Module 4.2 Eukaryotic

membrane fluidity

• refers to the physical state of the lipids in the bilayer

• 2 physical states

  • 2D liquid crystal in which lipids can move within the plane of one leaflet of the bilayer

  • frozen crystalline gel in which the movement of the lipids is greatly restricted

transition temperature

• the temperature in which membrane states change

• lipids may exist in either state, and the temperature at which they pass from one state to the other

• very quick change from one state to the other

liquid crystal state

• individual lipid molecules in the membrane

• can rotate and move laterally in the plane of the leaflet

frozen crystalline gel state

• all movement of lipids is greatly restricted

determination of transition temperature

• transition temperature of a lipid bilayer depends on the particular lipids of which it is constructed

• proteins do not contribute much to transition temperature, mainly lipids contribute

• degree of unsaturation of the fatty acid: fewer double bonds, higher transition temperature

• length of the fatty acid chain: longer chains need higher transition temperature

• cholesterol presence: cholesterol disrupts close packing of fatty acyl chains and interferes with their mobility.

  • abolishes sharp transition temperature, becomes more gradual

  • creates intermediate fluidity, a state in between crystalline gel and liquid crystal (created due to the gradual change)

biological significance of membrane fluidity

• 2D liquid crystal is a compromise between a rigid, ordered structure that would lack mobility and a completely fluid, nonviscous liquid in which components of the membrane could not be oriented and structural organization and mechanical support would be lacking

  • most important reason why membrane needs 2D liquid crystal

• fluidity allows for interactions to take place within the membrane

• fluidity is essential to membrane assembly

  • the growth of membrane is accomplished by the insertion of lipid and protein components into the fluid matrix of the membranous sheet

• many of the most basic cellular processes, including cell movement, cell growth, cell division, and secretion, etc. depend on the movement of membrane components

maintenance of membrane fluidity

• cell wants to stay in liquid crystal state, not gel crystalline

• cells adapt to temperature changes

maintenance of membrane fluidity - cold response

1. Desaturating single bonds in fatty acyl chains to form double bonds

2. reshuffling the chains between different phospholipid molecules to produce ones that contain two unsaturated fatty acids, which lowers the melting temperature of the bilayer

• desaturating: desaturases

• reshuffling: phospholipases and acyltransferases

• increase amount of double bonds or have the same amount of double bonds but concentrate them

• reduces melting temperature

phospholipases vs acyltransferases

• phospholipases will cut the ester bond between fatty acids and glycerol

• acyltransferases: transfers fatty acid from one lipid to another

lipid rafts

• detergent: triton x-100 (nonionic)

  • extracts lipids from membrane by dissolving them, but patches of lipids are left behind

  • tritone x-100 cannot dissolve/break interactions between these lipids

• cholesterol, lipids with long saturated fatty acyl tails (particularly sphingolipids)

  • pack very tightly, which forms patches since they are very well organized

  • packed in a microdomain

• SDS can break up lipid rafts/microdomains since it is ionic (dissolves entire membrane)

function of lipid rafts

• semi-permanent structure that can concentrate functional units

  • functional proteins like receptors or single-end proteins

• keeps protein complexes in place as one functional unit

dynamic nature of membrane

• movement of membrane lipids and proteins in the 2D lipid crystal bilayer gives membrane its dynamic nature

• not dynamic in the crystalline gel state

  • if cells stay in this state for too long, they will die from lack of exchange

• no carbohydrate movement because they are attached to protein and lipids, so the movement of the latter will move the carbohydrates

movement of lipids in membranes

• in fluid state, phospholipids are free to move in the plane of membrane bilayers (laterally in their leaflet)

• movement between bilayers (leaflets) is restricted

• rotation/flex: lipids turn around, very quick (10^-9 sec)

• lateral shift: lipids move in same leaflet, quick, (10^-6 sec)

• Flip-flop: movement from one leaflet to another, very slow (10^5 sec), requiring enzyme flippase

confined diffusion of phospholipids in plasma membrane

• track movement of single lipid by labeling it and use high speed microscope to watch it

  • ex: tagging

• records movement of one lipid

• movement is not random, it is restricted

movement of membrane proteins

• the degree of protein movement in the plane of the membrane varies with individual proteins

  • some proteins move more than others

• some proteins are free to move within plane of membrame, but movement is very slow compared to lipids

  • proteins are bigger than lipids

• other protein movement is restricted by their attachment ot other proteins or structures in the cell, like attachment to cytoskeleton

• no transmembrane movement of proteins

  • membrane has sideness because proteins cannot move from one leaflet to another

cell fusion technique

• larry frye and michael edidin in 1970

• movements of proteins demonstrated

• fuse two cells (human and mouse cell) since they contain different proteins

  • labeled antibodies

• look at their fluorescence change before and after fusion

• i cannot understand his accent but he says there’s a name for the cell that is the human and mouse fusion something like “sick cell” ?????????????????????????

• cell starts out with proteins of human and mouse separated on both sides of the cell then they will fuse because there is movement

• fluidity of membrane determines movement of proteins

• presence of mosaic cells increases sharply as the temperature passes transition temperature

  • movement of proteins has been enhanced

fluorescence recovery after photobleaching (FRAP)

• experiement to measure membrane fluidity and test if proteins can move laterally within the cell membrane.

• label: Tag membrane proteins with a fluorescent marker.

• bleach: Use a intense laser beam to destroy fluorescence in a tiny spot (photobleaching).

• recover: Monitor how fast fluorescence returns to the bleached area.

• unbleached, glowing proteins from adjacent (surrounding) areas diffuse laterally into the bleached spot, replacing the zapped proteins.

• if the bleached spot never recovered its fluorescence, it means the tagged proteins are anchored/fixed in place and cannot move laterally through the membrane

findings of FRAP

• membrane proteins move much slower in plasma membrane than in artificial bilayer

  • in real PM, membrane proteins are physically restricted by underlying cytoskeleton

• 30-70% of membrane proteins were not free to diffuse back to the irradicated circle

  • movement of these proteins are restricted

limitations to FRAP

• can only follow the average movement of a large number of labeled molecules as they diffuse over a large distance

• cannot distinguish between truly immobile proteins and ones that can only diffuse over a limited distance

single-particle tracking (SPT)

• observes movement of single protein

• label single protein and use high-speed computer enhanced microscope to record movement of protein

movement patterns in SPT

• A: random movement

  • largest fraction of proteins exhibit random movement

• B: no movement (immobilized)

• C: highly directed movement

control of membrane protein mobility (SPT)

• B: tethered or restricted by membrane skeleton

• D: impeded by crowded neighbors

• E: restricted by membrane skeleton fences

• F: slowed by extracellular materials

membrane domains and cell polarity

• a specialized, distinct region within a cell membrane or protein structure that has a specific lipid and protein composition or a distinct functional role

membrane domains and cell polarity: epithelial cells

• the apical surface contains integral proteins functioning in ion transport and hydrolysis of disaccharides

• the lateral surface contains integral proteins functioning in intercellular interactions.

• the basal surface contains integral proteins functioning in the association of the cell with the underlying basement membrane.

differentiation of mammalian sperm plasma membrane

• highest differentiated plasma membrane

• protein distribution/domain is very specific per area of the sperm cell

red blood cell (erythrocyte) plasma membrane

The most studied and best understood PM

• the red blood cells are inexpensive and readily available;

• they are present as single cells

• they are simple, lacking nuclear and cytoplasmic membranes;

• purified erythrocyte PMs can be simply obtained by placing the cells in a dilute salt solution.

• when proteins are extracted from membrane, they are all different sizes

• SDS-PAGE extracts proteins, can show how they form membrane using a cartoon..?

• carbohydrates are only on the extracellular surface

• proteins form a network in the protein, which gives the red blood cells in properties

movement of band 3 protein and glycophorin A

• the Band 3 proteins are immobilized in the membrane by the network of spectrin and other proteins.

• glycophorin A is free to diffuse in the membrane, but because its cytoplasmic domain extends in the cytosol of the erythrocyte, its diffusion is restricted to a limited area created by the specctrin network on the inside of the erythrocyte membrane

• gives erythrocyte property of resistance to shearing forces (mechanical dragging force caused by blood or fluid rushing rapidly across the surface of a cell. the cell will not be torn apart from these forces by allowing the cell to bend and squeeze through narrow spaces)

dual functions of cell membrane

• retains dissolved materials of the cell so they do not leak out into the environment

• must allow the necessary exchange of materials in and out of the cell

types of movement of substances across membranes

• simple diffusion through the lipid bilayer

• simple diffusion through an aqueous, protein-lined channel

• diffusion that is facilitated by a protein transporter

• active transport

movement of substances across membranes

• influx: movement of substance into the cell

• efflux: movement of substance out of the cell

• net flux: result of influx and efflux

energetics of solute movement

• diffusion is a spontaneous process in which a substance moves from a region of high to low concentration, eventually eliminating the concentration difference between the two regions

• if [Ci]/[Co] < 1, delta G is negative, influx is thermodynamically favored

• [Ci]/[Co] = 1, delta G is zero, at equilibrium, no net flux

Ci]/[Co] > 1, delta G is positive, efflux is thermodynamically favored

gradients that control diffusion

• tendency of a charged solute (electrolyte) to diffuse between two compartments depends on two gradient

• the chemical gradient: determined by concentration difference

• electric potential gradient: determined by difference in charge

• these gradient differences are combined to form an electrochemical gradient

• formula: delta G= RT ln [Ci]/[Co] + zF(delta)E_m

diffusion of water through membranes

• the process of the movement of water through semipermeable membrane from a region of lower solution concentration to a region of higher solute concentration osmosis

• when separated by semipermeable membrane, the compartment of higher solute concentration hypertonic (hyperosmotic) to the compartment of the lower solute concentration that is hypotonic (hypoosmotic)

• hypertonic: higher solute concentration than the cell

  • water will flow out and cause cell to shrink

• hypotonic: has a lower solute concentration than the cell

  • causing water to flow in and the cell to swell

effect of osmosis on plant cells

• plants are generally hypertonic compared to their fluid environment

• tendency for water to enter the cell develops an internal pressure (turgor) to push the cell against its surrounding wall, which provides support for nonwoody plants

• plasmolysis: the process where a plant cell loses water and shrinks, causing the cell membrane to pull away from the rigid cell wall

  • plant will wilt and drop

diffusion of ions through membranes

• most of the ion channels identified can exist in either an open or closed conformation

• closed channels are gated:

  • voltage-gated channels: conformational state depends on the difference in ionic charge on the two sides of the membrane.

  • ligand-gated channels: conformational state depends on the binding of a specific molecule (the ligand), which is usually not the solute that passes through the channel

  • mechano-gated channels: conformational state depends on mechanical forces (like stretch tension) that are applied to the membrane

bacterial KcsA K+ channel

• diameter:

  • the four oxygen atom ring, 3 angstrom

  • dehydrated K+, 2.7 angstrom

  • dehydrated Na+, 1.9 angstrom

• hinge-bending model for the opening of the KcsA channel

  • Change causes conformational change in the channel, causing M2 to bend and open the channel

  • Pore is closed by M1 and M2 helix

• establishes the structural and mechanistic basis for potassium selectivity and conduction in all living cells

• polypeptide sequence: T, V, G, Y, G (will be on test)

• carbonyl groups of amino acids V, G, Y, G point into the pore, which form the oxygen ring

  • Per layer there is 4 oxygens. K will interact with all oxygens in all layers

• hydroxyl group of T can form an oxygen ring

• K+ binds to ion binding position, there are 4 positions which are all at each carbonyl O

  • Will go from 1-3 or 2-4 (jumps)

• Pore has specificity for K+

  • Allows big ions to go through but not small

• Ions are surrounded by water shields, which is too big for the pore

  • Water shield must be removed, which will be removed by the oxygen ring because it will interact with the ion, repressing the water shield

  • If the ion is too small, interaction is too weak so the shield cannot be removed. Potassium is big enough to have a strong interaction to repress water shield (size is optimal)

  • The attraction between oxygen and ion takes the ion out of the water and attracts it to the pore

the eukaryotic voltage-gated K+ (Kv) channel

• four subunit

• each subunits contain six transmembrane helices (S1-S6) and pore domain (P segement) which is a partial helix

• grouped into two functionally distinct domains

  • A pore domain: including helices S5 and S6 and the P segment. They are homologous to helices 1 and 2 and the P segment of the KcsA channel. The the inner end of S6 forms the gate.

  • A voltage-sensor domain: including helices S1-S4 that senses the voltage across the plasma membrane. S4 is the key element of the sensor.

    • S4 is positively charged, so when voltage changes it moves the position of S4

the models of movement of S4 helix

1. Change conformation of S5 and S6 to open channel (moves up and down)

2. Change conformation of S4 by swinging it to open and close pore

inactivation of the Kv channel

• Channel is open for very short time (a millisecond)

• inactivation of the channel is accomplished by movement of the N-terminal cytoplasmic portion of the polypeptide through the mouth of the pore.

• The inactivation peptide moves into the lateral window to inactivate the channel

• After inactivation, it will go back to rest (closed) conformation

• Once inactivated, it takes a while to go back to rest position, meaning it cannot be automatically or quickly reopened

facilitated diffusion

• Using a protein to transport from high to low concentration across a membrane

• No ATP required, it is passive movement that requires a transporter

kinetics of facilitated diffusion

• similar to an enzyme-catalyzed reaction

• specificity- only one solute can be transported by a specific transporter

• saturation-type kinetics

• regulation

active transport

• the gradients in the pic are generated by active transport

• requires ATP

• Against concentration gradient

• endergonic process that is coupled to an exergonic process, such as the hydrolysis of ATP, the absorbance of light, the transport of electron, or the flow of other substances down their gradients.

comparison between facilitated and active transport

model of Na+/K+ ATPase Transport

• Jens Skou in 1957 discovered an ATP hydrolyzing enzyme in the nerve cells of a crab that was active only in the presence of both Na+ and K+

• proposed this protein transporting the two ions.

• the enzyme was called Na+/K+ ATPase, or sodium-potassium pump.

  • 3 sodium out, 2 potassium in

  • Gives cell a resting membrane potential of -70 V

• phosphorylation of the transport changes the protein’s shape and affinity for the ions.

  • p-type pump

  • Phosphate group comes from ATP

• p-type pump: phosphorylation of the transporter

• v-type pump: Vacuolar transporter

• ABC transporter: ATP-binding cassette transporter

H+/K+ ATPase Transport

• epithelial lining of stomach contains this P-type pump

• Pump is within vesicles that sit in the cytoplasm

• Food induces secretion of histamine, which binds to a receptor to trigger fusion of the vesicle with the plasma membrane

  • Pump is not integrated into plasma membrane, making it active

  • Pumps protons out and potassium into the cell using ATP (active transport)

• Transporter must be phosphorylation to work

Helps prevent heartburn

Regulation of H+/K+ ATPase

• Acid-blocking Drug: Zantac is a drug that can bind to the receptor like histamine does, which prevents fusion of vesicles to the plasma membrane

• Pump-Inhibiting Drug: prilosec will inhibit H+/K+ ATPase

• Acid-Neutralizing Drug: neutralize pH in lumen of the stomach

• Zantac, Pepcid, and Tagamet do not inhibit the ATPase directly, they block a receptor on the surface of the cells that stop activation by the hormone

coupling active transport to existing ion gradients (in Na+/glucose cotransporter)

• the primary active transport system, the Na+/K+ ATPase, produces the Na+ gradient

• the potential energy stores in the Na+ gradient is utilized to transport glucose into the cell

• glucose is driven by secondary transport

energetics of the Na+/glucose cotransport

• primary Na+/K+ ATPase keeps the external [Na+] 10 times the internal

• the voltage across the membrane of a cell is typically –70 mV,

• the free-energy change for the movement of a mole of Na+ into the cell under these conditions would be 3.1 kcal/mol

• two moles of Na+ are utilized to cotransport 1 mole of glucose

• [Ci]/[Co] = 1/23,000

  • the Na+/glucose cotransporter is capable to transporting

    glucose against a concentration gradient > 20,000.

uniport vs cotransport

• uniport: transport of a single solute across a membrane (may occur with either facilitated diffusion or active transport).

• cotransport: the linked transport of more than one solute across a membrane.

  • symport: the transport of two solutes in the same direction.

  • antiport: he transport of two solutes in opposite directions.

irritability

• a property that an organism responds to external stimulation

• irritability in a single-celled amoeba depends on the same basic properties of membranes that lead to the formation and propagation of nerve impulses.

membrane resting potential

• a voltage or electronic potential difference between the inside and outside of the plasma membrane is the membrane potential (for nonexcitable cells).

• for a nerve or muscle cell, this same potential is referred to as the resting potential

watch video

• if you change the membrane potential, the voltage gated channel will open or close

• the picture is a voltage gated potassium channel

• domains will move to open or close channel

movement of K+ across membrane

• the movement of K+ across the membrane is the major factor in determining resting potential

  • cell is most permeable to K+

• the vast majority of the ion channels that are open in the plasma membrane of a resting cell are K+ leak channels that are selective for K+.

• these channels lack the S4 voltage sensor and fail to respond to changes in voltage

K+ at equilibrium

• equilibrium potential using Nerst equation: -91 mV

• measured resting potential: -70 mV

• differ because of membrane’s slight permeability to Na+

depolarization

• decrease in polarity between two sides of the membrane

• creates an action potential when it reaches the threshold

• Na+ comes in to decrease the polarity between inside and outside, which makes the cell reach threshold to create an action potential

action potential

1. Leak K+ channels keep the resting potential of the membrane (-70mV)

2. External stimulation opens some Na+ channels, allowing Na+ ions to move into the cell, which causes depolarization of the membrane

3. If the depolarization passes the threshold (-50mV), the change in voltage causes the voltage- gated Na+ channels to open, allowing Na+ ions diffuse freely into the cell, which reverse the potential to +40mV, approaching the equilibrium potential for Na+.

4. after 1 msec, the Na+ channel are inactivated. The change in membrane potential triggers the opening of voltage-gated K+ channels, allowing the K+ ions to diffuse freely out of the cell, which causes the potential to swing back to about –80mV, approaching the K+ equilibrium potential. The large negative potential causes the voltage-gated K+ channels to close and the membrane returns to the resting state.

• the changes in membrane potential collectively are called an action potential

two characteristics of action potential

• The all-or-none law: once the membrane is depolarized to the threshold value, a full-blown action potential is triggered without further stimulation.

  • Subthreshold depolarization is incapable of triggering action potential.

• An action potential process does not require energy: It results from the flow of ions down their respective electrochemical gradients.

  • The Na+/K+ ATPase requires energy to generate the steep ionic gradients across the plasma membrane.

propogation of action potential

• membranes enter a brief refractory period following an action potential because the Na+ channels cannot reopen for several milliseconds after their inactivation.

• membranes in refractory period cannot be restimulated

• this gives the propogation of an action potential a direction

• once triggered, an action potentials travels down the entire length of the neuron with the same strength and arrives at its target cell with the same strength as at its initiation. Stronger stimuli cannot produce “bigger” impulse than weaker stimuli

differences in strengths of stimuli

• Stronger stimuli activate more nerve cells than do weaker stimuli

• Stronger stimuli activate “high-threshold” neurons that would remain at rest if the stimuli were weaker

• stimulus strength is also reflected by the pattern and frequency by which action potentials are launched down a particular neuron.

• the stronger the stimulus, the greater the number of impulses generated.

structure of a nerve cell

saltatory conduction

• to increase speed of a nerve impulse:

  • increase in the diameter of the axon

  • wrap the axon with a myelin sheath

• saltatory conduction: propagation of an impulse by a myelin sheath-wrapped axon

synapses

• specialized junctions that link neurons with their target cells

• synaptic cleft: a narrow gap (20-50nm) between a neuron and its target cell.

• presynaptic cell: a sensory cell or a neuron that conducts impulses toward a synapse.

• postsynaptic cell: a neuron, muscle, or gland cell that lie on the receiving side of a synapse.

• synaptic vesicles: carry neurotransmitters into the synaptic cleft

• neurotransmitters: acetylcholine, norepinephrine

neuromuscular junction

• neuron whose target cell is a muscle cell

• the space the vesicles release the neurotransmitters into is called the synaptic cleft

• action potential goes from neuron to muscle cell

synaptic transmission

1. arrival of action potential to the axon terminal

2. depolarizes presynaptic membrane

3. voltage-gated calcium channels open

4. calcium rushes in

5. calcium willl trigger the fusion of synaptic vesicles and presynaptic membrane

6. neurotransmitters are released into the cleft (acetylcholine in the image)

7. diffuses across cleft to receptor on postsynaptic membrane

8. Voltage-gated sodium channels open (sodium rushes in) and depolarize it

9. nerve impulse gets sent down the axon

• or Cl- channels will open (chlorine rushes in) instead of Na+ channels, which will inhibit the action potential

  • desensitizes the membrane

• two types of responses can occur on the postsynaptic membrane depending on the voltage-gated channels opening

receptors on post-synaptic membrane

• number of receptors on postsynaptic membrane can change

• more receptors make target cell more sensitive since it can bind to the neurotransmitter more

• less receptors make target cell less sensitive

synaptic plasticity

• the physical structure of synapses are genetically encoded, whereas their dynamic quality can be affected by environment.

• the responses of the dynamic quality of synapses is called synaptic plasticity

• when hippocampal neurons are repeatedly stimulated over a short period of time, the synapses that connect these neurons to their neighbors become “strengthened” by a process known as long-term potentiation (LTP), which last for days, weeks, or even longer

• synapses have undergone LTP are able to transmit weaker stimuli and evoke stronger responses in postsynaptic cells.

• the synaptic plasticity plays a major role as newly learned information or memories are encoded in the neural circuits of the brain