Membrane Permeability, Cell Volume and pH Regulation

Question 1

Which molecules can diffuse freely through a lipid bilayer?

Answer 1

Hydrophobic molecules (e.g. oxygen, carbon dioxide, nitrogen and benzene) and small uncharged polar molecules (e.g. water, urea and glycerol).

Question 2

What are the units for membrane permeability coefficients?

Answer 2

cm per second

Question 3

What variables are passive diffusion dependent on?

Answer 3

Permeability and concentration gradient. Rate of passive transport increases linearly with increasing concentration gradient.

Question 4

How is the net rate of transport calculated?

Answer 4

J = P (C1 – C2)

Where: P = permeability coefficient C1 and C2 = concentration gradients on side 1 and 2

Question 5

How can permeability coefficients be increased in the plasma membrane?

Answer 5

By the presence of proteins- ion channels and transporters.

Question 6

What are the roles of transport processes?

Answer 6

• Maintenance of ionic composition
• Maintenance of intracellular pH
• Regulation of cell volume
• Concentration of metabolic fuels and building blocks
• The extrusion of waste products of metabolism and toxic substances
• The generation of ion gradients necessary for the electrical excitability of nerve and muscle

Question 7

What is the stupid name for the way carrier molecules work? And what does he actually mean?

Answer 7

‘Ping-pong transport’ This refers to the conformational change of the transport protein with a gated pore, which allows transport through the membrane.

Question 8

What mechanism of transport is mediated by ion carriers?

Answer 8

Facilitated diffusion.

Question 9

Give examples of ligand gated ion channels.

Answer 9

Nicotinic acetylcholine receptor, ATP-sensitive potassium channels, GABA A and C channels.

Question 10

How do voltage gated ion channels work? And give examples.

Answer 10

They are ion channels that contain a positively charged voltage sensor. When the membrane is depolarised, the outside of the membrane is relatively negatively charged compared to the inside of the membrane. This results in movement of the voltage-sensor residues in the protein to move toward the part of the protein near the outside of the membrane, resulting in a conformation change in the ion channel, allowing diffusion of ions through the channel pore. Examples include voltage-gated sodium channels, voltage-gated potassium channels, voltage-gated calcium channels and NMDA receptors (although NMDARs are also ligand gated- glycine).

Question 11

How does rate of facilitated diffusion compare to that of simple diffusion?

Answer 11

Facilitated diffusion gives a hyperbolic curve when plotted on a graph (Transport rate vs Concentration of substance). Facilitated diffusion occurs much faster than simple diffusion, and it is saturable- reaches Vmax when all ion channels are saturated. Simple diffusion occurs linearly at a much slower rate. Both processes are affected by concentration gradient- rate slows and eventually stops when concentration gradients on the inside and outside of the membrane are equal.

Question 12

What are aquaporins?

Answer 12

Water channels in the plasma membrane of some cells, e.g. kidney proximal tubule that facilitate the movement of water across the membrane.

Question 13

How does water move across a membrane?

Answer 13

Osmosis.

Question 14

Why can’t hydrophilic molecules or ions cross the cell membrane?

Answer 14

Energetically unfavourable- a large free energy change would be required for a small hydrophilic molecule or ion to transverse the hydrophobic core of the lipid bilayer. Movement of such molecules across an intact lipid bilayer would be a rare event.

Question 15

Which proteins in a membrane have gated pores?

Answer 15

• Ligand-gated ion channels - open or close in response to ligand binding to a receptor site
• Voltage-gated ion channels - open and close in response to the potential difference across the membrane (Sessions 3, 4 and 5)
• Gap junctions (connexin) - closed when cellular calcium concentration rises above 10 uM or the cell becomes acidic.

Question 16

What is active transport?

Answer 16

A method of transport that occurs against the electrochemical gradient across a membrane and therefore requires energy. It must be coupled to a thermodynamically favourable reaction.

Question 17

Where does the energy from ATP come from?

Answer 17

The free energy to drive active transport can come either directly or indirectly from the hydrolysis of ATP, electron transport or light.

Some cells may spend up to 30-50% of their ATP on active transport.

E.g. Na+-K+-ATPase (Na+ pump) pumps 3 Na+ ions outwards, 2 K+ ions inwards, against the respective concentration gradients, at the expense of one ATP molecule hydrolysed.

N.B. if the pump runs in reverse it can act as an ATP generator. In mitochondria, a gradient of H+ ions in employed to drive ATP synthesis via an ATP-dependent proton transporter (ATP synthase).

Question 18

What is secondary active transport?

Answer 18

When the transport of one substance is linked to the concentration gradient for another via a co-transporter. This is known as secondary active transport, as the primary energy source, e.g. hydrolysis of ATP, is used indirectly. Membrane transporters may be driven by gradients of ATP, phosphoenolpyruvate, protons and sodium ions, light and high-potential electrons. Often a sodium gradient across a membrane is employed.

Question 19

Name 3 co-transport systems in cells.

Answer 19

• Na+- glucose co-transport system of the small intestine and kidney (symport). Entry of sodium provides the energy for the entry of glucose.
• Na+- Ca2+-exchange - Inward flow of sodium down its concentration gradient drives outward flow of Ca2+ up its concentration gradient (antiport).
• Na+- H+- exchange - Inward flow of sodium down its concentration gradient leads to cell alkalization by removing H+ (antiport).

Question 20

What do uniport, symport and antiport mean?

Answer 20

When one solute molecule species is transported from one side of the membrane to the other, the transporter is called a uniport.

Other transporters are referred to as co-transporters, when the transfer of one solute molecule depends on the simultaneous or sequential transfer of a second solute in the same direction (symport) or in the opposite direction (antiport).

Question 21

What is the formula to determine whether active or passive transport has occurred?

Answer 21

RTloge(C2/C1)+ ZF

Question 22

What 2 factors influence membrane transport?

Answer 22

Concentration ratio and membrane potential (mV).

Question 23

What are the intra- and extracellular physiological concentrations of ions?

Answer 23

Sodium- 145mM extracellularly, 12mM intracellularly.

Potassium- 4mM extracellularly, 155mM intracellularly.

Calcium- 1.5mM extracellularly, 10^-7M (10uM) intracellularly.

Chloride- 123mM extracellularly, 4.2mM intracellularly.

Question 24

What is primary active transport?

Answer 24

Transport that directly involves the hydrolysis of ATP.

Question 25

What is cotransport?

Answer 25

More than one type of ion or molecule may be transported on a membrane transporter per reaction cycle.

Question 26

What is the sodium pump (Na+K+-ATPase)?

Answer 26

Plasma membrane associated pump, which uses ATP to exchange 2 K+ ions for 3 Na+ ions, creating the Na+ K+ concentration gradient.

It uses 25% of Basal Metabolic Rate.

It’s called a P-type ATPase.

ATP phosphorylates one of its Aspartate residues during the reaction cycle, producing a phosphoenzyme intermediate.

Contains 1 alpha and 1 beta subunit

Inhibited by ouabain, which binds to the alpha subunit. Also inhibited by digoxin. Both of these are called cardiac glycosides.

Alpha subunit contains potassium, sodium and ATP binding sites.

Beta subunits contain surface oligosaccharides- glycoprotein directs pump to surface.

Given appropriate gradients for Na+ and K+ the Na+ pump can operate in reverse as an ATP synthetase.

Na+ pump activity is necessary for absorption of Na+ through epithelia.

Na+ pump activity contributes to cell volume regulation

Na+ pump activity contributes to cellular pH regulation

Question 27

How is the membrane potential created across the plasma membrane?

Answer 27

Na+K+-ATPase (sodium pump) creates high intracellular [K+]

K+ diffusion through channels is mainly responsible for membrane potential (-70mV)

Sodium pump generates ONLY about – 5-10mV through electrogenic pump activity

Question 28

How is calcium ion transport across the plasma membrane mediated?

Answer 28

Na+-Ca2+-exchanger

• has low affinity and high capacity for calcium ions (used in high intracellular concentrations of calcium)
• secondary transporter that uses inward Na+ gradient established by sodium pump
• antiport mechanism

Question 29

What is the role of the Na+H+ exchanger?

Answer 29

Antiport ion carrier that is important in control of pH (H+ transport) and regulation of cell volume (Na+ has osmotic influence).

Inward flow of Na down its concentration gradient leads to cell alkalinisation by removing H+.

Exchanges extracellular Na+ for an intracellular H+.

Electroneutral 1:1 exchange.

Activated by growth factors and inhibited by amiloride.

Question 30

What is the role of the Na+-Glucose cotransporter, and where is it found?

Answer 30

Symporter found in the kidneys and small intestine.

Entry of Na+ down the concentration gradient provides the energy for the entry of glucose against concentration gradient.

Question 31

How is membrane transport disrupted in cystic fibrosis?

Answer 31

The Cystic fibrosis transmembrane conductance regulator is a chloride ion channel that is either misfolded or does not reach the plasma membrane in cystic fibrosis suffers.

The lack of chloride transport out of the cell results in lack of sodium and water transport to the outside of the cell, rendering mucus more viscous and dehydrated than normal.

This can have different effects in different parts of the body, e.g. pancreas (blocking of pancreatic duct, leading to pancreatitis) and in the small intesting (leading to malabsorption of fats).

Question 32

How is membrane transport disrupted in diarrhoea?

Answer 32

In diarrhoea, an increase in the activity of Protein kinase A on CFTR results in an increase in chloride transport out of the cell. This results in increased movement of sodium and water into the lumen of the bowel, resulting in excess water loss in faeces.

Question 33

What are the functions of Na+K+-ATPase?

Answer 33

• Forms Na+ and K+ gradients
• Drives many secondary active transport processes:

o Ion homeostasis

o intracellular Ca2+ ion concentration - [Ca2+]i

o pHi

o cell volume

o ion gradients underpinning resting membrane potential

o nutrient uptake

Question 34

Why is intracellular calcium kept at such a low level compared to the extracellular fluid?

Answer 34

There is an ~20,000 fold difference in Ca2+ concentration across the plasma membrane (~2 mM/50-100 nM (extracellular/intracellular).

High [Ca2+]i is toxic to cells and therefore needs to be controlled.

Several important cellular processes rely on a small increase in intracellular calcium concentration in order to work, e.g. neurotransmitter release, some signalling cascades and muscle contraction.

Question 35

How is the resting intracellular concentration of calcium controlled?

Answer 35

– PMCA expels Ca2+ out of the cell

• Primary active transport

– SERCA accumulates Ca2+ into the SR/ER

• High affinity, low capacity (removes residual Ca2+)

• Secondary active transport – Na+-Ca+-exchange (NCX)

• Low affinity, high capacity (removes most Ca2+)

– Mitochondrial Ca2+ uniports

• Operate at high [Ca2+]I to buffer potentially damaging [Ca2+]

Question 36

What is the sodium calcium exchanger?

Answer 36

The NCX exchanges 3 Na+ for 1 Ca2+ and is, therefore, electrogenic with current flowing in the direction of the Na+ gradient.

In depolarised cells, the normal mode of operation of NCX is inhibited and its mode of operation reverses, i.e. to bring Ca2+ into the cell.

In this way NCX makes a contribution to Ca2+ influx during the cardiac action potential and can contribute to Ca2+ toxicity during periods of ischaemia.

Question 37

How does the sodium calcium exchanger contribute to calcium ion toxicity in ischaemia?

Answer 37

ATP depleted –sodium pump inhibited [Na+]in accumulates

Cell depolarised

NCX reverses- [Na+]in exchanges for [Ca2+]out

High [Ca++]in toxic

Question 38

How is intracellular pH controlled in cells

Answer 38

When cellular buffering capacity is exceeded, cellular pH is controlled by the activity of a variety of plasma membrane transporters.

Acidification can be opposed by expelling H+ ions or the inward movement of bicarbonate ions.

Alkalinisation is opposed by expelling bicarbonate via the anion exchanger.

Question 39

Which ion transporters are involved in control of intracellular pH and what do they do?

Answer 39

All cells: Na+-K+-ATPase- Creates Na+ gradient to provide energy for following transporters:

• Most cells: Na+-H+- Exchange (Acid extrusion)
• Some cells: Na+-Cl–HCO3–H+-co-transporter (NBC) (coupled Na+-H+- and anion exchange) (Acid extrusion and alkali influx).
• Some cells: Na+-HCO3–co-transport (Alkali influx)
• Most cells: Anion exchange (AE) (Band 3) (Alkali extrusion).

Question 40

Draw a diagram showing an overview of the control of intracellular pH.

Answer 40

Question 41

How is cell volume regulated?

Answer 41

Electroneutral transport of ions allows the osmotic strength of the cytoplasm to be varied without effect on the membrane potential.

Cells extrude ions in response to cell swelling and influx ions in response to cell shrinking.

Water follows.

Different cell types use particular combinations of transporters to achieve the regulation they need.

Question 42

Draw a diagram to show how cells resist cell shrinking.

Answer 42

Question 43

Draw a diagram to show how cells resist cell swelling.

Answer 43

Question 44

Why is it important for ion transporters to work together?

Answer 44

By working together, ion channels can achieve physiological endpoints that would not be possible if they worked in isolation, e.g. bicarbonate reabsorption in the proximal tubule of the kidney, Na+ reabsorption in kidney tubules.

Combinations of transporters to achieve the regulation they need.

Question 45

Why, under normal circumstances, does the kidney reabsorb all the bicarbonate filtered into the proximal tubule?

Answer 45

The main reason is to retain base for pH buffers.

Question 46

How are sodium and bicarbonate reabsorbed by the proximal tubule?

Answer 46

Cells in the proximal tubule contain many ion channels that work together to drive reabsorption. In the lumen, NaHCO3 is broken down to Na+ and HCO3-. Na+ is transported by NHE into the cell and reabsorbed into the capillaries through the sodium pump. When transported by NHE, the sodium is exchanged for a H+ ion, which enters the lumen, combining with HCO3- to make H2CO3. This is broken down by carbonic anhydrase to produce CO2 and H2O, which readily diffuse through the membrane into the epithelial cell, where it is catalysed by carbonic anhydrase back into H2CO3 (H2O and CO2 from metabolism of epithelial cell and CO2 from blood also undergo this reaction). H2CO3 is broken down to H+ (reused for NHE transport) and HCO3-, which is transported back into the capillaries by AE in exchange for a chloride ion.

Question 47

Why is renal control of Na+ uptake clinically important?

Answer 47

Renal control of circulating Na+ concentration is often a first line treatment for mild hypertension (diuretics (‘water tablets’)). Where fluid loss is required to treat oedema or hypertension, block of one or more of the Na+ reabsorption mechanisms with diuretic drugs can be used to increase Na+ excretion to produce a hyperosmotic urine and, hence, the excretion of water.

Question 48

Where does reuptake of sodium occur in the kidney?

Answer 48

The distal convoluted tubule, thick ascending limb and the cortical collecting duct.

Question 49

Which drugs target the thick ascending limb of the kidney?

Answer 49

Loop diuretics.

Question 50

How is sodium reuptake regulated in the thick ascending limb of the kidney, and how is this clinically important?

Answer 50

In the thick ascending limb of the kidney, magnesium and calcium ions are transported between epithelial cells to be reabsorbed into the capillaries. On the lumenal side of the epithelial cell, the NKCC2 transporter transports Na+, K+ and 2Cl- from the lumen into the epithelial cell. K+ and one Cl- are transported into the capillaries by KClCT. The second Cl- is transported into the capillaries by ClC-Kb. The sodium ion (along with 2 others) is extruded from the cell into the capillaries by the sodium pump in exchange for 2K+ from the blood (using ATP). The K+ ions from this are extruded from the epithelial cell through ROMK (a K+ channel) on the lumenal side of the plasma membrane.

Loop diuretics target and inhibit NKCC2, thereby inhibiting resorption of Na+ and other osmotic ions into the blood, producing hyperosmotic urine, leading to increased excretion of water in the urine.

Question 51

How is sodium reuptake regulated in the distal convoluted tubule, and how is this clinically relevant?

Answer 51

On the lumenal side of the epithelial cells in the distal convoluted tubules, there are 3 ion transporters: NCCT that transports Na+ and Cl- into the epithelial cell from the lumen, TRPM6- a channel that allows Ca2+ and Mg2+ into the epithelial cell from the lumen and ENaC- a non voltage gated sodium channel that allows Na+ into the epithelial cell from the lumen.

On the opposite side of the plasma membrane nearest the capillary, there are 4 ion transporters: ClC-Kb- a channel that allows Cl- through the plasma membrane into the capillary, KClCT- a cotransporter that transports K+ and Cl- into the capillary, NCX- an exchanger that transports Ca2+ into the capillary in exchange for 3Na+ from the capillary and Na+K+ATPase, which uses ATP to transport 3Na+ into the capillary in exchange for 2K+ against the concentration gradient.

Reuptake of sodium is inhibited by blockade of NCCT by Thiazides and ENaC by Amiloride (Side effects on NHE in bicarbonate transport).

Question 52

How is sodium reuptake regulated in the cortical collecting duct, and how is this clinically relevant?

Answer 52

On the lumenal side of epithelial cells in the cortical collecting duct, there are 4 transporters: an aquaporin channel that allows water to pass more easily into the epithelial cell from the lumen (from there diffusing through the plasma membrane on the other side into the blood), a ROMK channel that allows K+ from the epithelial cell into the lumen, ENaC- a channel that allows Na+ into the epithelial cell from the lumen and ClC- a chloride channel that allows Cl- into the epithelial cell from the lumen.

On the capillary side of the epithelial cell, there are 3 transporters: a ROMK channel that allows K+ into the capillaries from the epithelial cell, a sodium pump that transports 3Na+ into the bloodstream in exchange for 2K+ against the concentration gradient and ClC, which allows Cl- into the bloodstream.

Antidiuretic hormone (ADH/Vasopressin) agonises the aquaporin channels in the epithelium of the cortical collecting duct, increasing the reuptake of water into the bloodstream.

Aldosterone agonises ROMK, increasing K+ movement into the lumen and capillary from the epithelial cell, ENaC, increasing uptake of Na+ from the lumen, and Na+K+ATPase, further increasing Na+ resorption into the bloodstream. This movement of sodium increases the resorption of water into the bloodstream, decreasing urine volume.

Spironolactone is a glucocorticoid receptor antagonist, and opposes the effects of aldosterone. It inhibits ROMK, decreasing K+ movement out of the epithelial cell, ENaC, decreasing movement of Na+ from the lumen into the epithelial cell, and Na+K+ATPase, inhibiting Na+ transport out of the cell into the bloodstream and K+ transport into the epithelial cell. Spironolactone is often given in conjunction with other treatments to increase efficacy.

Amiloride inhibits ENaC, decreasing uptake of Na+ from the lumen into the epithelial cell.

Question 53

What is hyperaldosteronism?

Answer 53

An excess of aldosterone secretion, which causes the retention of more sodium than normal, which may lead to hypertension.

Question 54

How are intracellular calcium levels raised in a cell?

Answer 54

• Facilitated diffusion

– Receptor-operated Ca2+ channels (ROC)

– Voltage-operated Ca2+ channels (VOCC, VGCC (gated))

– IP3-gated Ca2+ channels (IP3R)

– Ca2+ induced Ca2+ release (CICR) (Ryanodinesensitive Ca2+ channels)

– Store-operated Ca2+ channels (SOC)

– Mitochondrial Ca2+ uniporters

• Secondary active transport

– Na+-Ca+-exchange (NCX)- Reverse mode in depolarised cells

Question 55

Draw a diagram to show how NCX works in ventricular myocytes.

Answer 55

Question 56

What does passive transport depend on?

Answer 56

It depends on the free energy change of the transported species from a higher energy state to a lower energy state

Question 57

Indicate FOUR mechanisms that utilise carrier proteins.

Answer 57

Noradrenaline uptake into noradrenergic presynaptic varicosities
Noradrenaline uptake into secretory vesicles within noradrenergic presynaptic varicosities
Uptake of Na+, K+, Cl-ions from the renal tubule into cells of the thick ascending limb of the kidney
Uptake of Na+ and Cl-ions in the distal tubule of the kidney