Membrane mechanisms and epithelial transport

Question 1

What is diffusion?

Answer 1

Diffusion is the process by which random (Brownian) motion of the molecules of a substance results in the net redistribution of the substance towards a state of uniform distribution; i.e. the state of maximum entropy and thus minimum free energy.

Question 2

What is the formula for the relationship of the movement of an uncharged solute across a membrane?

Answer 2

the relationship known as Fick’s law:

φ = (D/x) (C2 -C1)

Where:
layer of thickness x
separating solutions of concentrations C1 and C2
φ is the rate of solute movement per unit cross sectional area (or the flux in the x direction) and has the units mmoles x cm-2 x sec-1 (# of mmoles going through a surface area per second) and D is the diffusion coefficient, a constant which applies to a particular solute particle in a particular medium at a given temperature.

φ = (Dapparent/xm) (C2 -C1) = (DmKmw/xm) (C2 -C1)

where Dm is the diffusion coefficient within the membrane and xm is the membrane thickness

Question 3

What is the value of D for small molecules in aqueous solutions?

Answer 3

1 x 10-5 cm2/sec

Question 4

What is the partition coefficient?

Answer 4

ratio of the solute concentration in the membrane to that in the aqueous phase

Question 5

Which studies found a relationship between permeability and partition coefficient

Answer 5

The studies of overton

Question 6

Which formula is used in real life considering many factors in fick’s law are not known with much precision?

Answer 6

using the “black box approach” it is often found that solute fluxes are proportional to the transmembrane concentration difference:
Φ = P (C2 -C1)
The constant of proportionality, P, is the permeability coefficient, an empirically determined constant
commonly used to express “membrane permeability” to a given solute. (replaces (DmKmw/xm))

Question 7

As solutes are less polar, what happens to the permeability through the membrane?

Answer 7

It increases

Question 8

What factors does the permeability coefficient rely on?

Answer 8

that for a given membrane, the relative permeability coefficients for different solutes will depend on

• the relative membrane/water partition coefficients
• the size of the solute molecules
• The polarity of the molecules

Small, uncharged molecules and larger molecules that are lipophilic may cross biological membranes at appreciable rates by simple diffusion through the lipid bilayer.

Question 9

Name molecules that cross biological membranes by simple diffusion through the lipid bilayer.

Answer 9

all gases (O2, CO2), water, urea, glycerol, some hormones (eg T3), and many drugs (eg. digoxin).

Question 10

What is the formula for net water flux in the kidneys

Answer 10

JH20 = P (πeff2 - πeff1)

where P is the water permeability (typically 10-4-10-5 cm/sec) and πeff is the effective osmotic pressure

Question 11

How is πeff calculated?

Answer 11

πeff=RTσC,

where
R = gas constant (l·mmHg·mOsm-1·deg-1) 
T = temperature (deg K)
σ = reflection coefficient (no units; 0 1) which depends on the relative permeability of the membrane to solute and water: σ = 0 when the solute is freely permeant through the membrane and σ = 1 when it is impermeant.
C = osmotic concentration (mOsm·l-1)

Question 12

Which hormone increases P of water by ~ 4 folds?

Answer 12

ADH

Question 13

How is water reabsorbed in the proximal tubule?

Answer 13

In the proximal tubule, passive water reabsorption occurs around the cells through the tight junctions, and also through the cell membranes via CHIP28, a specialized membrane protein which serves as a water- selective channel.

Question 14

How is filtered bicarbonate reabsorbed?

Answer 14

Most bicarbonate filtered is reabsorbed as gaseous CO2.

Question 15

How is CO2 made in the lumen from bicarb?

Answer 15

by the combined effects of H+ extrusion and the enzyme carbonic anhydrase (c.a.)

Question 16

Which carbonic anhydrase performs this work of H2CO3 to CO2?? where is it located?

Answer 16

C.A4 is bound to the apical surface of the proximal tubules; catalyzes reaction of production of CO2 from filtered bicarb

Question 17

Which CA reconstructs bicarb in the cell?

Answer 17

CA2

Question 18

What happens when CO2 and HCO3 are added to the kidney tubules in terms of pH changes?

Answer 18

a) 5% CO2 and 25% HCO3- infused –> acidification of intracellular pH
b) To c) Spontaneous recovery of the pH
c) Removal of perfusion (no bicarb or CO2 present) –> strong alkalinisation
d) To e) spontaneous recovery

Question 19

What happens to the cell pH when NH4+ is added in tubules?

Answer 19

NH4+ enters the cell as NH3 and thus gives out a proton in the tubule
NH3 is a base and non-polar, thus it can enter the cell and pick up a proton
NH3 alkalinizes the inside of the cell until NH4+ is slowly produced

Question 20

Name examples of uncharged lipophilic molecules that can permeate through cell membranes

Answer 20

Drugs that are weak acids (e.g. salicylic acid from aspirin) can permeate through cell membranes in their undissociated acid form. Undissociated acids are usually written as HA
Many drugs diffuse through the lipid bilayer by non-ionic diffusion, e.g. salicylic acid as the undissociated acid

Question 21

What happens to filtered weak acids?

Answer 21

A) Weak acids can escape through nonionic diffusion; thus secreted H+ can titrate filtered A- to form HA (equilibrium) which can be reabsorbed

Question 22

What happens to filtered neutral weak bases?

Answer 22

B) Non-ionic diffusion of neutral weak base: secreted H+ can be titrated by filtered and secreted B to form BH+ and be trapped in –> increased excretion

Question 23

How does a neutral weak acid and its conjugate base’s clearance change as pH of urine decreases?
Name an example of a neutral weak acid drug. what can we do do excrete it?

Answer 23

Clearance decreases
e.g. of neutral weak acid drug = Salicylate

Thus if we want to excrete it we need to induce an alkaline diuresis

Question 24

How does a neutral weak base and its conjugate acid’s clearance change as pH of urine decreases?
Name an example of a neutral weak base drug

Answer 24

D) Clearance of neutral weak base and conjugate acid increases as pH decreases (more acidic)

e.g. of neutral weak base: Quinine (treatment of malaria)

Question 25

Name 3 characteristics that carriers and channels exhibit. Explain them.

Answer 25

• Specificity
  means that only one substance, or a small group of related substances, can bind to the transporter and permeate the membrane.
• Saturation
  the fact that transport rate reaches some maximum (Vmax) when the concentration of solute is elevated.
• Competition
  a second solute may also bind to the transport site, although it doesn’t necessarily have to be transported.

Question 26

Which equation can describe carrier and channel-mediaed transport?

Answer 26

the Michaelis-Menten equation

Initial flux rate = Vmax / (1+(Km/[A]))

Question 27

What is the most popular explanation for carrier-mediated translocation?

Answer 27

“gated pore” mechanism: When a solute molecule (or an ion) binds at one side, there is a conformational change in the protein which opens a pore to the other side. This cycle is repeated each time a molecule passes through the protein. The conformational changes of the protein may be quite subtle but are not instantaneous and may set a limit on the turnover number - the number of times one transporter can do its thing in one second.

Question 28

What is the turnover number of anion exchangers? Of Na-glucose cotransporters?

Answer 28

Turnover numbers vary depending on the type of carrier;

• anion exchangers have turnover numbers up to 50,000/sec (because one conformational change can lead to the movement of millions of ion, not only 1-2) …
• The Na-glucose cotransporter has a turnover number of only ~5/sec.

Question 29

Compare turnover number of carriers vs. channels and explain why there is a difference.

Answer 29

Channels are also intrinsic membrane proteins that contain a pore and a gate. However, each time a channel gate opens, millions of ions pass through rather than just one or two. Consequently, channel turnover numbers are typically 2-100 million/sec, several orders of magnitude higher than the fastest carriers. Turnover number has been used as a criterion for distinguishing between carriers and channels. When ions flow passively through an open channel, they can produce electrical currents that are large enough to be measured experimentally.

Question 30

Why does thiourea not lyse RBCs as fast as urea despite it being nonpolar?

Answer 30

Urea clear because urea is taken up rapidly by RBC membrane (UT2) carries urea very efficiently
When put in isotonic urea solution, then urea goes quickly inside cell —> osmosis —> quick lysis
Thiourea takes 5-6 seconds longer for cells to lyse. Even though less polar (should go more easily through membrane); because of specificity of UT2 transporter for urea over tiourea

Question 31

What happens to RBCs if put in isotonic Na solution?

Answer 31

Nothing, no lysis