Lecture 17: Membrane Channels and Transporters

Question 1

Membranes as Barriers

Answer 1

• Biological membranes serve as semipermeable barriers
• > They separate aqueous environments but some things can still get through
• Nonpolar molecules can diffuse freely, but polar/charged solutes are unable to cross without assistance
• Transmembrane protein channels and transporters serve to allow polar/charged solutes to cross membranes

Question 2

Channels

Answer 2

Allow diffusion down a concentration gradient (open up space to allow molecules to diffuse across a concentration gradient)

Question 3

Transporters

Answer 3

Use conformational changes to move substrates across the membrane, and may transport down (passive transport/facilitated transport) or up (active transport/pumps) a concentration gradient

Question 4

Diffusion

Answer 4

• HIGH concentration –> LOW concentration
• DOWN concentration gradient
• The process or movement of any molecule or ion moving down or up a concentration gradient requires a change in free energy
• There is a change in entropy (ordered area to disordered area)
• Net movement of charged solute down its electrochemical gradient

Question 5

Concentration gradient

Answer 5

Difference in concentration of a solute in one region compared to another

Question 6

Electrochemical gradient

Answer 6

• Concentration gradient
• Electrical gradient (charge) – electrical potential difference between 2 sides of membrane (there is a voltage across the membrane; one side is more positive than the other)
• Also represents a lower entropy state because there is more organization

Question 7

Electrochemical differences are additive

Answer 7

• If there’s no potential across the membrane, then only the concentration gradient is left
• If there is potential across the membrane and positive ions are on the positive side, then they can diffuse across more easily via additive forces (electrical and concentration gradient)
• If there is potential across the membrane and positive ions are on the negative side, then the attractive forces between the positive and negative charges will reduce the number of ions going across the membrane

Question 8

Diffusion of CHARGED substances across cell membranes

Answer 8

• So, ΔG can tell us whether a substance will move passively (no energy needed) or actively (energy input required) across a membrane.
• If positive, the value of DG (net flux INTO cell) is the amount of energy required to move a mole of solute up its electrochemical gradient and into the cell.
• If negative, DG (net flux INTO cell) is the maximum amount of extractable energy available to drive another process that is coupled to the diffusion of a solute down its electrochemical gradient and into the cell.

Question 9

Simple Diffusion

Answer 9

• Diffusion across lipid bilayer (independent of proteins)
• > For solutes that don’t require assistance to diffuse across the membrane
• Limited by:
• > Polarity
• > Charge
• > Size
• Can occur in either direction depending on the electrochemical gradient and the transport of molecules
• Cannot be saturated – there is no limit to the net flux as the electrochemical gradient increases

Question 10

Diffusion across membranes: Permeability

Answer 10

• In the absence of proteins, the lipid bilayer allows free diffusion of select types substances down their concentration gradients:
• > Hydrophobic molecules
• > Small polar molecules (not as much as hydrophobic molecules)
• Restricts the diffusion of other types of substances:
• > Ions
• > Larger polar molecules
• The membrane = semipermeable barrier.
• The membrane must be able to maintain concentration differences between the internal and external environments.
• Health of the cell requires that material transport be a regulated process
• > So the appropriate concentration and electrochemical gradients are maintained

Question 11

Transport Proteins

Answer 11

• Cells have evolved membrane proteins whose function is to transport small molecules and ions
• Membrane proteins mediate 2 basic types of movement across cell membranes:
• > Passive Transport
• > Active Transport

Question 12

Transporter-mediated diffusion

Answer 12

• Directional (usually work in one direction)

- Saturable (Similar to Enzyme-substrate kinetics)

Question 13

Passive transport (facilitated diffusion)

Answer 13

• Move spontaneously down their electrochemical gradient (–Delta G)
• Moves down the concentration gradient

Question 14

Active transport

Answer 14

• Energy (ATP) is consumed to move molecules up their electrochemical gradient – similar to a “coupled reaction”
• Moves against the concentration gradient

Question 15

Ion channels

Answer 15

• Allow net flux of specific ions (e.g. Na+, K+, Ca2+, Cl- ) across a membrane down their electrochemical gradients ONLY.
• Only undergo one conformational change when they open or close.
• Unlike transporter proteins, they do not need to change their conformation to move each ion across the membrane.
• So they are capable of maintaining solute fluxes that are about a 1000-fold higher than those of transporters – up to about 10^6 ions per second.

Question 16

Ion channel structure

Answer 16

• One or more membrane-spanning protein subunits surrounding a central pore lined with hydrophilic R – groups. (aqueous)
• The subunits around the pore are often (but not always) formed from membrane-spanning alpha-helices – as many as 4 subunits with 6 alpha helices each in some channels (can be complicated)

Question 17

Ion channel general structure

Answer 17

• Ion channels are often highly selective – mostly allowing only one type of ion to pass through
• The pore appears to consist of a double funnel, with wide openings at the surfaces of the membrane and a narrow region further in the lipid bilayer.
• The narrow region is the place where ion selectivity is believed to occur.

Question 18

Ion channel regulation

Answer 18

• Ion channels are not always open - the central pore can be “gated” open and shut by changes in channel conformation.
• The conformational change that opens a channel is generally initiated in some region remote from the pore, eventually being transmitted to the pore domain so as to open it
• The change in conformation that opens the channel can be driven by different things depending on the channel type

Question 19

Ligand-gated ion channels

Answer 19

Change in conformation that opens the channel is driven by the binding of ligands (small molecules or other proteins) to the channel

Question 20

Voltage-gated ion channels

Answer 20

Change in conformation that opens the channel is driven by changes in membrane potential

Question 21

Mechanosensitive channels

Answer 21

• Change in conformation that opens the channel is driven by distortion of the bilayer
• Responsible for sense of touch or pain

Question 22

Temperature - sensitive channels

Answer 22

Change in conformation that opens the channel is driven by temperature

Question 23

Passive Transport – Facilitated Diffusion

Answer 23

• Transport of neutral, polar molecules larger than water or urea, such as glucose - or charged molecules such as amino acids
• Not coupled to any external energy source, such as ATP
• > The direction of net flux follows the electrochemical gradient for whatever molecule is transported.
• Solute molecule binds tightly to a highly specific site on the protein and causes a conformational change in the transporter protein (one side of the membrane to the other)
• > Conformational change may occur only when the solute binds to the membrane protein OR it may happen randomly regardless of if the solute is bound
• Transition between A and B is random/reversible, does not depend on whether solute binding sites are occupied.

Question 24

Facilitated transport proteins

Answer 24

• Like enzymes, do not alter deltaG for transport – movement is always down the electrochemical gradient for the solute - they just speed up movement
• Have a relatively slow turnover compared to channels
• > Maximal transport rate ~1000 molecules per transport protein per second
• > Compared to 1 million molecules for channels

Question 25

Facilitated transport - Glucose transport

Answer 25

• Humans have 5 related glucose facilitated transport proteins, One of these, GLUT4 is common to insulin-responsive cells
• Glucose in higher concentration in blood than in cells
• Insulin promotes the insertion of GLUT4 transporters into the membrane of target cells, promoting glucose uptake
• Passive transport

Question 26

Active transport

Answer 26

• Movement of solutes UP their electrochemical gradients.
• Can create and maintain concentration gradients of solutes across membranes.
• Can sometimes directly contribute to membrane potentials (can be “electrogenic”) if the transported solutes carry a net charge across the membrane.
• 3 types of active transport

Question 27

ATP-ase pumps

Answer 27

• One type of active transport
• Couple movement of solutes to ATP hydrolysis
• 3 types of ATP-ase pumps

Question 28

One type of active transport

Answer 28

Other pumps that use diverse energy sources (e.g. light, oxidation of NADH)

Question 29

Coupled transporters

Answer 29

• Link the movement of one solute up its electrochemical gradient to the movement of another solute down its electrochemical gradient
• Their energy source is therefore an existing electrochemical gradient
• 2 types of coupled transporters

Question 30

Uniport

Answer 30

Moves just one substance across the membrane

Question 31

Symport

Answer 31

• One type of coupled transporter

- Moves two substances across the membrane in the same direction (regardless of electrochemical gradient)

Question 32

Antiport

Answer 32

• One type of coupled transporter

- Moves two substances across the membrane in opposite directions (regardless of electrochemical gradient)

Question 33

“P” type pumps

Answer 33

• One type of ATPase pump
• “P” stands for the use of a high energy phosphoprotein intermediate in this pump type
• Pump H+, K+, Na+, Ca2+

Question 34

P-type Pump Example 1: Ca2+ ATPase

Answer 34

• Like Na+, Ca2+ ions are kept at very low concentrations in the cell relative to the extracellular environment:
• > Inside the cell: [Ca2+] ~ 0.1µM
• > Outside the cell: [Ca2+] ~ 103 µM
• Even the smallest Ca2+ cytosolic influx can lead to a massive increase in cell levels.
• Regulated Ca2+ influxes form the basis of a variety of cell signaling events.
• Helps maintain a very low resting Ca2+ concentration in the cytosol.
• Transports one Ca2+ ion out of the cell for every ATP molecule consumed.
• Plasma membrane, smooth ER, sarcoplasmic reticulum (muscle ER)
• Creates a calcium ion store within the smooth ER

Question 35

P-type Pump Example 2: Na+/K+ Pump

Answer 35

• Smaller concentration of Na compared to K inside the cell vs smaller concentration of K compared to Na outside the cell
• Each cycle of the sodium/potassium pump (Na/K ATP-ase) transports:
• > 3 Na+ out of a cell
• > 2 K+ into a cell (Causes a net +1 charge out of the cell per cycle & electrogenic)
• > All for the hydrolysis of only one ATP molecule
• The pump has specific binding sites for sodium and potassium.
• The binding and release of sodium and potassium ions ion either side of the membrane is coupled to ATP-hydrolysis
• Each step in the hydrolysis of ATP and the binding of the transported ions drives conformational changes that create a pumping cycle by alternately exposing the ion binding sites to one side of the membrane and then the other.
• Antiporter: pumps 2 different ions in opposite directions

Question 36

F-type pumps

Answer 36

• One type of ATPase pump
• F type pumps are found in membranes of:
• > Bacteria
• > Mitochondria
• > Thylakoid (chloroplast)
• They generate ATP through use of H+ gradient, so not necessarily pumps
• Essentially V type pumps working in reverse
  If the concentration gradient waas reversed, then the pumps could use ATP to pump molecules up the concentration gradient
• Are often called ATP synthases because they use H+ gradients to drive the synthesis of ATP from ADP + Pi.
• > Electron Transport Chain in Mitochondria (last stage of cell respiration)
• > Electron Transport in Chloroplasts (light reaction step)
• > Light-activated Bacteriorhodopsin

Question 37

V-type Pumps

Answer 37

• One type of ATPase pump
• Made up of multiple subunits
• Use ATP, but not via a phosphorylated intermediate.
• V type pumps are found in membranes of:
• > Lysosomes
• > Synaptic vesicles
• > Plant vacuoles
• Regulate the pH environment by pumping H+ into these compartments to make them more acidic
• ATP hydrolysis doesn’t phosphorylate the pump, but drives a rotary motion that pumps molecules across the concentration gradient

Question 38

ABC Transporters

Answer 38

• One type of ATPase pump
• ATP Binding Cassettes
• Multiple domains (usually dimeric meaning 2 subunits)
• Many pump small molecules, rather than ions
• Some pump ions

Question 39

ABC Transporters Example: MDR proteins

Answer 39

• The gene responsible for multidrug resistance in cancer cells is an ABC transporter.
• Cancer cells overexpress this protein, which pumps hydrophobic molecules, including some drugs, out of the cytosol, making them resistant to toxic anticancer drugs.
• Up to 40% of human cancers may develop multidrug resistance