:21.28 and GLUT1 has been a target for several decades.
00:13:25.12 Why were we able to crystallize
00:13:28.19 and determine the structure
00:13:30.17 of this very intriguing protein?
00:13:31.15 In retrospect, there are three key elements
00:13:35.23 that contributed to the crystallization of GLUT1
00:13:38.26 and gave us the diffracting crystals.
00:13:41.21 First, we actually introduced point mutations.
00:13:45.01 first is to eliminate glycosylation,
00:13:47.10 which really represents major troubles
00:13:50.29 for crystallization.
00:13:52.12 And the other point mutation,
00:13:54.17 glutamate-329 to glutamine,
00:13:57.10 this one is a disease-related mutation
00:14:00.18 originally identified in GLUT4,
00:14:03.02 and it was suggested to l
00:14:05.21 ock the protein in the inward-open conformation,
00:14:08.24 which was exactly the case, as seen in our structure.
00:14:12.09 And second, on the detergent we used for crystallization
00:14:16.01 is nonyl-glucoside.
00:14:17.03 I will come back later with
00:14:19.01 why this was important.
00:14:20.04 And third, you know, for glucose transporters,
00:14:22.05 they are highly mobile,
00:14:23.14 so we would try to slow them down,
00:14:25.08 to lock them at certain conformations,
00:14:27.08 so we did all the experiments at low temperature,
00:14:29.21 at 4 degrees Celsius
00:14:31.08 -- that helped a lot.
00:14:33.06 And to cut a long story short,
00:14:35.25 one particular day my student showed me these crystals,
00:14:39.06 these tiny crystals.
00:14:40.21 I thought, probably,
00:14:43.02 they were contaminations from insect cells,
00:14:45.08 however, you know,
00:14:47.07 it doesn't hurt to send them to the synchrotron
00:14:49.29 for data collection
00:14:51.08 and we sent this single crystal
00:14:53.08 to the Shanghai synchrotron,
00:14:54.24 and several hours later
00:14:56.12 we solved the structure
00:14:58.13 that was exactly our target, GLUT1.
00:15:00.16 As shown here, this structure
00:15:04.00 exhibits a very typical MFS fold,
00:15:08.02 remember, major facilitator superfamily.
00:15:10.11 It contains 12 transmembrane helices,
00:15:13.10 with the first 6 named the
00:15:17.14 N-domain or the N-terminal domains,
00:15:19.03 shown in silver,
00:15:20.26 and the C-terminal one in blue.
00:15:23.07 And very unexpectedly,
00:15:25.01 we also see an intracellular helical domain,
00:15:28.25 we named it ICH.
00:15:30.07 Actually, this domain.
00:15:32.03 this little domain harbors
00:15:34.03 a lot of serine or threonine or lysine,
00:15:36.27 so these residues are probably
00:15:38.28 important for their post-translation modification.
00:15:42.01 Now, with this structure,
00:15:43.27 we really can provide the answer to many questions.
00:15:49.05 So, as I asked at the beginning
00:15:52.03 -- so, what is the mechanism of substrate selectivity?
00:15:55.07 For this purpose, we actually examined,
00:15:57.18 through a biochemical approach,
00:16:01.13 the sugar selectivity by GLUT1 and GLUT3,
00:16:04.09 shown here are the results for GLUT3.
00:16:06.14 As you can see, indeed,
00:16:08.13 this protein has kind of a stringent selectivity,
00:16:14.13 and one.
00:16:15.16 wherever you see these lower,
00:16:17.09 these shorter bars,
00:16:19.02 that means these sugars
00:16:21.09 can inhibit the uptake of glucose,
00:16:23.27 meaning that they can be recognized by GLUT3,
00:16:25.23 to compete for glucose binding.
00:16:28.19 And when we examined these chemicals,
00:16:31.06 very interestingly
00:16:33.09 we found one common feature,
00:16:34.23 that is, their C3 hydroxyl group
00:16:37.22 all points to one orientation,
00:16:39.23 so that means that C3 hydroxyl group is important.
00:16:43.02 That's the conclusion from biochemistry,
00:16:46.29 from our biochemical characterizations.
00:16:49.20 Then, how is one sugar molecule
00:16:52.29 recognized by the protein?
00:16:55.00 So, the answer is from the
00:16:56.24 very high resolution structure of GLUT3.
00:16:59.06 So, we determined GLUT3
00:17:02.10 in complex with its substrate, D-glucose,
00:17:04.10 at 1.5 Angstrom resolution,
00:17:08.13 and shown here is the omit electron density map.
00:17:10.19 As you can see, it's beautiful.
00:17:12.21 To our surprise, we identified.
00:17:15.11 although we just, you know,
00:17:17.20 add glucose to the protein
00:17:19.11 and we identified two different
00:17:22.05 anomeric forms of glucose,
00:17:26.01 simply by the electron density map.
00:17:27.23 As you can see, both alpha and beta glucose
00:17:31.20 are present in the structure.
00:17:33.26 I mean, I have to clarify.
00:17:35.15 so, one protein can only bind to one glucose,
00:17:39.03 but for crystallography, you know,
00:17:41.15 this is the average of many billions of molecules,
00:17:44.18 so you know some proteins bind to the alpha form,
00:17:47.16 some bind to the beta form.
00:17:49.16 And this observation,
00:17:51.07 this structural observation
00:17:53.02 actually settled down one long-term controversy,
00:17:55.13 that is, whether glucose transporters
00:17:58.19 can recognize the alpha form of glucose,
00:18:01.26 because we know the beta form is the prevailing one,
00:18:04.18 the dominant form in solution.
00:18:05.22 And this observation shows, yes,
00:18:07.28 GLUT1 or GLUT3,
00:18:09.20 they can bind and transport
00:18:12.15 both anomeric forms of D-glucose.
00:18:16.05 Alright.
00:18:17.28 Another interesting discovery is that,
00:18:19.20 as I told you,
00:18:21.04 glucose transporters have two distinct domains,
00:18:24.08 N-domain and C-domain.
00:18:26.08 However, in the structure of GLUT3
00:18:28.19 in complex with glucose,
00:18:31.17 as well as in the structure of GLUT1
00:18:34.03 in the presence of this detergent molecule NG.
00:18:37.27 what is NG?
00:18:39.04 It is actually a derivative of glucose,
00:18:41.21 so that's why NG is important for us
00:18:44.09 to capture the structure of GLUT1
00:18:46.09 -- it mimics the substrate binding.
00:18:48.13 And if you compare these two structures,
00:18:50.11 a common feature is
00:18:52.08 the C-domain provides
00:18:54.08 the primary accommodation site for glucose,
00:18:58.22 so the C-domain
00:19:01.09 is the primary substrate binding site.
00:19:04.04 Then, what does the N-domain do?
00:19:07.08 Alright.
00:19:08.14 So, before that, you know,
00:19:10.10 we tried to complete the alternating access cycle
00:19:13.09 by, you know.
00:19:16.11 in the attempt to capturing another conformation,
00:19:19.09 that is, the outward-open,
00:19:20.28 because now we have GLUT3
00:19:23.01 in complex with glucose
00:19:25.01 in the occluded conformation,
00:19:26.10 that is, the substrate is trapped
00:19:28.10 in the center of the transporter
00:19:31.20 and isolated from either side of the membrane.
00:19:34.22 And GLUT1 is open to the inside of the cell,
00:19:38.02 so it's called inward-open.
00:19:39.11 Now, we still need this outward-open conformation.
00:19:44.00 In order to capture the outward-open structure,
00:19:46.18 we really had some rational thinking.
00:19:49.04 So, people always say that
00:19:51.01 crystallization is an art,
00:19:52.12 it seems like you have to do a lot of screening,
00:19:54.12 but in this case we really did
00:19:57.10 some rational thinking.
00:19:58.09 That is, when we obtained the structure of GLUT1,
00:20:00.22 I told you NG is important, right?
00:20:04.02 So we introduced several factors,
00:20:05.22 like the mutation E329Q,
00:20:08.28 that is, to lock the inward-open conformation,
00:20:10.19 and then when we see the binding of NG to the protein,
00:20:13.28 as you can see on the tail,
00:20:16.01 the aliphatic tail of this detergent,
00:20:17.29 it actually is
00:20:20.20 lining down the intracellular vestibule,
00:20:24.18 when the sugar moeity is
00:20:27.19 specifically coordinated by the C-terminal domain.
00:20:30.06 So, along.
00:20:31.18 so, basically, the presence of this aliphatic tail
00:20:35.00 precludes the closure of these two domains
00:20:39.00 on the intracellular side,
00:20:40.22 that is, to stabilize this inward-open conformation
00:20:44.08 -- with this aliphatic tail,
00:20:46.19 it cannot close, right?
00:20:48.10 So, along this line of thinking,
00:20:50.27 we thought, if we can find a chemical,
00:20:53.07 a glucose derivative,
00:20:55.07 that has some chemical groups
00:20:57.14 on the other side,
00:20:59.08 on the upper side of the sugar ring,
00:21:01.06 probably that can preclude
00:21:04.12 the closure of the protein on the extracellular side,
00:21:07.01 that is, to capture
00:21:09.24 an outward-open conformation.
00:21:11.02 Do we have these kind of chemicals?
00:21:12.29 Yes, we have a lot of disaccharides
00:21:15.22 that are derivatives of glucose.
00:21:17.28 As shown here, we selected a few
00:21:20.15 and we examined their ability to inhibit glucose uptake.
00:21:24.22 As shown here, it turns out that
00:21:26.27 maltose was a potent inhibitor,
00:21:28.26 and when we checked the literature
00:21:30.22 this was really consistent,
00:21:32.00 because maltose was regarded.
00:21:34.06 was suggested as the exofacial inhibitor,
00:21:37.26 that means it can inhibit glucose uptake
00:21:40.16 from the extracellular side.
00:21:44.10 To cut a long story short,
00:21:45.25 in the presence of maltose
00:21:47.29 we actually crystallized the protein
00:21:50.17 using lipidic cubic phase.
00:21:52.14 It gave us two different structures.
00:21:56.01 One is almost identical to.
00:22:00.17 shown on the left, it's almost identical
00:22:01.28 to the glucose-bound GLUT3,
00:22:04.17 and is occluded from.
00:22:06.19 so, maltose is bound in the center,
00:22:08.16 occluded from either side of the membrane.
00:22:11.01 But the other crystal form
00:22:15.03 gives us this outward-open conformation,
00:22:18.22 so this was really serendipity,
00:22:21.02 I mean, we just mixed them together
00:22:22.15 and it gave us two different crystal structures.
00:22:25.12 So, I will focus on the illustration
00:22:27.25 of this outward-open conformation,
00:22:29.02 with comparison of the inward-open GLUT1
00:22:32.11 and the occluded GLUT3.
00:22:34.10 So, now we have these three conformations
00:22:36.28 I showed before.
00:22:38.06 We could generate a morph
00:22:40.03 that illustrates the whole transport process.
00:22:44.17 As you see here,
00:22:46.25 outward-open, arrival of glucose,
00:22:48.24 and it undergoes this alternating access
00:22:52.00 by the relative rotation of these two domains,
00:22:55.23 and the substrate is released
00:22:57.15 into the inside of the cell.
00:23:01.06 And, very interestingly,
00:23:02.13 remember this small domain,
00:23:05.12 shown in yellow,
00:23:06.27 is the ICH, intracellular helical domain,
00:23:09.17 and during the conformation change,
00:23:11.13 we can see it also
00:23:14.04 undergoes interdomain rearrangement.
00:23:15.26 In a way, it restrains
00:23:18.07 the N- and the C-domains
00:23:20.01 from opening too much,
00:23:21.17 so this ICH domain,
00:23:23.20 we named it the latch,
00:23:25.17 to secure this intracellular gate.
00:23:31.07 Alright.
00:23:33.03 From the movie you might think, hmm.
00:23:34.21 these two domains undergo a rigid body rotation,
00:23:36.21 but close examination of the structures
00:23:39.27 of the outward-open and occluded GLUT3
00:23:44.21 suggest, no, it's not rigid body.
00:23:46.20 Actually, we can see very sophisticated
00:23:50.06 local rearrangement of the C-domain elements.
00:23:53.27 As shown here, the one shown in cyan
00:23:57.06 is the C-domain
00:24:01.08 and the one in green is the N-domain.
00:24:02.03 Please pay attention to this TM7 motif.
00:24:06.02 You can see it undergoes a bending, a bending.
00:24:10.09 Right? This is TM7.
00:24:13.02 Not only a bending.
00:24:15.07 so, when the sidechains are shown,
00:24:17.06 you will see it actually also undergoes a rotation,
00:24:21.28 so this TM7 undergoes
00:24:24.11 very complicated local rearrangement
00:24:27.20 by bending.
00:24:29.25 the combination of bending and rotation.
00:24:32.03 So, whether this is induced by substrate binding
00:24:34.28 or this is the so-called dynamic equilibrium,
00:24:38.15 remains to be further characterized,
00:24:40.22 and our preliminary MD simulations
00:24:43.06 suggest that this is dynamic equilibrium.
00:24:47.00 even in the absence of substrate,
00:24:49.09 you can see this kind of conformational change of TM7.
00:24:53.00 Now, here's the question.
00:24:55.18 why the C-domain, shown in cyan,
00:24:58.04 is so flexible,
00:24:59.28 whereas the N-domain is just so rigid, as a stone?
00:25:03.25 And when we examine the interior of these two domains,
00:25:08.08 the answer is really clear.
00:25:09.22 So, as shown here,
00:25:12.10 the red dashes represent hydrogen bonds.
00:25:16.07 As you can see,
00:25:18.14 the interior of the N-domain is really hydrophilic,
00:25:22.28 so the high-resolution structure of GLUT3
00:25:25.18 allowed us to identify
00:25:29.02 seven water molecules within the N-domain of GLUT3,
00:25:32.12 and these water molecules, together,
00:25:34.02 interact with a set of groups of many polar residues
00:25:38.10 as a strip of hydrogen bonds,
00:25:40.01 and this stabilizes the N-domain,
00:25:45.12 so it makes it very rigid during conformation change.
00:25:48.04 In contrast, the interior of the C-domain
00:25:51.14 is highly hydrophobic,
00:25:54.12 as shown here,
00:25:55.29 so these hydrophobic residues,
00:25:57.12 they just contact each other
00:26:00.12 through Van der Waals interactions,
00:26:02.07 so they make the interior relatively greasy,
00:26:05.11 and that's easier for bending and rotation.
00:26:08.04 So, the structural analysis
00:26:10.08 really provides a good answer
00:26:12.05 to account for the distinct features
00:26:14.12 of the N-domain and the C-domain
00:26:16.04 during the alternating access cycle.
00:26:19.19 Alright.
00:26:21.06 Now, I'll.
00:26:23.01 shown here is the very simple diagram
00:26:25.03 of alternating access.
00:26:26.14 With our structures, the three structures,
00:26:27.28 we are able to
00:26:30.15 update this model with more sophisticated features.
00:26:34.24 As you can see, TM7
00:26:36.27 and also TM10,
00:26:38.18 they undergo local conformational change,
00:26:40.21 and the overall relative rotation
00:26:42.20 of the N- and the C-domains
00:26:44.11 results in the alternating exposure
00:26:48.15 of the substrate to either side of the membrane.
00:26:50.08 And, besides, please pay attention
00:26:52.19 to these yellow bars,
00:26:54.07 they are the intracellular ICH domain,
00:26:56.08 we call them the latch,
00:26:57.16 the intracellular latch.
00:26:59.05 Okay, now with the structure.
00:27:01.23 We were able to map the disease-related mutations.
00:27:06.12 Shown her is an example of the mutations
00:27:08.29 identified in patients
00:27:11.05 with the so-called GLUT-1 deficiency syndrome.
00:27:14.04 So, in total, more than 40 mutations were identified.
00:27:17.10 So, when we mapped them
00:27:19.06 onto the structure of GLUT1,
00:27:20.24 very interestingly we realized that
00:27:24.03 they clustered to three areas,
00:27:26.25 as shown here.
00:27:27.29 So, area 1 is really
00:27:31.11 involved in substrate binding
00:27:34.03 and it's easy to understand how mutations of this cluster
00:27:37.19 would affect substrate recognition or substrate binding,
00:27:40.13 hence compromising the transport activity.
00:27:44.11 And cluster 2, as shown here,
00:27:46.29 highlighted by this cyan circle.
00:27:50.22 the cyan circle.
00:27:53.09 so, basically they mapped to the interface
00:27:58.09 between ICH, N-domain, and C-domain,
00:28:01.14 and they together constitute the intracellular gate.
00:28:03.10 And, not surprisingly,
00:28:05.11 cluster 3 maps to the extracellular gate.
00:28:08.13 So, the structure really provides
00:28:11.01 a beautiful answer to
00:28:13.26 understand most of these disease-associated mutations,
00:28:16.24 so they either affect substrate binding
00:28:19.19 or the two gates,
00:28:21.03 hence affecting the alternating access cycle
00:28:24.06 of the protein.
00:28:25.18 Alright. Now, with these results,
00:28:27.12 we can address the questions
00:28:29.05 asked at the very beginning, right?
00:28:31.22 So, we know the architecture
00:28:33.16 and we provide a basis
00:28:35.20 to see the substrate selection
00:28:38.06 and we revealed three conformations of GLUT1 and GLUT3
00:28:43.08 during the transport cycle, the alternating access cycle,
00:28:48.07 and we provided some answers
00:28:49.29 to the disease-related mutations.
00:28:52.10 And, with regard to the mechanistic difference
00:28:56.10 between symporters and facilitators,
00:28:58.12 we are now doing some MD simulations
00:29:00.03 and biochemical characterizations,
00:29:02.02 and we have some tentative clues,
00:29:04.16 but this really requires further characterizations.
00:29:07.29 And now our focus has shifted to
00:29:11.02 the modulation of the transport activity
00:29:14.07 by lipids, as well as, you know,
00:29:16.09 the kinetic study of transport cycles.
00:29:18.25 And finally, we are very interested in
00:29:21.13 structure-based ligand design,
00:29:23.05 because these proteins are important drug targets.
00:29:27.06 So, with this, I would like to conclude my talk
00:29:29.15 by acknowledging the people
00:29:32.22 who made this work possible.
00:29:34.04 So, Dong, he was my postdoc
00:29:37.17 who has been the primary driver of this project,
00:29:40.17 he was leading this team of Tsinghua undergraduate students
00:29:44.00 and graduate students
00:29:45.13 to elucidate the structures
00:29:47.17 of both GLUT1 and GLUT3,
00:29:49.01 and he's now a professor in Tsinghua University.
00:29:51.23 And this work was in collaboration with many colleagues,
00:29:55.23 in Tsinghua or in the US,
00:29:57.10 as shown here.
00:29:59.21 And I'd like to thank you for watching this online seminar.

  • Part 1: Introduction to Membrane Transport Proteins