Lecture 14: Membrane transport 2

Question 1

Are all types of channels integral or peripheral proteins

Answer 1

Note that channels, facilitative transporters and active transporters are ALL integral membrane proteins

Question 2

Electrochemical gradients

Answer 2

are dispersed by diffusion of ions across membranes

Question 3

Define conductance in relation to biological membranes

Answer 3

The rapid movement of ions across a membrane

is important in processes such as:

• Nerve impulses
• Muscle contraction

These rapid movements are from high to low concentration

Question 4

How does conductance occur

Answer 4

The movement is done through ion channels (another example of B, simple diffusion thru a channel)

• these are integral membrane proteins
• the movement is selective. Each ion channel will only pass one type of ion
• the movement is bidirectional dependant on concentration

Question 5

Give an example of a membrane protein that functions in conductance

Answer 5

The voltage gated K+ channel is an example of a membrane protein that functions in conductance.

Question 6

Ion channels

Answer 6

can be gated
These channels can be maintained in either open or closed states (gated). This allows the channel to be closed until they exactly need to be opened.

Question 7

Describe three different ways how gated ion channels can be triggered to open and close

Answer 7

1. Voltage gated channels (e.g. voltage gated K+ channel)
   • Monitor the voltage around them and open/close if there’s a change
   • What happens along nerve axons
2. Ligand-gated channels (e.g. neurotransmitter receptor/channels)
   • Open/close if a specific ligand binds to them… the ligand is not what goes through them
3. Mechano-gated channels
   • Monitor mechanical forces and open/close with the appropriate signal

Question 8

Ligand-gated ion channel

Answer 8

The binding of the neurotransmitter acetylcholine at certain synapses opens the Na+ channel. This initiates a nerve impulse or muscle contraction.

Question 9

Mechanically-gated ion channels

Answer 9

Sound waves bend cilia-like projections on the hair cells of the inner ear; the bending opens up ion channels, leading to the creation of nerve impulses to the brain.

Question 10

The K+ channel

Answer 10

Voltage gated ion channel
- involved in action potential transduction in nerves, muscles, etc.
- Opens after Na+ channels first open
2 homodimers, 6 membrane spanning domains each

Question 11

Describe how the K+ ion channel senses changes in membrane polarity and opens

Answer 11

The S4 helix is + charged and senses the charge state across the
membrane (keeps channel closed)
- When the membrane charge state changes from when the Na+
channels open, S4 shifts configuration that moves S6 out of the way to
open the pore

Question 12

Describe how the K+ ion channel is selective for K+ ions and excludes Na+ ions

Answer 12

An X-ray crystallography structure of a bacterial K+ channel shows it’s selectivity.

It’s based on stabilizing the pore structure in an ‘open’ state.
Carbonyl oxygen atoms of the peptide backbone are held just right inside the pore bind K+ ions as they go through. This the pore shape and allows passage, and is stabilized by the binding of K+ ions.
Pore = 3 Å K+ ion = 2.7 Å
Na+ ions are smaller and cannot stabilize the pore shape to open for them, so they’re not passed.

Question 13

Describe how the K+ channel closes itself

Answer 13

has an ‘auto-shutoff’ feature
After a few milliseconds, the cytoplasmic inactivation peptide domain of the channel slides into the pore area, blocking the ion flow

Question 14

Dendrites

Answer 14

branched projections that receive neurotransmitter signals from other neuron’s synaptic terminals. Each neuron is connected to many, many other neurons!

Question 15

Axon

Answer 15

much longer projection that transmits signals towards neurons (or effector cells)

Question 16

Synaptic terminals

Answer 16

branched projections at the end of the axon where the signal is passed (as neurotransmitters) to other neurons/effector cells.

Question 17

Glia

Answer 17

cells that support the nervous system (e.g. Schwann cells). 98% of brain cells are glia!

Question 18

Myelin sheath

Answer 18

insulating material that allows the signal to go waaaay faster down an axon

Question 19

Multiple sclerosis

Answer 19

autoimmune disease where the myelin sheaths are slowly destroyed

Question 20

Describe how a nerve impulse is conducted down the axon

Answer 20

A single neuron receives signals from hundreds of other neurons. Depending on the type of receptor that is triggered to open, different ions are let in that tell that cell whether it should generate an action potential.

For example:
If sodium (Na+) ions are let in, it’s excitatory (promotes action potential; green) If chloride (Cl-) ions are let in, it’s inhibitory (suppresses action potential; red)
To generalize, the sum of these signals determines whether the neuron will generate an action potential and send it down the axon to interact with other neurons.

Question 21

Describe how neurotransmitters are released at the neuron terminals and trigger (or surpress) nerve impulses on the next neuron

Answer 21

1. Na+/K+ pumps move 3 Na+ to the outside of the cell and 2 K+ to the inside of the cell, setting up an electrochemical gradient. (more on this later!)
2. When a signal is perceived in the neuron cell body, voltage-gated Na+ channels open up and allow the Na+ ions from the outside of the axon to the inside of the axon. The depolarization is recognized by adjacent Na+ channels, and they then open.
3. K+ ion channels recognize the change in polarity of the membrane, open up and allow the flow of K+ ions from the inside of the axon to the outside. Together with the Na+ movement, this causes a wave of depolarization to go down the neuron.
4. At the end of the neuron, voltage-gated Ca+ channels recognize the depolarization and open, allowing Ca+ into the cell. This causes fusion of neurotransmitter- containing vesicles to fuse with the PM.
5. The neurotransmitter (acetylcholine here) diffuses across the synaptic cleft and
6. binds to it’s receptor (a ligand-gated ion channel) on the next neuron. It opens and lets Na+ ions across the membrane to initiate the signal in the next neuron. If this is a large enough depolarization, the next neuron ‘fires’

Question 22

Describe how facilitated transporters operate

Answer 22

Larger molecules can be transported across membranes by facilitative transporters - We’re still talking about passive diffusion from high to low concentration!
- These are still bidirectional, flow will go from [high] to [low]

1. The glucose molecule binds to the transporter (i.e. directly interacts with the transporter amino acids)
2. This causes a conformational change in the transporter shape
3. This change exposes the glucose to the other side

Question 23

Compare facilitated transport to enzyme catalysis

Answer 23

Because there is actual binding of the solute to the transporter, these behave a bit like an enzyme. Thus, the binding and transport can saturate at high substrate concentration!

Question 24

Cystic fibrosis

Answer 24

is caused by a defective chloride channel encoded by the CFTR (1 in 25 people of Northern European descent are carriers)

Question 25

Describe how mutations in the CFTR gene can cause cystic fibrosis

Answer 25

many tissues affected, but the lungs show the most severe effects. Chloride ions draw water out of the cells by osmosis to make mucous. CF patients have thicker sticky mucous causing impaired breathing and recurrent infection

Question 26

Treatments for cystic fibrosis

Answer 26

Gene therapy has been attempted to cure CF: - Pack a ‘good’ CFTR gene into a virus that incorporates its genes into the human genome.. Then infect the cells - Unfortunately, the first patient died from this procedure - Good candidate for CRISPR/CAS9 therapy!!

Question 27

Active transport

Answer 27

is the pumping of ions or molecules through a membrane against their concentration gradient - it always requires a trans- membrane (integral) protein – usually called a transporter or pump

Question 28

active transport is:

Answer 28

• specific for a given chemical or ion • coupled to the consumption of energy • unidirectional (only pumped in one direction, as opposed to ion channels which flow in whatever gradient is set up) • controlled by the cell

Question 29

Explain the difference between direct and indirect active transport

Answer 29

The energy of ATP may be used directly or indirectly. • Direct Active Transport - the transporter binds ATP directly and uses the energy of its hydrolysis to drive active transport. • Indirect Active Transport - a transporter uses the energy of ATP to set up a concentration gradient of one molecule (from x to X in the example below). The energy of this concentration gradient is used to transport another molecule against its concentration gradient (from s to S in example below

Question 30

Explain why the Na+/K+ ATPase pump is electrogenic

Answer 30

* cells maintain high internal K+ (100 mM inside, 5 mM outside) * cells simultaneously maintain high external Na+ (10-20 mM inside, 150 mM outside) * sodium-potassium ATPase is the enzyme and a trans- membrane protein that establishes this gradient of these ions * this transporter uses ATP energy to pump out 3 Na+ and pump in 2 K+ , against their concentration gradients * electrogenic pump, unequal transport of charged particles (3 Na+ for every 2 K+) for each ATP burned, important in maintaining normal trans-membrane electrical potential

Question 31

List the sequence of events through which the Na+/K+ ATPase pump moves ions across the membrane a. describe what happens at each step

Answer 31

1. To begin with, the transporter has a high affinity for sodium and low affinity for potassium - sodium ions bind on the inside of the membrane (at a low conc. on the inside of the cell) 2. ATP is hydrolyzed, transfers terminal phosphate on to the protein 3. The protein changes its conformation and changes ion affinity • Exposes Na+ to extracellular side • affinity for sodium lowered and they diffuse away 4. Change in conformation increases affinity for potassium ions on outside of membrane (where the conc. of the ion is low). 5. phosphate removed from protein - energy from this causes conformational change... 6. protein returns to original conformation • lower affinity for potassium, so the K+ ions are released • Nowhasahigheraffinityof sodium

Question 32

What type of pump is Na+/K+ ATPase

Answer 32

Note: this pump is known as a P-type ion pump because it gets phosphorylated by ATP

Question 33

Describe why the Na+/K+ ATPase pump is important for cellular function

Answer 33

1. It creates 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 nerve impulses and muscle contraction. 2. The accumulation of sodium ions outside of the cell draws creates an extracellular isotonic solution, and thus enables the cell to maintain osmotic balance (otherwise it would swell and burst from the inward diffusion of water). 3. The gradient of sodium ions is harnessed to provide the energy to run several types of indirect pumps. 4. The crucial roles of the Na+/K+ ATPase are reflected in the fact that almost one-third of all the energy generated (i.e ATP) in animal cells is used just to run this pump.

Question 34

Describe how the H+/K+ pump is regulated to move protons into the gut

Answer 34

Active transport 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 (pH 7.2) within the cell to a concentration of about 0.15 M in the gastric juice (giving it a pH close to 1). • This three-million fold concentration of protons needs huge amounts of ATP energy! Food in the stomach triggers histamine to bind to parietal cells H+/K+ ATPase is translocated from vesicles to cell’s membrane to initiate pumping of protons into stomach

Question 35

a. Describe ways in which drugs can prevent high stomach acid

Question 36

List two locations in cells where the Ca+ ATPase functions

Answer 36

On cell membrane | SER

Question 37

List the 3 states of the K+ ion channel

Answer 37

1. Rest (closed)... S4 and S6 helices keeps the pore closed 2. An open state... after the change in membrane polarity has been perceived by the S4 helix and S6 has moved 3. An inactivated state where the inactivation peptide is blocking the pore

Question 38

describe how the Ca 2+ ATPase removes Ca2+ from the cytosol of muscle cells after contraction

Answer 38

* In resting striated muscle, there is a much higher concentration of calcium ions (Ca2+) in the sarcoplasmic reticulum (ER) 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 actively pumped back into the sarcoplasmic reticulum (= ER of muscle cells). * This is done by a Ca2+ ATPase that uses the energy from each molecule of ATP to pump 2 Ca2+ ions.

Question 39

Where else does the Ca2+ ATPase function?

Answer 39

• A Ca2+ ATPase is also located in the plasma membrane of all eukaryotic cells. It pumps Ca2+ out of the cell helping to maintain the ~10,000-fold concentration gradient of Ca2+ between the cytosol and the ECF.

Question 40

Describe the difference between V and P type pumps?

Answer 40

P-type”, transporter gets phosphorylated | also uses ATP, but transporter itself not phosphorylated)

Question 41

“P-type”, transporter

Answer 41

* Four-subunit structure * Na+/K+ ATPase is an example of this * Includes Ca++ pump on cell membrane and SER (maintains low cytoplasmic [Ca++]) * Includes stomach acid (H+) secretion pump

Question 42

V-type,

Answer 42

(also uses ATP, but transporter itself not phosphorylated) – these pumps are found on vacuoles • H+ pump only • Complex 7-subunit structure (a bit like ATP synthase backwards!) • Plant cells use V-ATPase pumps to fill vacuoles with H+, also they pump H+ outside of roots to bring things in • Animal cells have V-type pumps on lysosomes, endosomes, vesicles, and in the kidney (regulating pH in blood)

Question 43

ABC type (ATP-Binding Cassette)

Answer 43

▪Share a unique phosphorylated region: “ATP-binding cassette” ▪Human genome has 48 ABC transporters ▪ABC transporters are transmembrane proteins that have: a substrate-binding domain at one surface of protein; and a ATP-binding domain at another surface (or domain). ▪The substrate-binding domain can be restricted to a single type of molecule, or can be more general. Often used to pump ‘unwanted’ chemicals out of the cell ▪The ATP bound to its domain provides the energy to pump the substrate across the membrane. ATP binding causes conformational change, which helps push substrate through

Question 44

Describe how ABC transporters can lead to chemotherapy resistant drugs

Answer 44

Cancer cells can become resistant to chemotherapy drugs by over- producing ABC transporters that expel the drug from the cell

Question 45

Indirect active transport

Answer 45

cotransport • Indirect active transport uses the flow of an ion from [high] to [low] to pump some other molecule or ion against its gradient. The driving ion is often sodium (Na+) with its gradient established by the Na+/K+ ATPase.

Question 46

Explain the difference between symporters and antiporters

Answer 46

Symport Pumps • the driving ion (Na+) and the pumped molecule pass through the membrane pump in the same direction. * 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.

Question 47

Explain how glucose is absorbed in the small intestine into the blood stream using the Na+/K+ ATPAse pump, the Na+/glucose transporter, and a facilitated glucose transporter

Answer 47

* glucose transport from the small intestine into bloodstream: * utilizes a sodium gradient to transport glucose against its concentration gradient * two Na+ and one glucose are transported together * Glucose moves from low conc. of glucose in gut to a higher conc. within the cells lining the gut * the energy source: Na+ gradient is created by the Na+/K+ ATPase on the plasma membrane * GLUT2 is a passive channel then allows glucose to move into the blood, down its concentration gradient via facilitated diffusion (previous lecture!) 1. 2. 3. The Na+/K+ ATPase sets up an extracellular Na+ gradient The energy from this gradient brings in one glucose from the lumen for every two Na+ also brought in GLUT2 then transports the glucose into the bloodstream. This works because the concentration in the blood is lower than the epithelial cell.

Question 48

Explain how ingesting simple sodium/glucose mixtures can alleviate diarrhea

Answer 48

With cholera or other diseases that cause diarrhea, water loss is serious Treatment to prevent water loss can be as simple as feeding sugar-salt solutions The Na+/glucose symporter does its job and moves lots of Na+ and glucose into the intestinal cells. This the cytosol of the cell hypertonic and this sucks water back into the body by osmosis.

Question 49

Describe how the Na+/Ca+ can maintain a low intracellular concentration

Answer 49

Ca2+ ions are pumped to the extracellular fluid by active transport in two different ways: 1. an ATP-driven pump like the Ca2+ ATPase of skeletal muscle and 2. a Na+/Ca2+ exchanger. This antiport pump harnesses the energy of 3 Na+ ions flowing DOWN their concentration gradient to pump one Ca2+ against its gradient.

Question 50

Explain how two different transporters together lead to this low intracellular Ca+ concentrations

Answer 50

calcium is pumped out of the cell by 2 main active transport mechanisms - the Na+/Ca2+ exchanger and the Ca2+ ATPase pump (described in earlier slide)

Question 51

Describe how plant cells maintain a high H+ concentration in the vacuole, and how this concentration can be used to sequester Na+ ions also in the vacuole

Answer 51

• Plant cells have a V-type H+ pump that pumps H+ ions into the vacuole * The Na+/H+ antiport pump in the vacuoles of some plant cells couples: * facilitated diffusion of protons (H+) out from the vacuole with: * the active transport of sodium ions into the vacuole * This sodium/proton antiport pump enables the plant to sequester sodium ions in its vacuole. * Transgenic tomato plants that over- express this sodium/proton antiport pump are able to thrive in saline soils too salty for conventional tomatoe