Lecture #1 - Common Principles in Signal Transduction and Overview of Membrane Physiology Flashcards
(101 cards)
1
Q
Signal Transduction
A
The processes by which chemical or physical information from the extracellular environment is detected, transferred into the cell, amplified, and integrated to produce change in cell activity
Example - Extracellular signal is transduced into a cell –> changes gene expression/differentiation OR moves the cell/changes the cell shape/ contracts the cell OR drives an action potential OR cells live/die OR secretes something from the cell
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Q
Key features of signaling pathways
A
- Reception of a signal
- Discrimination
- Information Transfer
- Amplification
- Adaptation
- Integration
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Q
Key features of signaling pathways - Reception of a signal
A
Need to be able to detect the signal in order to transduce the signal
Signal Reception (signal detection) usually occurs using:
1. A cell-surface receptor (protein on the cell surface)
2. An intracellular receptor
4
Q
Key features of signaling pathways - Reception of a signal
A
Discrimination = Only certain signals will activate a given pathway
Cell are surounded by MANY molecules and MANY forces –> MEANS for cell to respond appropriately to its envirnment it needs to be able to discriminate between ALL the signals
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Q
Key features of signaling pathways - Information transfer
A
Information is transferred across the plasma membrane into the cell
- Transfer is needed so extracellular signals can impact intracellular events
The form of information may change during transfer
- Ex – neuron converts chemical signals (Nuerotransimtter) into electrical signals (AP)
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Q
Key features of signaling pathways - Amplification
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Signal needs to be amplified l because the cel needs to produce macroscopic changes in response to small amounts of extracellular signals
Amplification occurs using receptor or Intracellular enzymes
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Q
Key features of signaling pathways - Adaptation
A
Adaptation = Activity of the pathway is decreased upon continuous exposure to the signal
- Need to be able to turn the pathway off
Need to be able to turn the pathway off so the cells cells don’t get saturated by a signal and consequently can’t respond to new signals
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Q
Key features of signaling pathways - Integration
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Integration = Convergence within a signaling pathway OR cross-talk between signaling pathways
Cells need to take in information and coordinate the response to multiple signals
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Q
How can signal transduction processes by classified
A
- Based on the type of signal
- Based on Signal transudation molecules involved
- Based on Site of detection
- Based on Origin or route of signal
10
Q
Types of signals
A
- Chemical Signals
- Physical Signals
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Q
Chemical Signals
A
Cells can respond to a wide range of chemically diverse signals (requires many receptors)
Includes:
1. Small Molecules – Ions + Lipids (Ex. Steroids) + Nucleic Acids (Ex. ATP) + Amino Acid derivatives (Ex. Glutamate) + Small organic molecules (Ex. Tastants) + Electrophils (Ex. Isothiocyanates)
- Carbohydrates (Ex. Glycoproteins)
- Peptides/proteins (Ex. Insulin or Collagen)
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Q
Physical Signals
A
Need to be able to respond to physical stimuli in a QUNATATIVE way (Ex. warm vs. hot hazardous surface)
- Need to be able to distinguish hazardous physical stimuli vs. Non-hazardous physical stimuli
Physical signals include:
1. Electromagnetic force (Visible or UV or infrared)
2. Thermal (Hot Vs. Cold temperature sensors)
3. Mechanical forces (Pressure and sound)
4. Electrical Voltage
13
Q
Components that carry out signal transduction
A
- Ligands - Molecule that binds to the sensor (receptor) on the surface
- Receptor - Sensor for stimuli
- Adaptor proteins – Couples the receptor/ligand complex to downstream output molecule (to the effector)
- Effector - Act on proteins in the cell to affect cell behavior OR can catalyze the production of a second messenger that will ultimately drive the downstream activities
14
Q
Way the components of signal transduction can exist
A
Components can exist in separate or a single entity
Example configurations:
1. Receptor carries out the downstream catalytic activity (no separate effector)
2. No adapter molecule (effector binds to the receptor)
3. One receptor can interact with multiple adapters (each adapters binds to different effector) to independently activate multiple downstream effectors
4. Receptor binds to one adapter and the adapter binds to the effector
15
Q
Sites of detection - Where can the signal is being detected
A
- Plasma membrane (many hydrophobic ligands + some lipids)
- Most receptors are located at the plasma membrane = many signaling molecules are detected at the plasma membrane
- Intracellular by receptors INSIDE of the cytoplasm OR in the nucleus
- Receptors of Steroid hormones or Viral dsRNA are inside the cell
16
Q
Origin or route of signals - where can signals come from
A
- Diffusable signals - Signals diffusing from one place to another
- Includes: Endocrine + Paracrine + Autocrine - Anchored signals - Signals are stuck somewhere
- Includes: Cell-cel interactions (homotypic and heterotypic) + Cell-substate interactions)
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Q
Endocrine Vs. Paracrine Vs. Autocrine
A
Endocrine - Signals produced by a distant source an uses the blood to move
- Example – hormones travel through the blood and act on a distant tissue
Paracrine - Cell responds to a signal that is produced/released by a nearby cell
- Example - Nueronal synapse/neuromsuclar junctions - Nerves make a synpase –> pre-synaptic neuron releases a Neurotransmitter to the cleft between the cells –> Neurotransmitters are detected by a receptor on the post synaptic neuron
Autocrine – Signal is made by a cell and that same cell will detect that signal
- Allows cells to gauge their own activity
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Q
Anchored signal - Cell-Cell Interactions
A
Anchored signal on one cell and receptor on the other cell –> two cells come together –> signal and receptor interact with one another –> change behavior in one or both cell
Includes:
1. Homotypic – same molecule on both cell (ex. Adhesion molecules)
2. Heterotypic – Different molecules on each cell (Ex. Delta notch interaction –> delta on one cell and notch on other cell)
- Example - Immunologic synapse - Antigen presenting cell comes close to the T cell –> binds the antigen presenting cells to the T cell –> signals to the T cells and T cell decides to respond or not
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Q
Anchored signal - Cell-Substrate Interactions
A
Cell responds to the surface that they are on
Example – cells respond to the ECM they are on using intergins
- Integrins on the cell surface bind to ECM –> binding causes a confirmation change of the integrin –> confirmation change of the intergin changes cytoskeletal behavior inside the cell (BECAUSE the integrin binds to the cytoskeleton)
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Q
What do receptors do
A
- Bind ligand to detect stimulus (saturable and specific)
- Transduce signal across plasma membrane or across membrane within the cell
- Catalytic activity (some receptors have enzymatic activity)
- Activity is often in the cytoplasmic domains
- Complex with other subunits
- Occupancy induced changes in activity - Receptor Adaptation
- Internize a ligand
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Q
Receptors transducing signal across plasma membrane or across membrane within the cell
A
Often transduce signals via a conformation changes or oligomerization state of the receptor
Example – Ligand binds to a receptor causes confirmation change in the receptor –> Change in confirmation allows two receptors (both bound to a ligand) to bind –> binding of the two receptors leads to:
1. ANOTHER confirmation change
2. Two receptors to phosphorylate one another
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Q
Receptor Catylytic Activity
A
Receptor Catalytic Activity can:
1. Form or break covalent bonds (Example – Kinases phosphorylate target)
2. Activate intracellular proteins (Ex. receptor activates kinases or TF)
- In NOTCH - receptor itself is cleaved and acts as TF in nucelus
3. Allow ions to flow down electrochemical gradient in or out of a cell (shows receptor as ion chanel)
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Q
Receptors complexing with other subunits
A
Peptides that form receptors are often part of a multimer
Can be constitutive multimeric complex (receptor is always inmultimer) OR inducible (ex. complex is onlyformed once the ligand binds)
Complex can be homomeric (copies of same peptide come together) or heteromeric (Different peptides)
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Q
Why have multimeric complex?
A
1 – Multimeric complex increases binding avidity = increases the receptor sensitivity
- Having a multimeric complex allows for multiple ligands biding sites in close proximity (because have multiple copies of the same peptide each with a ligand binding site) –> THIS increases the sensitivity of the system because if a ligand leaves a binding site THEN the ligand will bind to the nearby ligand binding sites
3. Multimeric complex increases the specificity
- Increases specificity because the ligand binding pocket in multimerc receptor is ONLY formed when the subunits come together –> ligand can only bind when the SU come together
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Effect of forming multimeric complex
Formation of a multimeric complex enhances signaling repertoire
WHY - because the receptor is able to signal different downstream pathways when form a multimeric receptor
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Receptor Adaptation - What do signaling systems do when they have been signaling for a while (when presented with signal for a long period of time)
After time the signaling system needs to change activity
Example – Over time the ability to signal might have to increase or decrease
- When increase or decrease ability to signal the affinity for the ligand itself might change (due to confirmational changes)
Occupancy induced changes = Increases or decrease in signlaing due to chnages in affinity for ligand to bind
- Often triggered by phosphorylation or a post tranlsational modifications
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Receptor Internizing a ligand
Receptor can internalize ligands through receptor mediated endocytosis
Effect of internilizing a ligand:
1. Desensitizes systems (turn off signaling) --> cells stop responding to continued presence of a stimulus
2. Deliver ligand to intracellular targets
- Brings the ligand into the cell where it can preform new functions
3. Bring the receptor ligand complex into the cell
- NOW complex can signal in a new compartment
- After receptor mediated endocytosis the complex goes into MVB --> MVB is transported and signals in new place (shows signaling does not always stop at the plasma membrane)
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Affinity of receptors
Receptors need affinity for a ligand that is appropriate for the concentration of the ligand
Example – If you have a small amount of ligand --> the rectors affinity for the ligand needs to be in the same range in order for the cell to be able to respond qunataivley to the abundance of the ligand
Ex. Receptor affinity for ligand needs to be in nm range of the concetration of the ligand is in the nm range (Want the Kd of the receptor to be in the nm range)
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Ligand binding assay
Use - Measure ligand binding affinity
Process:
1. Start with a labeled ligand
2. Mix the labeled ligand with a receptor
- Receptor can be from intact cells or purified proteins or membrane preparation from cells
3. Allow the binding reaction to come to equiliborum
4. Remove the unbound ligand
- Wash cells to get rid of non-specific binding
5. Count how much ligand is there (ligand is all bound to a receptor)
END – only have the receptor ligand complex
Challanges:
1. Small number of receptors on cell
2. Rapid dissociation of bound ligand from the receptor
3/4. Non-saturatable sites and non-specific binding
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Challanges in Ligand Binding Assay - Small number of receptors on cell
Overall – often have few receptors on the cell
- Means detecting ligand binding to the receptors needs to be sensitive
Solution:
1. Use radioactive or fluorescent labeled ligand
2. Overexpress the receptor heterologously or purify the native receptor
- Overexpress so there are more of the receptor
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Challanges in Ligand Binding Assay - Rapid dissociation of bound ligand from the receptor
Problem when receptor ligand intercations are low affinity (10-100 um range)
Issue - On/off rate of ligand might be fast --> ligand might dissociate from the receptor very fast
- When ligand dissociates off of receptor fast --> ligand will leave the receptor while you are wash the unbound ligand off the cells --> can't truly know how many receptors are there (won’t know how much ligand was binding to the receptors when at equiliborum because the ligand came off while washing the cells)
To solve - Need to trap the ligand and receptor before the ligand can leave
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Challanges in Ligand Binding Assay - Rapid dissociation of bound ligand from the receptor SOLUTION
Solution:
1. Rapid filtration of receptor/ligand complex through membrane that will retain the complex and rapid wash
2. Centrufugation through mineral oil
- Ligand molecules stay at top and ligand bound to receptor go to the bottom
3. Rapid size chromatography (size exclusion)
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Challanges in Ligand Binding Assay - Non-saturatable sites
Non-saturatable sites = ligand binding to non-receptor
Labeled ligand gets stuck in the cell membrane OR can get trapped between cells
- When isolate what you think is the receptor ligand complex it is ACTUALLY the ligand bound somehwere else
Solution:
1. Competetion with excess unlabled ligand
- In a separate binding reaction add excess of unlabeled ligand --> unlabled ligand will displace the labeled ligand from the receptor BUT won’t displace the ligand stuck between cells or in the plasma membrane
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Challanges in Ligand Binding Assay - Non-specific sites
Non-specific = Ligand binds to other receptors
When you isolate what you think is the receptor ligand complex you might isolate the ligand stuck to a difefrent receptor (two receptors bind to the ligand BUT you only want to isolate one of those receptors)
Solution: Competition with excess site-specific ligand
- In a separate binding reaction add excess of molecule that binds to one of the receptors (binds to receptor you are not interested in)
- Eliminate binding to one of the receptors --> NOW ligand will only bind to the receptor of interest
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Experimental Design of a binding assay
Start - Cells express the receptor of interest AND have or express the radio labeled ligand
Two sets of reactions:
1. Fixed number of cells and an increasing concertation of the labeled ligand BUT have no unlabeled ligand
2. Fixed number of cells with increasing concentration of the labeled ligand AND have an excess of unlabeled ligand in every tube
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Binding Assay – Results
Low concertation of labeled ligand ; no unlabeled ligand – some of the labeled ligand binds to the receptors AND some saturatble binding (between cells or in membrane)
High concertation of the labeled ligand ; no unlabled ligand – All of the receptors are bound by a ligand AND there is more non-saturatable binding
Low amounts of labeled ligand WITH unlabeled ligand – unlabeled ligand displaces the labeled ligand from all receptor sites (no receptors have labled ligand) AND have some non-saturatable binding
- Have non-saturable binding because the unlabeled ligand doesn’t affect if the label ligand gets trapped between cells or in the membrane
High concentrations of labeled ligand WITH unlabeled ligand – the unlabeled ligand has displaced the labeled ligand from all of the receptors AND there is more non-saturatable binding
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Binding Assay – Results Chart (Total binding)
Total binding is based on high concertations of labled labled WITHOUT unlabeled ligand
Point A = Binding at a high concertation of labeled ligand with NO unlabled ligand
Point C = Higher concreation of labled ligand
- Shows that when increase the concertaion of the the labled ligand the amount of bound labeled ligand goes up (more labled ligand trapped inside of the cell)
- At C the amount of bound labeled ligand continues to increase slowly
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Binding Assay – Results Chart (Non-saturatble binding)
Bottom line – Nonsaturatable binding based on Low concertation of labeled ligand WITH unlabeled ligand
B – The unlabled ligand displaces the labeled ligand from the receptor = have 0 bound labled ligand
- Bound label = 0
D – There is no labeled ligand on the receptpor (because the unlabeled ligand displaces the labled ligand)
- BUT have a slow increase in the non-saturatable binding (more labled ligand trappped betrween the cells/in the plasma membrane) --> Have an increase in labled ligand BUT that labled ligand is NOT bound to receptor
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Binding Assay - Results (ALL together)
Plot – shows total binding and non-specific binding and saturable binding
Subtract the non-saturable binding curve from the total binding curve --> gives the saturable binding
- Saturable bindning = binding of the labeled ligand to the receptor
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Quantitive Features of the saturable binding
1. Line plateaus because have a limited number of receptors per cell
2. The peak of the line is the Binding maximum (Bmax)
- Bmax tells you how many receptors there are on the cell surface
3. Get the Dissociation constant (Kd)
- Kd = Ligand concertation where half of the receptors are occupied
- Kd tells you about the affinity of the receptor for the ligand
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Determining the Receptor Structure and how it functions
1. Biochemical Purification/Characterization
2. cDNA cloning
3. Topology mapping on intact cells
4. Heterologous expression studies
5. Structural analysis of purified receptor protein
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Biochemical Purification/Characterization
If you have a ligand binding site in a cell --> can fractionate the cells --> isolate the protein that the ligand binds to
Once have the purified protein you can determine:
1. Molecular Mass
2. Phosphorylation state
3. Subunit composition
4. Partial sequence (sequence the isolated protein to find the gene)
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cDNA cloning
Overall - Based on the partial peptide sequence from biochemical purification use functional expression cloning and homology with known receptors
Use - Get cDNA encoding receptor
- Get cDNA based on the protein sequence from biochemical purfication
Other ways to get the cDNA that codes for the receptor:
1. Functional expression cloning or homology cloning
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What can you do once have cDNA
1. Find membrane topology
- Can predict membrane topology based on cDNA and verify model with proteases or epitope acceibility
2. Can see if the sequence has functional domains (Ex. kinase domains)
3. Use computational methods to find relatedness to other known receptors
- Can use computational methodsand similatries to other proteins to predict the 3D structure and consequentely infer function
4. Alphafold and related computational prediction tools
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Topology mapping on intact cells
Methods to refine the topology map (learn more about membrane topology):
1. Protease accessibility
2. Epitope accesibility (+/- cell permeabilization)
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Heterologous expression studies
Once have cDNA can do heterologous expression in cells
Use of heterologous expression and mutationegenis/functional studies the receptor to find the functionally important domains
- Use heterologous expression to determine the Subunit composition required for function
- Use mutagenesis to find the functional importance of different domains
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Structural analysis of purified receptor protein
Methods used for structural analysis of purified receptor protein:
1. CryoEM --> find receptor structure AND the different confirmation states of the receptor
- Example – can see the receptor structure when it is OR is not bound to the ligand
2. X-Ray crystalography
3. EPR or NMR for small domains
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Hydropathy plots
Hydropathy plots are used to predict membrane topology
- Uses the cDNA sequnece and computational methods to see the probability that a domain is a transmembrane domain
Examples:
Left graphs – cDNA predicts 3 transmembrane domains --> have two options for membrane topology because don't know if the N terminus is inside or outside
Right graphs – cDNA predicts 4 transmembrane domains --> have 3 options for membrane topology because the first domain might be a cleavable signal sequence AND don’t know if the N terminus is inside vs. Ootside
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How do you know which predicted membrane topology is right
Use epitope mapping further determine membrane topology (test which prediction is correct)
Process: Fuse epitope onto the N terminus or the C terminus of cDNA --> Express the fusion peptide in cells --> immunostain cells in 2 conditions (without detergent or with detergent)
Stain W/O detergent (non-permeable cells) --> antibody ONLY binds if the epitope tag is facing the extracellular space (Ex. Epitope taged N termus only binds to the AB if the N termus faces the outide of the cell)
Stain With the detergent (permeabilize cells) --> antibody should be able to bind in both configurations
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Example predicted membrane topology - Rhopospin
Predicted membrane topology of Rhodopsin using topologic mapping
Rhodospin = GPCR (has 7 transmembrane domains with the N terminus is in the extracellular fluid and C terminus is in the cytoplasm)
- Rhodopsin ALSO has extra anchor in the plasma membrane (Red loop in image)
Rhodopsin function uses Retinal (visual pigement)
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Rhopospin as a Receptor
Rhodopsin by itself does NOT act as a receptor – only acts as a receptor when it is covalently linked to 11-cis retinal
Retinal binds to a Lys in the 7th transmembrane domain Rhodopsin --> tucks Retinal into the plane of the plasma membrane
When retinal is bound to the receptor – the retinal molecule can absorb certain wL of light
- Light hits the receptor retinal complex --> causes confirmation change in the double bond in retinal (cis to trans) --> confirmation change in retinal causes a confirmation change Rhodopsin (opsin molecule) --> confimation chnage in GPCR allows the GPCR to signal --> can see
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Crystal structure of Bovine Rhodospin at 2.8 Angstromes
Crystal structure shows the alpha helices of the transmembrane domains
- 7 Transmembrane domains are alpha helicies
NOTE - Internilized proteins maintain their membrane topologies
- N terminus stays in the equivilent of extracellular fluid ; C terminus stays in the cytoplasm
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Growth hormone receptor complex
2 SU that make the growth hormone receptor --> growth hormone binds at the interface between the two SU
Only if the SU come together can they bind to the growth hormone
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Use of CryoEM
CryoEM can show the different confirmations of the receptor
Image – shows the glutamate receptor (has large extracellular domain)
- CryEM shows the receptor extracellular domain has a different confirmation in the active vs. Inactive state
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Major classes of receptors - GPCRs
Example – Rhodopsin or Beta Adrenergic receptors
Key features:
1. Have 7 transmembrane domains
2. signals through intracellular adapters (Intracellular adapters = heterotrimeric G proteins)
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Major classes of receptors - Ligand gated ion channels
Often multimeric proteins with multiple Su that have to come together
- Can be homomeric or heteromeric
When molecule binds to the extracellular domain of receptor a pore opens up --> pore allows ions to flow down their electrochemical gradient
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Major classes of receptors - Intracellular receptors
Example – Steroid hormone receptors (in cytoplasm)
Hormone (Ligand) diffuses across the membrane --> ligand binds to the receptors in the cytoplasm --> binding triggers translocation of receptor into the nucleus --> in the nucleus the receptor will act as a transcription factor
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Major classes of receptors - Tyrosine kinase receptor
Types:
1. Intrinsic Tyrosine kinase domain --> tyrosine kinase activity is in the receptor subunit (Ex. EGF Receptor)
2. Interaction with separate tyrosine kinase --> kinase activity is from a different protein that associates with an intracellular SU on the receptor (Ex. Cytokine receptors + T cell receptor complex)
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Adapter molecules Functions
1. Act as scaffolds --> tethers the receptor cytoplasmic domains to downstream signaling components OR tether the receptors to each other OR tether the receptors to the cytoskalaton
2. Interact with the receptors BUT then dissociate from the receptors to activate effector proteins (activate enzymes or ion channels)
- Example - G Proteins
Receptors can have complex cytoplasm domains --> multiple adapter proteins to interact with 1 receptor
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Structure of Adapter Proteins
Adapter proteins often contain modular interactions or functional domains (similar to receptors and effectors)
Examples of domains used in adapters (ON Slide):
1. SH2/PTB domains --> Binds to phsphotryosines in specific sequence context
2. PDZ domains --> binds to E-S/T-X-V/I animo acids at carboxyl terminus
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PDZ domain Example
InaD --> Adapter proteins that has 5 PDZ domains
InaD = acts as scafold to form a multiplex structure that includes the PDZ protein AND receptors AND ion channels AND enzymes AND adapters AND the cytoskeleton (actin)
- InaD keeps these molecules close together to facilitate rapid phototransduction
Example - allows flies to see light stimulus when swat then and triggers fast response
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Effectors
Effectors - downstream protein that does things in response to the signaling pathway
- Effectors = part of the pathway
Effectors includes:
1. Enzymes that catalyze the genration of second messenger
2. Enzymes that phosphorylate dephosphorylate proteins
3. Transcription factors that alter gene expression
4. Proteins that regulate cellular processes (Ex. modulate the cytosklaton and secretion)
Examples (have more on slide):
1. Phospholipase C --> cleaves membrane lipids into other signaling molecules
2. Guanine nucleotide exhange factor --> exchanges GTP for GDP on guanine nucleotide regulatory protein (important for regulating GTPase activity)
3. Adenylate cylase
4. Kinases
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Second messengers
Effectors can make the second messenger --> second messenger will go to a cellular target
Second Messengers: diffusible hydrophilic or amphipathic molecules that modulate the activities of downstream”cellular targets
- Second messenger leaves the receptor and regulates the activity of other proteins
Example (see more in chart) - cAMP + Inositol Triphosphate (IP3)
- Phospholipase C cleaves the membrane lipids in 1,2diaglycerol and IP3 (both signaling molecules)
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Example second messenger - cAMP
cAMP can binds to a regualtory subunit on a protein kinase (PKA) --> binding triggers release of the catylyztic subunit of PKA --> catalytic SU will phosphorylate other proteins
cAMP is made using adenylate cyclase and is broken down using cAMP phosphodiesters
- adenylate cyclase makes ATP --> cAMP
- cAMP phosphodiesters makes cAMP --> AMP (needed to turn pathway off)
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What allows for temporal and spatial control of signaling pathways
Membrane pathways can be self limiting --> allows for the temporal and spatial properties of cellular response to be controlled
Way pathways turn off (BOTH allow for precise kinetic control):
1. Have 2 branches of the pathway --> One branch leads to cell response ; Second branch leads to a different response that leads to a molecule that turns signlaing off
2. End product of the pathway can feedback and inhibit earilier parts of the pathway
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Kinetics of pathway regulation
Chart - Red is the time course of the activation arm of the pathway ; Green is desensitation/feedback inabition arm of the pathway
Desentization arm (green) lags --> initially get big response BUT then activation arm decreases and desentizatoion arm increases --> response is turned off
- In chart - Desensitation arm/feedback inhibition increases throughout then active arm--> THEN when dissemnination/feedback inhibition arm peaks active arm decreases
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Feature of Signlaing Pathways
Signaling pathways can cross talk
Cross talk allows for:
1. Reciprocal regulation of competing cellular responses (two pathways can inhibit one another)
- Example - Apoptosis death receptor and survival factor
2. Coincidence detection --> produce a response ONLY when more than one condition is satisfied (only respond when BOTH pathways are active at the same time)
- Example - Release of NT upon simultaneous activation of NMDA receptor and Cannaboid receptor
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Synthetic reprogramming of signaling pathways
Signaling pathways can be synthetically reprogramed
Synthetic biology is using the properties of signaling pathways and re-engineering signaling pathways to get a useful outcome
- Example - Using Srp pathways to change the logic of outcomes
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History of Chemiosmotic circuits
1. Found steps of pathways --> thought that the membrane encloses enzymes that do everything (cell = bag of enzymes)
2. Looked at how cells make ATP --> found ETC proteins in the membrane that take electrons from NADH and pass them between ETC commplexes (movement of electrons produces water and moves H+ across gradient)
3. Knew ATP synthese is also in the membrane (knew ATP synthese uses a lot of energy ; knew ATP synthase is coupled to the exergonic reactions of the ETC BUT didn't know what couples ATP synthetics and the ETC)
- Thought a energy intermediate goes from the ETC to ATP synthase to give energy to ATP synthase
- NEW idea (know is correct) - high energy intermediate is the H+ that are transported across the membrane
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What couples ATP synthase and the ETC
H+ pumped across the membrane is the high energy intermediate that couples the ETC and ATP synthase
ETC pumps H+ across the membrane --> creates a electrochemical gradient --> H+ go back down their gradient ‘downhill’ through ATP synthesis to drives formation of ATP
- Electrochemical gradient itself is the high energy intermediate
Shows that H+ gradient can do chemical work (making ATP)
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Work H+ gradient can do at a membrane
1. Chemical work --> H+ gradient can make ATP
2. Osmotic work --> H+ can go through a transporter and drag solutes with it (bring solution in the same direction as H+ OR push solutes in opposite direction)
- Transporter moves H+ down its gradient and ALSO using the energy of H+ going down its gradient to trasport something else
3. Mechanical work
- Example - H+ gradient goes through proteins and causes the bacterial flagella to rotate
SHOWs that the seperation of ions across the membrane is a form of energy
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Seperartion of ions across membrane
Seperation of ions across the membrane constitutes a chemiosmotic circuit
- Chemiosmotic circuits across all membranes do work (osmotic or mechanical or chemical work)
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Variations on the chemiosmotic Scheme
Example 1- Anerobic bacteria --> use glycolysis to make lactic acid to make ATP
- Use ATP to drive ATP synthase backwards --> ATP synthase will break down ATP to make a H+ gradient --> H+ gradient can do work in the membrane
Example 2 – Vibrio --> can't maintain a H+ gradient because live in alkiline water
- Replace H+ with Na - ETC moves Na and H and the Na will go through ATP synthase --> makes ATP
Example 3 - Modestum Bacteria --> links decarboxylation exothermic reaction to pushing Na ions across the membrane --> THEN Na will drive formation of ATP
Example 4 - Oxalobacteria exchanges oxycylate (-2) for Formate (-1) --> generates a difference in ions (charge) across membrane to drive chemiosmotic circut
- Virtual H+ pump
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What is the chemiosmotic made of
1. Pumps
2. carriers
3. Chanels --> allow one type of ion to go down their concentration gradients across the membrane quickly
ALL together make a chemosomotic circut across the membrane
- Can have circuit at the plasma membrane or membrane of organelles
- Circuit =can do many types of work
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Pumps
Pumps - mechanisms of transport that are driven by energy (moves ions to create a gradient)
- Often use ATP as energu (ATPase pumps) OR can be driven by light (Ex. Bacterial Rhoposin using Retinal which completes signaling cascade AND pumps H+ across the membrane)
Once pumps establish the gradient --> the gradient can be used to move molecules across the membrane through carriers or channels
- Carriers and channels utilize the gradient made by pumps to move other molecules (molecule moved by the pump will move through the carrier along with a new molecule)
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Example circuts built using H+
1. H+ pumps that use ATP to drive H+ across membrane
- Use the pump to make the H+ gradient THEN use the gradient to move sugar or amino acids into the cell OR use the H+ gradient to move ions through ion chanels
2. Chlororplats --> harness light to make ATP
3. Organelles can have pumps + carriers + ion chanels
There are chemisomotic circuts across eveyrthing membrane
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H+ Vs. Na Chemostatic circuts
H+ circuits are in: Prokaryotes plasma membrane + Lower Eukaryotes plasma membrane + Plant plasma membrane + Major organelles
Na Circules are in: Animal cell plasma membrane
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Development of membrane potential - Example
Example – Have a compartment that has 150 mM KCl inside (compartment is sitting in solution with less KCl (1.5 mM)
Have more KCl inside than outside = gradient of K and Cl is facing outwards + have a K chanel in the membrane
Start: Charge across the membrane is 0 (Charge gradient = 0) BUT the concentration gradient is not
1. K Chanel will open and K ions will flow down the gradient from inside to outside (because lots of K inside and less K outside) AND some of the K will go inside due to simple diffusion
- END - more K goes outside than the K going inside BUT some goes inside
2. Unequal movement of K creates a charge gradient across the membrane (+ outside and - inside) --> have negative membrane potential
3. After some time amount of K going outside decereases and the amount of K going inside increases
4. Eventually the rate of K going out is equal and oposite to the rate of K going in --> get eletrochemical equiliborum where the rates are equal and opposite
- NOW keep a constant membrane potential value (Ek)
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What drives the rate of K in vs. K Out
Rate of K going out is driven by the concentration gradient
Rate of K going in is driven by the electrical gradient
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Why is the charge gradient 0 at the start
At the start the charge across the membrane is 0 because for every K+ inside there is Cl- outside and for every K+ outside there is a Cl- inside --> MEANS that everything is electrically balanced and neutral
At the start the charge gradient is zero BUT the concentration gradient is NOT zero
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Why is there a charge difference when K+ leaves compartment
Get + charge outside because more K moves outside (Have K+ outside) AND get negative charge inside because Cl- that is left inside --> NOW have a charge across the membrane (negative charge)
- Get negative charge inside because when K+ leaves there is more single Cl- without any + K to balance it
NOW have a membrane potential (negative membrane potential because negative inside of the compartment)
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Why does the amount of K going outside decreases and the amount of K going inside increases
Because K+ starts to get repelled by the + charge that is building outside of the compartment
- + charge builds outside of the cell because K+ is moving outside of the cell
K+ going inside increases because the negative charge that has been built inside of the compartment pulls K+ in
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Chart of membrane potential in K+ example
Establishment of negative membrane potential due to uneven movement of K+ (More K+ leaving than going inside) --> begining of the red line going down
- Negative because building a negative charge inside the compartment)
Less K going out than before and more K going in --> line goes down but not as fast
- Membrane potential continues to get more negative because inside of compartment continues getting more negative BUT not as fast
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Ek
Ek = Value that the membrane potential stays at when at equilroium
- K is for potasium (would be Na doe Sodium etc.)
- At Ek the current is zero
When the membrane is at Ek the concetration gradient and the elctrical gradeint are equal and opposite
- Ions are moving BUT there is no net change in the voltage and no net flux
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Flux and current
Flux = difference between things going out and in
Current = rate of movement of charge over time
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Using the Nernst equation to Calculate Ek
Ek is the charge across the membrane
Can simplify larger equation (RT/F = 60mV) --> Ek = -60mV(log[Ki]/[Ko])
Example – have 150 mM inside and 1.5 outside --> Ki/Ko = 100 --> Log of 100 = 2 --> -60 X 2 = -120 mv --> Ek = -120mv
- K = +1 --> Z = +1
- IF did for Cl STILL get 120 BUT now it is +120 because z is –1 (neg X neg)
- IF did this for Ca Z = +2 --> Ca gradients are large BUT the membrane potential does not increases as quickly because divide by 2 (because divide by z)
NOTE - MAKE sure know basic logs (ex. Log 100)
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Capacitance
Charge moving across the membrane (Q) is proportional to the voltage gradient being made (V) (Q is proportional to V)
C = capacitance --> Q= CV
- C = 1/Resistance
- C determines the membrane potential built for the charge that is moved
IF Something has big capacitance Ex. ions moving across a barrier filled with water) --> Water shield the charges that builds up --> need to move a LOT of ions to build up the membrane potential
LOW capacitane --> very few charged ions need to move across barrier to build membrane potential
- Lipid Membranes are low capacitance because have no conductivity
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What happens to the concentration gradient at Ek --> does it change (DID we lose the concentration gradient)
When reach Ek the concentration gradient does NOT change significantly
WHY - because membranes have low capacitance --> moving a small amount of ions creates a membrane potential
- Few ions creates potential because Q is proportional to V
Because don’t need to move many ions to developed a membrane potential (difference in charge) --> MEANS that the concentrations won’t chnane when get to Ek
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How many ions have to move to get Ek
Can calculate how many ions moved from inside to outside to get to Ek --> number of K+ moved across the membrane to get to Ek = 0.25% (calculation on slide)
- SHOWS very few K lost = have little change to concentration gradient
As radius increases the proportion of ions that move across the membrane to generate Nerst potential/charge difference becomes smaller
- END - making the charge difference does NOT change the concertation gradient that much
Good that it does not take moving many ions because it takes a lot of energy to make a concetration gradient = would not want to keep losing the gradient
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Rules of thumb for concentration/charge gradients
1. Number of ions moved to generate dV is small
2. Concentration gradients remain largely unchanged
3. Electrical potentials change faster than concentration gradients
4. Must shunt the potential to generate a concentration gradient
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How can you make a concentration gradient
IF you are trying to get a concentration gradient and the gradient also creates an ion gradient then need a way to deal with the electric potential so you can keep moving ions to make the concentration gradient
- Because moving few ions would get electric potential faster --> once have electrical gradient then can't move more ions = you would not be able to move enough ions to get a concentration gradient
Example - Lysosome
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Acidification of the lysosome - Set up
Lysosome has a H+ pump that hydrolyzases ATP and pumps H+ inside the lysoome to make lyososme acidic
IF moved H+ inside you quickly build a positive charge inside of lyososme --> can't move more H+ inside once have + charge --> can't get a concentration gradient because build a charge difference faster (can't move enough H+ to get a concentration gradient)
- Charge gradient will build faster than the concentration gradient
- Need a concentration gradient to get pH difference
- Once have a charge gradient can't move H+ inside
END - Get charge difference but not pH different
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Acidification of the lysosome - Solution
1. Lysosome has the ATP pump and the K+ chanel (need high K+ inside lysosome)
- Each time a H+ goes into the lysosome a K+ goes out --> for every +1 (H+) that comes in a +1 (K+) leaves --> no charge gradient --> more H+ can come in --> can get concentration gradient --> get acidic lyososme
2. Use transporter that allows a Negative ion to go into the lysosome
- Each time H+ goes in Cl- goes in (+1 and -1 go in) --> no charge gradient --> more H+ can come in --> can get concentration gradient --> get acidic lyososme
- Cl- goes in because the + charge inside due to the H+ attracts the Cl-
- Cl- and H+ are using transporters
- NEED both Cl- and H+ makes the lysosome acidic
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Ionic gradients inside of mammalian cells
Ratios of In/Out shows if the Ion is higher outside vs. Inside
- More inside than Outside = Whole number (>1)
- More outside than inside = Decimal (<1)
More K+ inside Vs. Less K+ outside
More Na outside the cell than inside (Less Na inside)
More Cl outside of the cell than inside (Less Cl inside)
More Ca outside of the cell than inside (Less Ca in the cell)
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How to know if get negative or positive membrane potential
Example - K+ --> More K+ inside Vs. Less K+ outside
K+ chanel opens the membrane potentila becomes negative (-90mV)
- Negative membrane potential because K+ goes from inside to outside --> lose + charge inside = inside becomes more negative
Example #2 - More Na+ outside than inside
- Na+ chanels open --> Na goes inside the cell = get + charge inside (Ena = +69)
Membrane potential (Eion) is always with respect to the inside
- Negative inside = negative membrane potential ; Positive inside = positive membrane potential
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What happens when open one set of channels
When open one set of channels the voltage goes to E of the ions
When K chanel opens then the membrane postential decreases to Ek (Neg membrane potential because K+ leaves cell)
Close K and open Na --> Membrane potential goes up to Ena (Positive membrane potential because Na+ goes into the cell)
Close Na and open Cl --> Membrane potential goes down to Ecl (Negative because Cl- goes into cell)
Close Cl chanel and Ca opens --> Membrane potential goes to Positive Eca (Positive because Ca+ goes into
cell)
Shows that using f ion chanels AND the gradients --> the membrane potential can change quickly
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Why is change in membrane potential important
Change in membrane potential is important because the change in voltage is the signal that drives action potentials
Action potentials is the signal that is sent along nerves to the muscles AND Action potential is the signal that the muscles will use to contract
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Complicating factor for changing the membrane potential
When two chanels open at once
Example - Na and K chanels both open --> Na+ going in is balaced by the K+ is coming out --> no Nernst potential scenario because the gradients could cancel out and you would run down and burn out the gradients
Solution – ion channels have evoloved to open and shut quickly in a highly regulated fashion
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Summary
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