L15 - L19: Cellular Processes Flashcards
cell membrane function (extra)
movement in and out of cell governed by physical forces which allows:
- maintenance of conc. grad.
- spatial organisation of chemical/physical processes within cell
- development of membrane potential
- controlled uptake of nutrients, discharge of waste products and secretion of molecules
membrane structure
thin, 8nm, flexible, sturdy barrier surrounding cell cytoplasm
- 50% lipid, 50% protein held together by hydrogen bonds
membrane lipids structure
two back to back layers of 3 types of lipid molecules:
- phospholipid
- cholesterol
- glycolipids
membrane lipids function
barrier to entry/exit of POLAR substances
phospholipids
- 2 parallel layers of molecules
- each amphipathic: charged polar and uncharged nonpolar region
- membrane thickness determined by tail length
phospholipid abundance
75%
cholesterol
steroid with attached -OH
cholesterol abundance
~20%
glycolipids
lipids with attached carbohydrate group
glycolipid abundance
~5%
glycolipid and cholesterol location
scattered among phospholipid bilayer
membrane protein function
‘gate keepers’ - regulate traffic
- different cells => diff. proteins => diff. properties
peripheral protein features
easily removed
- only bond attaching protein needs to be broken e.g by changing salt conc., washing with other molecules
peripheral protein function example
may attach integral protein to cytoskeleton to bend and give cell shape
integral protein features
not easily removed
- bilayer needs to be broken up e.g by using detergent
transmembrane proteins are
amphipathic
- hydrophobic regions with nonpolar amino acids coiled into helices spanning hydrophobic core of lipid bilayer
- hydrophilic ends interacting with aqueous soln.
amphoteric
- react as both acid and base
membrane protein transport function
- ion channels
- transporter proteins
membrane properties
- fluidity
- selective permeability
- gradients
fluidity
lipids/proteins move within plane but rarely between leaflets => lipid composition of leaflets can be asymmetric
factors affecting fluidity
chemical structure of looped molecules:
- lipid tail length: longer => less fluid
- no. double bonds: more => more packing is upset => more fluid
- amount of cholesterol: more => less fluid
permeability
ability of particular molecules to cross cell membrane
- governed by laws of diffusion
permeability depends on
- size
- charge
- lipid solubility
lipid bilayer is permeable to
- nonpolar, uncharged molecules
- lipid soluble molecules
- small uncharged polar molecules
lipid bilayer is impermeable to
- large uncharged polar molecules
- small ions (due to charge)
transport mediated by membrane proteins
membrane gradients
- conc. grad
- electrical grad
conc grad
- uncharged molecules diffuse down conc grad
- established by selective permeability
electrical grad
- ions influences by membrane potential in addition to conc grad => net ion movement influences by electrochemical grad (effect of both conc and electrical grad)
effect of membrane potential
- maintains difference in charged ions between inside/outside membrane
- membranes mimic capacitors (separate and store charge/energy) => energy generated when ions allowed to flow
energy required to maintain gradients
~30% of resting energy used
- gradient represents stored energy
hyperpolarisation
cell becomes more negative
depolarisation
cell becomes less negative
typical extracellular ion conc
positive
typical cytoplasmic ion conc
negative
Na+ conc
high outside - low inside cell
K+ conc
high inside - low outside cell
- equilibrium at -80mV membrane potential (if less, K+ moves out)
- moves down conc grad until elect grad stops it as leaving -ve charge every time K+ leaves make it more difficult for next K+ to leave
Cl- conc
high outside - low inside cell
- at equilibrium no net movement
- depolarised => Cl- into cell, conc grad takes over
- hyperpolarised => Cl- out, elect grad takes over
passive and active transport classification
- use of energy (passive only uses their kinetic energy)
- direction in relation to grad. (down or against grad)
vesicular transport
moves materials across membranes in small vesicles by exocytosis or endocytosis
non-mediated transport
doesn’t directly use a transport protein (diffuse through bilayer)
diffusion
- random mixing of particles in a solution as a result of particles’ kinetic energy
- more molecules move down conc. grad (net diffusion)
- eventually reach equilibrium - evenly distributed - no net movement
factors affecting diffusion
- steepness/magnitude of diffusion/conc grad: greater difference => faster
- temperature: higher => faster
- mass/size of diffusing substance: larger => slower
- surface area: larger => faster (e.g invaginations in membrane)
- diffusion distance: small => faster
a. cell size: rate sets limit on size of cells at ~20µm
b. membrane thickness: thicker => slower
diffusion function
- nutrient absorption
- waste excretion
- gas exchange
diffusion transports
nonpolar, hydrophobic molecules
osmosis
only occurs if membrane is permeable to water but not to certain solutes
semi-permeable
more permeable to water than solute
osmosis net movement
only in one direction and if an osmotic gradient exists as water moves to eliminate it
ion movement and water movement
closely linked
ion movement => create osmotic difference => stimulate water movement
Pw =
Pd + Pf
Pd
through lipid bilayer
- small
- insensitive to mercury (more to cholesterol)
- temp. dependent (more fluid => more water goes through this way)
Pf
through water channels
- large
- mercury sensitive
- temp. independent (not blocked by lipids in bilayer)
aquaporins
water channels that mediate Pf
- 9 different isoforms each with different permeabilities to water => varying Pw between cells
aquaporins location
- sweat glands have many
- some cells have none:
- very low water flow (Pw) as it only relies on small (Pd) even with osmotic difference
- ion movement without water movement (decoupling)
osmotic pressure
Hydrostatic pressure (acting against) applied to a soln. To prevent inward flow of water across semi-permeable membrane
- opposing force
- colligative property
- proportional to conc. of solute particles that can’t cross membrane
colligative property
depends only on number of solute not types/nature of particles in soln.
osmolarity
measure of solute (molecules/ions) in soln
= øic
- physically measured by osmometers
comparing osmolarity to reference soln
- hyposmotic: lower os
- isosmotic: same os
- hyperosmotic: higher os
water movement based on osmolarity
high to low osmolarity
osmolarity of body fluids
- 280 mOsmol
- maintained as isosmotic => no net water movement => no change in cell volume
- if solute leaks (water leaks) pump throws out again: if leak > pump = cell swelling
change in cell volume is generally
unfavourable esp. in some cells (e.g brain cells)
- body systems are thus involved in controlling osmolarity
tonicity
measure of soln’s ability to change cell volume by altering water content
- depends on membrane permeability of solute
osmolarity and tonicity
not always same thing
- osmosis when solutes impermeable but if they pass, different effect
- e.g urea: isosmotic, hypotonic
tonicity of soln
- isotonic: no change in cell vol
- hypotonic: cause cell swelling, eventually lysis (haemolysis in RBC)
- hypertonic: cause cell shrinkage/crenation
mediated transport
moves materials with help of transport proteins (ion channels, transporters)
ion channel structure
- water-filled pore
- lined by hydrophilic amino acids (ion charges) that shield ions from hydrophobic core
- hydrophobic amino acids facing lipid core of bilayer
- specific amino acids lining pore determine selectivity of channel to specific ions
ion channel mechanism/properties
- gating
- energy harnessing
- non-binding
- electrical current
ion channel gating
control opening/closing of pore controlled by different stimuli (can be coupled)
- voltage
- ligand binding (e.g neurotransmitter)
- cell volume (stretch/shrink)
- pH (measure/function of cell metabolism)
- phosphorylation of channel itself (using second messenger system: phosphorylate => ATP binds => opens, dephosphorylate => closes)
is the ion channel gate always open?
no, otherwise no electrochemical gradient ever due to diffusion maintaining equilibrium
energy harnessing
closed gate separates ions into ion conc grad => stores energy
- current/energy generated when ions allowed to flow
selectivity to particular ion and energy harnessing
harness energy stored in different/particular ion grad
non-binding
ions do NOT bind to channel => no restrictions once open => very rapid, nonstop transport/movement as long as electrochem grad present
electrical current
- diffusion of over 1 million ions per sec generates measurable current (~10^-12 amp) - larger if several channels synchronised
- fluctuations represent conformational changes in channel structure associated with gating
patch clamp technique
electrically isolate ion channel from the rest in the membrane to record current through an individual channel
carrier mediated transport
substrate directly interacts with transporter protein
- binds specific solute -> conformational change (series of events)
- moves molecules but does not change molecule itself
- slower than channels
transporter protein aka
carrier, permeases, transporters
transporter protein properties
similar to enzymes
- chemical specificity: need strong fit
- inhibition: blocking of binding pocket
- competition: competitive inhibition when two substrates bind to same pocket and are present together
- saturation (transport max.): limited no. binding pockets no matter increase in solute conc. (display classic enzyme kinetics)
while similar to enzymes, transporter proteins do NOT
catalyse chemical reactions, instead they mediate transport at faster than normal rates which would otherwise be too slow and cause cell death
passive carrier mediated transport
facilitated diffusion
- down grad. which in most cells is INTO cell but could be reversed as in epithelial cells
facilitated diffusion of glucose
uncharged => only conc grad and no elect grad
1) glucose binds to transport protein (GLUT)
2) transport protein shape change
3) glucose moves down grad. Across membrane
4) kinase enzyme reduces glucose conc. Inside cell by transforming glucose into glucose-6-phosphate
- maintains conc. grad for further glucose transport (otherwise stopped due to reaching equilibrium)
GLUT
glucose transporters
- 14 types
active carrier mediated transport
utilises metabolic energy to drive substances (molecules/ions) AGAINST conc./electrochem. grad
primary active transport
- energy directly derived from hydrolysis of ATP
- sets up ion grad (forms of stored energy) that secondary active transporters use
primary active transport example
Na/K ATPase, Ca/K ATPase (muscle sarcoplasmic reticulum), H/K ATPase (stomach- establish very low pH)
ATPase
transport proteins binding/hydrolysing ATP to provide enough E to cause conformational change and perform active transport
Na/K ATPase mechanism
1) Na+ binds
2) ATP split/hydrolysed by nucleotide binding domains to produce ADP and phosphate group
3) P (large -ve charge) binds to ATPase
4) conformational change
5) Na+ pushed out of cell
—
6) K+ binds
7) phosphate released
8) K+ pushed into cell
Na/K ATPase overall movement
3 Na+ removed, 2 K+ brought into cell
- net charge difference of 1 -ve charge
- maintains low Na+ and high K+ conc in cytosol
- pump generates net current and is electrogenic
electrogenic
produce change in electrical potential of cell
pump-leak hypothesis
ions continually leak back into cell (via channels etc.) down their respective grad (Na+ into, K+ out of cell) so pump continuously
- fine as long as pump rate keeps up with leak
importance of setting up ion conc grad
- Maintain resting membrane potential
- Electrical excitability
- created by electricity generated by ion channels etc. - Muscle contraction
- Maintenance of steady state cell volume
via secondary active transporters: - Nutrient uptake
- Maintenance of intracellular pH
secondary active transport
utilises energy stored in ionic conc grad
- indirectly uses energy obtained by ATP hydrolysis
- many powered predominantly by Na+ grad initially established by Na pump as it’s a more powerful grad than K, Ca etc. => lots of E stored
secondary active transporter examples
- Na+ antiporter/exchanger: Na+ inward, Ca2+/H+ pushed out
- Na+ symporter/cotransporter: Na+ inward, glucose/amino acid rush inward
antiporter/exchanger
ions in different directions
symporter/cotransporter
ions in same direction
ion channel vs. carrier mediated transport
- transport rate/speed
- mechanism
- properties associated with transport
Neighbouring epithelial cells are separated by
Intercellular/paracellular space and held together at luminal edges by tight junctions
Luminal edge
Closest to luminal side - exposed to outside/lumen
Tight junction appearance
- electron microscopy: membranes appear fused together
- freeze fracture: junction appear as interlocking network of ridges in plasma membrane
Tight junction structure
Composed of thin bands encircling the cell and make contact with those of adjacent cells
- membrane proteins interact across adjacent lipid bilayer to weld two cells together
Tight junction function
Separate epithelial cells into two distinct membrane domains
- barrier
- fence
Tight junction barrier function
restrict movement of substances through intercellular space
- good for stopping bacteria/viruses from entering across surface and into body
- allows certain classes of small ions, water etc.
- varied by varying structure of junctions
Tight junction fence function
Prevent membrane proteins from diffusing in plane of lipid bilayer
- implications/involvement in membrane domain formation and transport in epithelial cells
Epithelial cell membrane domains
- apical/luminal/mucosal: face lumen of organ/body cavity
- basolateral: adheres to adjacent basement membrane and interfaces with the blood
Tight junction tightness
Electrical resistance to ion flow through junction can be measured via measurement of voltage and current
- higher resistance = more tight junction strands holding cells together
Tight junction tightness trend
Changes (increases) in a proximal to distal direction in gastrointestinal tract and kidneys
Functional classification of epithelial tissue
1) leaky epithelium
- paracellular transport dominates
2) tight epithelium
- trans cellular transport dominates (junctions so tight very little movement occurs via paracellular pathway)
Types of epithelial transport
- paracellular transport
- transcellular transport
Paracellular transport
Through tight junction
- governed by laws of diffusion (no pump/transporters to mediate active transport) AND tightness of junctions
Transcellular transport
Through cell, using primary/secondary transport (set up conc grad) often in combo with passive diffusion through ion channels
- absorption: lumen -> blood
- secretion: blood -> lumen
Proximal tight junctions
- leaky epithelium
- low electrical resistance
- low no. Strands
- bulk transport (paracellular)
Proximal tight junctions examples
Duodenum, proximal tubule
Distal tight junctions
- tight epithelium
- high electrical resistance
- high no. Strands
- hormonally controlled (transcellular)
- less transport than leaky epithelium
- last 10-20% of transport (regulation of ion absorption etc. under hormonal control, can determine blood pressure/osmolarity)
Distal tight junctions examples
Colon, collect duct
Rules of transcellular transport
1) entry/exit steps
- entry for absorption = apical
Secretion = basolateral
2) electrochemical gradient
- forces driving movement in entry/exit steps passive (diffusion) or active (transport)
3) electroneutrality
- movement of positive/negative ion will attract counter ion
4) osmosis
- net movement of ions establish difference in osmolarity which causes water flow by osmosis
Glucose absorption in small intestine process
1) tight junctions divide into apical/basolateral, leaky epithelium
2) Na/K ATPase sets up ion gradient
3) sodium glucose symporter (SGLT) uses energy of Na gradient => actively accumulate glucose above its conc. grad.
4) facilitative glucose transporter (GLUT) mediates glucose exit across basolateral membrane via passive diffusion
5) Na taken up with glucose exits via basolateral Na pump
6) accumulation of positive charge (due to Na+) and increase in osmolarity (due to glucose) in blood side induces paracellular Cl- and water fluxes => isotonic fluid absorption (same osmolarity inside/outside epithelial tissue)
- high water permeability => water moves quickly
Net effect of glucose absorption in small intestine
Glucose, NaCl and water absorption
If no glucose in lumen
Glucose absorption doesn’t happen
Oral rehydration therapy
In babies, diarrhoea -> dehydration -> lack of water -> lack of electrolytes (NaCl) composition
- glucose enhances Na+ absorption and thus Cl- and water absorption so simple sugar soln. Is a solution
Glucose-galactose malabsorption syndrome
Caused by mutation resulting in defective SGLT in small intestine
=> sugar (glucose/galactose) retained/accumulated in small intestine lumen
=> increases lumen osmolarity
=> water effluent (osmotic imbalance attracts water)
=> osmotic diarrhoea due to increased water flow
SGLT
Sodium glucose transporter
Glucose-galactose malabsorption syndrome treatment
- remove glucose/galactose from diet
- use fructose as a carbohydrate source
Fructose absorption
Utilises facilitative transporter (GLUT5) specific for fructose
- fructose exits using GLUT2
Transporters and treatment
Multiple different types of transporters involved in doing different transport processes can be utilised to design therapy to offset problems
Glucose reabsorption in kidney
Glucose in plasma filtered and reabsorbed in kidney
Glucosuria
Accumulation of glucose in urine due to transport max. Of SGLT protein (only site of glucose absorption in kidney) being exceeded (too high glucose conc.) or impaired
Glucosuria common cause
Diabetes mellitis
=> deficient insulin activity
=> too high blood sugar (>200mg/mL)
=> SGLT can’t absorb glucose fast enough despite correctly functioning
Saturation of SGLT
All filtered glucose reabsorbed until renal threshold reached when glucose appears in urine
Renal threshold
Reflects transport max. Of SGLT
Chloride secretion locations
Small intestine, colon of bowel
Chloride secretion process
1) tight junctions divide into apical/basolateral
- note: tight junctions change selectivity to allow Na+ to flow in chloride secretion
Cl- to flow in glucose absorption
2) Na pump sets up ion gradient
3) NaK2Cl symporter uses energy of Na gradient => actively accumulate chloride above electchem. grad.
4) Cl- leaves cell by passive diffusion through ion channel (CFTR)
- Opening (strictly regulated/gated) of channel is the rate limiting step in Cl- secretion as Cl- cannot
Leave unless channel open even if accumulated above electrochemical equilibrium
5) Na+ exits via basolateral Na pump
6) K+ exits via channel
- leaving K+ makes membrane potential more negative (hyperpolarises) => more negative inside cell
=> more favourable for Cl- to leave
7) accumulation of negative charge in lumen induces paracellular Na and water fluxes to preserve electroneutrality => isosmotice/isotonic fluid secretion
Cystic fibrosis transmembrane conductance regulator (CFTR)
Cl- channel
CFTR structure
Identified by cloning gene believed to be responsible for cystic fibrosis
- unique as it has both a gated channel AND ability to hydrolyse ATP
- transmembrane region has alpha-helical cells in them
- hydrophobic amino acids interact with hydrophobic core
CFTR mechanism
Protein kinase A dependent phosphorylation of R domain
=> Bind and hydrolyse ATP at NBD
=> energy used to open pore (conformational change)
=> ion channel
R domain
Ball that gates CFTR channel
NBD
Nucleotide binding domain
- where ATP binds
CFTR overstimulation
Secretory diarrhoea (for people without CF) due to overstimulation of secretory cells in crypts of small intestine/colon
Causes of CFTR overstimulation
- abnormally high conc. Of endogenous secretagogues
- secretion of enterotoxins (more common)
Endogenous secretagogues
Neurotransmitter/small peptide hormone that stimulate cells to secrete
- overproduction caused by tumours/inflammation
From bacteria such as vibrio cholerae (e.g from drinking contaminated water)
- cholera toxin binds so tightly that it irreversibly activates adenylate cyclase
CFTR overstimulation consequences
Maximal stimulation of CFTR
=> secretion overwhelms absorptive capacity of colon
=> diarrhoea
=> dehydration
=> could lose up to 2kg of body weight and if not replaced, fatal (body shuts down)
CFTR overstimulation treatment
Oral rehydration therapy for secretory diarrhoea caused by cholera by counteracting oversecretion
CFTR dysfunction
Cystic fibrosis
- CFTR gene defect => defective ion transport => airway surface liquid depletion => defective mucocillary clearance => mucus obstruction - infection - inflammation (cycle)
Cystic fibrosis
Complex inherited disorder in an autosomal recessive fashion
- heterozygous have no symptoms but are carriers
- child of two carriers have a 1 in 4 chance of getting CF
Cystic fibrosis affected PEOPLE
children and young adults (not older people as most people die in middle age)
varies among ethnic groups
- Northern Europeans (Caucasians): 1 in 2500 newborns affected, 1 in 25 carriers
- less common in other ethnic groups (e.g Asian, Africans etc.)
Cystic fibrosis affected ORGANS
- diverse range of symptoms as wide effect on many organs
- common theme: involvement of epithelial tissue
- most cases of mortality due to respiratory failure
Cystic fibrosis survival rate
median survival WAS 38 years of age but now increased, CF suffers born today can expect survival into late 50s
Cystic fibrosis clinical management
- chest percussion to improve clearance of infected secretions
- antibiotics to treat infections
- pancreatic enzyme replacement
- attention to nutritional status
Cystic fibrosis solutions
- gene therapy
- restore transport
- combat infection
Normal lung epithelial transport
Balance between secretion and absorption keeps lung surface moist but prevents excessive fluid build up (drowning)
Normal lung surface/airway
- Wet, thin mucus traps inhaled particles; cilia push mucus to throat for removal
- airways stay clear for breathing
CF lung epithelial transport
Defective Cl- channel prevents isotonic fluid secretion and enhances Na+ absorption (Na channel open for longer => remove more water) to give dry lung surface
CF lung surface and airways
- mucus becomes thick and difficult to remove (stuck)
- bacteria proliferate (environ. Is good breeding ground) and attract mine cells which can damage healthy tissue
- DNA released from bacteria and lung cells adds to stickiness
- airways become plugged and begin to deteriorate
CF ability to do gas exchange
Decreases
=> eventually lose respiratory capacity
=> mortality, death
Sweat formation
1) primary isotonic secretion of fluid by acinar cells
2) secondary reabsorption of NaCl NOT water produces hypotonic soln. (Decoupling of ion and water transport)
Acinar cells
Exocrine cells
Primary secretion stimulation
Heat, nervousness etc. causes two pathways (parasympathetic and sympathetic stimulation) to converge to produce primary secretion
Primary secretion mechanism
Cl- accumulated INSIDE cell via pumps => Cl- wants to leave
- leaves via standard CFTR and CLCA
CLCA
Calcium activated chloride channel
Secondary reabsorption mechanism
Depolarised duct cells => Cl- wants to come in
- ONLY through CFTR
No water as duct cells impermeable to water due to lack of aquaporins
Sweat formation result
Water with reduced salt conc. Flow out to wet skin
=> increase conviction of heat from underlying blood supply
=> radiate off heat to cool body
Sweat formation in CF patients
1) primary secretion still happens via alternative pathway (CLCA)
2) no secondary reabsorption of Cl- due to defective CFTR
Consequence of no secondary reabsorption in CF patient sweat formation
=> accumulation of Cl-
=> no reabsorption of Na+ due to staying attracted to Cl-
=> no reabsorbed NaCl in combo with water released during primary secretion produces SALTY SWEAT
