Lecture #5 - Ion Channel Structure and Function

Question 1

Why do we need ion chanels

Answer 1

Ion channels are needed to be able to assess the environment + decide to to respond + needs to be able to execute that resonse fast

Question 2

Timescale of electrical signaling

Answer 2

Electrical signaling + nerve conductions + muscle contraction _ sensory (vision + hearing) - works on a millisecond time scale

Need molecular machines that are able to accommodate for the spectrum of function

Question 3

Ion channels overall

Answer 3

Ion chanel = forms a pore in the membrane

Function – allows the flow of selective ions across membranes (acts like a gate)

Found at the plasma membrane or membranes of intracellular organelles (Ex. lysosomes and mitocondira)
- Lysosomal/endosome need channels to maintain homeostais of the organelle

Question 4

Ions chanell Vs. Transporter

Answer 4

1. Trasnport speed
   
   • Ion chanels moves ions 1000X faster than trasnorters (Uniporter + sympoter + antiporter)
     - Transporters are slower because only one side of the transporter is open at a given time Vs. ion chanels both sides are openso ion conduction is faster
2. Ion chanels are always passive (move ions down gradient)
   
   • Some transporters use passive (uniporter) but most directly or indirectley use energy from an already build gradinet to do work (active - symperters and anti porters)

Question 5

What direction do ions move through channels

Answer 5

Ions can move through channels in both directions
- The direction of ions moving is determined by the concentration gradient and the electrical gradient

In vacule example – have K that leaves the compartment BUT some of the K goes back in because of simple diffusion

Question 6

Two basic properties of ion chanels

Answer 6

1. Chanels have ion selectivity –> open chanels are selective to which kinds of ions pass
   
   • Example Na chanel is selective to Na and not K (allows Na to pass not K)
   • Because of selectivity there are different types of chanels (Na Vs. K Vs. Ca)
2. Chanels are gated –> means they need to receive a signal in order to open (chanel opens when the signal is present)

Question 7

Directionality of ions

Answer 7

Na, Ca into cell
K flows out of cell
Cl can go in or out of the cell

Question 8

How are ion channels selective (why are some channels permeable to Na or K (because K and Na both have a +1 charge AND Na is smaller than K so Na should be able to fit through a K channel)

Answer 8

Using the Selectivity filter (filter works based on the hydration shell around the ion)
- Ions are surrounded by water (form bonds ith O groups)
- K channel selectivity filter has O –> the O in the selectivity filter aligns around the K in the same way that the O in the hydration sheel normally aligns around the K in the hydration shell = K can pass through the filter
- Vs. Na going through selectivity filter of the K channel would not be energetically favorable

Found how selectivity works by looking using biochemistry to solve the structure of the K channel

Question 9

Types of ion chanels

Answer 9

Bevause channels are selective there are many kinds of channels:
1. K
2. Na
3. Ca
4. Non-selective cation chanels (Na, K, Ca)
5. H+
6. Cl-
7. Non-selective anoin channels (move large anions (Cl-) or glutamate- or ATP4-)
8. Large conductance non-selective (Na, K, Ca, Cl-, ATP4-, small metaboloites)
9. Aquaporins – water chanels (Ex. CHIP28)

Question 10

Discovery of Aquaproin

Answer 10

Did SDS-PAGE on RBCS –> Found a 28kD protein and wnated to know what it was
- Thought it was a membrane protein AND that it could be a water chanel because RBCs are permable to wtaer

Experiment to see if the protein is a water chanel –> expressed the cRNA in Xenopus oocytes to express the protein and study its function
- put oocyte expressing CHIP28 into a hypoosomotic solution –> cell swells

Control experiment - Add Gaba chanel (cl- channel) to oocyte –> add Gaba to activate the channel –> cell does NOT swell
- Means that it is not an ion causing the cell to swell (water is causing it to swell) means that cRNA expressed codes for a water chanel
- Needed contol becaue any channel that passes ion can leak water

Question 11

Why was the Aqporin Experiment Lucky

Answer 11

Just so happened that they used oocytes that are resistant to osmotic shock = the cell does not swell naturally in a hyposomotic solution (needs to express the cRNA for CHIP28 in order for the cell to swell)
- If did on human cells they would burst without the cRNA

Deisgned the expeirment by guessing (vs. Looking for selectibility filter used biochemistry to find strcture)

Question 12

Types of gating

Answer 12

1. Voltage gating (Kv, Nav, Cac, CLC) - voltage chnages –> the channel opens
2. Ligand gated (Ca, IP3, cAMP, H+, ATP, glutanate, Actylycholine, G-Porteins) - Ligand binds –> chanel open
3. Mechanical (physical) force and cell volume gate the chanel (MscS, MscL, Piezz, and swell1) - Chanel opens/closes due to chnages in mechanical force/ cell volume
4. Temperture and chemicals (TRP) gate channels - temperature chnages/add chemical –> Channel opens

Question 13

How ion channel gate works

Answer 13

Image - Shows structure

Top = selectivility filter
Bottom = Gate (physical barrier/gate)
- Gate is closed ions can’t pass ; Gate is open
= channel is open = ion can pass
- Domains will pull and the gate will open = have open channel

Question 14

Open Vs. Closed states of the gate

Answer 14

Open. Vs. Close states correlates to the physical movement of proteins in those 2 confirmations (protein is moving from one state to the other)

Question 15

Applications of Patch Clamp

Answer 15

1. Cell attached patch clamp –> Look at the current if a single protein (single molecule expreiment)
   
   • Membrane stays intact
2. Whole cell recording –> break the membrane –> have acecss to all of the chanels in the cell
   - Gives a bigger current because looking at many channels

Question 16

Summary of basic properties of ion channels

Answer 16

Question 17

Na/K ATPase pump

Answer 17

Use ATP to pump Na out and K into the cell –> maintains the Na and K gradients (needed grdaients for nerve cells to conduct signals and muscle contraction)
- Uses 25% of the total ATP (70% of total ATP in nuerons)

Question 18

Equilibrium potential

Answer 18

Membrane potential where the net flow thorugh any open chanel is 0

Find Eqrilibroiam potnetial based on nerst equation (RT/ZXLog(i/O))

Equilibrium potential (Ek) = reversal potentila

Question 19

Equilibrium potential (K)

Answer 19

K gradient – high inside of cells ; low outside of the cell
- K chanel opens –> K leaves the cell –> inside of the cell becomes negative

Equilibrium potential for K = -90 mV (when inside of cell is -90mV net ion flow is 0)

Why do the ions stop flowing –> Because when the K ions leave a negative change buidls in the cell –> at Ek there is enough netaive charge in the cell to pull the K ions back into the cell (rate of K in is equal and opposite K out)

Question 20

Equilibrium potential (Na)

Answer 20

Na graduent – High Na outside ; Lower Na inside
- Have high out/low in because of the Na/K ATPase pump
- Na chanel opens –> Na goes into the cell –> builds a positive charge inside of the cell

Equilibrum potential or Na = +66mV (movment of Na in and out is equal and oppsosite)

When Na goes into the cell = builds a positive charge inside of the cel = over time less Na goes into the cell becaue Na+ is repleled by the psotive charged that is building inside of the cell –> Na stops going into the cell –> get equillirvoun potnetial

Question 21

Equilibroum potential (Ca)

Answer 21

Ca concentration gradient ( 20,000 fold gradeint) – More Calcium outsid ; less caclium inside
- When Calcium chanel opens –> Ca goes into the cell

Equilirium potential for Ca = +130 mV

Why is Caclium low in a cell - becuase phosphogroups precipitate with Caclium –> when life first develped it was important for the cells to exclude Caclium (evoloved to have many Calcium pumps to rmeoce Ca and keep Ca low)

Question 22

Use of the Ca gradient

Answer 22

Having a large gradinet makes Ca ideal to be a second messenger

Calcium can be a second messnger because cells can easly sense an increase in Calcium
- A little bit of Ca going into the cells allows the cells to do a lot of things
- Can increase the dynamic range of concentration (good for second messnger ; can increase the calcium a ton)

END – can incerase the calcium a ton because there is so little calcium inside the cell (makes it a good second messnger) vs. Na can’t increase the Na as much because there is a higehr strating concetration of Na in the cell

Question 23

Stimulus in Action potential

Answer 23

1. Opening of a few volatge gated Na channels in the adjancebnt membrane of an axon (Na chanels open nearby and the + charge drifts)
2. Stimulus in the nerve terminus that cause the sensory channels (transmitter gated cation chanels) to open –> cation (Na and Ca) goes into cell –> get depolariation

Question 24

His definition of thershold

Answer 24

Threshold = when the voltage gated Na chanels are triggerd to open (Whether or not you reach threshold = determins if all or none of the Na chanels open)
- Threshold mmebrane potentila = -55mV

HE says - stimulus cuases the membrane potentila to reach a threshold –> Na chanels open –> Na Ions go into cell –> triggers Action potential

Rajni - Stimulus –> na chanels open –> reach thershole

Question 25

Steps of the action potential in the chart

Answer 25

1. Stimulus 2. Depolarization (Na chanels are opening --> Na goes inot the cell) (At peak ALL of the Na chanels are open) 3. Repolarization --> Na chanels close and Delayed K chanels open (K leaves teh cell = membrane repolarizes) 4. Hyperpolarization- the membrane potential drops below the resting potential as K+ continues to leave 5. Voltage-gated K+ channels close; resting state re-established by Na+/K+ pump & K+ leak channel

Question 26

Diagram of ball and chain model

Answer 26

Bottom right = have the open confirmation --> THEN the N temrinal swings and plugs the pore (N temrinal goes back into the pore) - Chanel is not stable in the open confirmation

Question 27

Cardiac Action potential

Answer 27

Uses a voltage gated Ca channel to mediate the contraction AP - voltage gated Na chanels open --> trainets outward flow of K --> Voltage gated Calcium chanels open (Ca flows into cell) --> calcium channels close and volatge gated K chanels open --> K continues to leave (cell repolarizes) AP is borader (no sharp peak) - Broader part (cell is depolarized) = where the volatge gated Ca channels open Muscles Vs. Nerves AP --> nerves have a shorter period of depolarization Vs. Muscle have a broad (longer) period of depolarization

Question 28

What are the molecules that ar responsible for the voltage gated channels

Answer 28

Goal - Find molecules needed to have a voltage gated channel - Genetic/molecular biology = allowed people to cloned new proteins Experiment studied a fly with mutaion in shaker gene (shakes and falls when temperature changes) - Did recording experiments --> found it is DUE to change in voltage gated K chanel conductance - To find the gene that causes shaker mutation --> did positional cloning --> found the cDNA of the voltage gated K chanel protein - Once have the cDNA --> looked at the protein sequence (found 6 transmembrane regions)

Question 29

Compoennts of a voltage gated ion chanel –

Answer 29

When found structure based on cDNA the protein aligned with what people understood about channels 1. Pore domain - Corresponds to the selectivity filter 2. Pore domain - Contains gate that is pulled by the voltage sensing domain 3. Voltage sensing domain (S4-S5 domain) 4. Inactivtaion domain in the N terminus

Question 30

How does a channel sense voltage across the membrane

Answer 30

Idea of how voltage gated channels can open/close in response to voltage - Voltage gated chanel is in the membrane has a sensor (S4) in the membrane domain Positive charges in the S4 sensor is draws it towards the negative charge from the negative membrane potential inside of the cell When the cell becomes depolarized (more +) --> membrane expels the positiev charged S4 helix out of the membrane --> S4 helix leaving causing a confirmation change --> Chanel opens - SHOWs chanel confirmation is coupled to membrane potential Here they knew nothing about a molecule used basic observtaions from physiology to predict the features of the molecules

Question 31

Basic structure of a voltage gated ion channel

Answer 31

Voltage gated K ion chanels = Teromeric --> have pore that is permeable to ions in the middle of the tetromer - Can be Homomeric or hetromic complex Volatge gated Na and Ca channels = fuses 6 SU together (24 transmembrane domains total)

Question 32

Volatge gated K chanels (overall)

Answer 32

Most diverse voltage gated chanel because every cells has a K channel (needed because it regulates membrane potential) Diversity of the K Chanel (have unique properties): 1. Calcium cativted K chanels - K chanels can hook with a Ca binding domain - Ca binds to teh K chanel --> K chanel opens --> K ions go through 2. Leak K chanels - K chanel can lose the voltage sensing domain 3. Inward rectfied K chanels - fuse 2 pore domain

Question 33

Example diversity + signal convergence in K channels

Answer 33

G protein gated K channels - shows signal convergence G portein is actiavted using Calcium and cAMP --> Beta/Gamma Subunit of the G protein binds to the K chanel --> opens the K chanel --> K leaves --> cells stops signlaing - Causes negative feedback --> slows down the heart rate

Question 34

Example of K channel diveirsty #2

Answer 34

Katp channel - has SUR domain responds to ATP:ADP ratio (K chanel fusing with teh pore domain of other subunits to sense metabolic state of cells) Process – When eat glucose levels rise in the blood --> beta cells in the pancreus express GLUT2 --> GLUT2 brings glucose into the beta cells --> bringing glucse into beta cells increase the metabolsim of glucose and ATP levelas (decreases ADP levels) --> chnage inATP:ADP ratio closes the Katp chanel --> block K from leaving --> cell becomes depolarized --> depolarizaton opens the voltage gated Ca channels --> Ca goes into the cell --> Ca causes the synpatomagin to has confrmation change --> V and T smare on the insuline vesciles and membrane form a complex --> release insulin - Beta cells have insuline ready to be relased using V and T Snares and synpatomagin

Question 35

Mutation in kATP chanels

Answer 35

LOF of Katp --> get hyperinsulinemia (low blood sugar ) GOF of Katp --> Neonatal diabetes - Not secreting insulin because not getting membrane depolarization Katp is a target for diabetes treatment in Katp GOF (Ex. Sulfonylurea: Gilbenclamide)

Question 36

Voltage gated Na channel family

Answer 36

Voltage gated Na channel is only expressed in excitable tissue (nuerons and muscle) - Because only expressed in few cells types there are is little diversity - Expressed in PNA + CNS + cardiac/skelatal muscle

Question 37

Toxins that target the Na channel

Answer 37

Na chanels are important for firing an action potentiial --> animal kingdome has evoloved toxins that targets the Na chanels Toxins can target the voltage gated Na channel to paralyze Prey (Cone snails use the toxin to paralyze fish)

Question 38

Use of toxins in lab

Answer 38

Looking for Na channel proteins using toxin that poisons Na chanels and biochemistry Cloning channels - - radiolable the toxin (has high affinity for Na chanel) --> use the radiolabled toxin to purify the Na chanel - Found Na channel has an big protein (alpha SU) and accesory beta subunits Used a similar apprach to identify the opoid recpetor (used radiolabed opioid to map where the receptor is

Question 39

Voltage gated Na channel beta subunits function

Answer 39

Roles of the accessory beta subunits: 1. Regulate channel expression 2. Modulate channel gating 3. Form links to cytosklatal network and exracellular matrix Beta subunits = lipid modulator protein that is regulating the function of the chanel

Question 40

Chanelopathies

Answer 40

Diseaes related to dysfunctions of ion chanels - Can be congenital or aquired - Affects many systems

Question 41

Looking for the pain receptor

Answer 41

Looked for the pain receptor to be able to make new pain killers Found the Na chanels involvement using genetics: - Sequenced people that don't feel pain --> LOF of the Na chanels (Congentical Insensitivity to Pain ) - Sequences from people with increases sensitivity to pain - found GOF in the Na channels - Suggess the Voltage gated Na chanels can be a target for pain --> made a pain killer that inhibites Na1.8 because it is the main conducting chanel and 1.7 didn't work (was able to reduce pain score by 1 point)

Question 42

Brake to excitability

Answer 42

Voltage gated K channel is the brake to excitability (makes the AP go down or decrease excitability in Katp) Because when K chanel opens --> K leave --> the membrane repolarizes (more negtaive)

Question 43

Mutation in the K chanel K1.1

Answer 43

LOF in the K chanel (K1.1) --> Can't repolarize after AP --> get episodic ataxia and epilepsiy Person shakes after small amounts of physical activity (similar to shaker mutation in flies)

Question 44

Summary of voltage gated ion channels and AP

Answer 44

Question 45

Sensory receptors Overall

Answer 45

Sensory system uses sensory receptors Sensory receptors ARE non-selectve cation channels - Sensory recetors are used to quicly respond to stimuli - Usualy the sensory receptor channels are not active (usually closed) Example – stimuli --> Sensory receptor non-selectve cation channels opens --> Na AND Ca goes into the cell (cell becomes depolarized) - Ca going into the cell = acts a second messenger (drive excitability or gene expression)

Question 46

Signal transduction in the somitosensory system

Answer 46

Sensory receotors sense: 1. Chemicals (chemosensation) 2. Machanical sensation (mechanosensation) 3. Temperatre (thermosensation) Pathway - Sensory receptors (ex in the skin) sense chemicals or mehcnical inputs or tempertures --> sensation/signal goes to the cell body in the dorsal root gangiion --> informatoion (signal) goes to the brain --> have response In research want to identify the sensory receptors

Question 47

Step 1 in finding the sensory receptors

Answer 47

Experiment - measure the membrane potential in a DRG nueron with is it stimultaed with a chemical - When add casacin or habanero (chemical stimulus) --> measure the membrane potential --> get electrical activity of an inward current (Na and Ca goes into the cell) --> Shows neurons can detect chemical in vitro Wanted to add a tempertaure simulus and measure the membrane potnetilas to see if nuerons are senstaive to temperture BUT temperture is hard to study --> Solution was to use chemicals that can cause the sensation of heat

Question 48

Step 2 in finding the sensory receptors - Add chemicals that cause the sensation of heat

Answer 48

Use pepper or capsacin Experiment - Add hapabero (spicy peper) stimulus to DRG nueron --> measure the membrane potentials --> there is a large inward current - When add a not spicy pepper there no current Found the inward current is due to capcacin in the spicy peppers --> if add capsacin to the DRG neuron get a large inward current Showed that nuerons can be activated by these ligands in vitro (nuerons have sensory receptors for capsacins)

Question 49

Step 2 in finding the sensory receptors - Finding the sensory receptor (ways to do it)

Answer 49

Idea 1 - Knockout genes in the nuerons and see which Knockout causes a reduced respnse to capsacin Idea 2 - Add cDNA into cell and express that gene

Question 50

Step 2 in finding the sensory receptors - Finding the capsacin sensory receptor

Answer 50

To find the capscacin receptor – isolate ALL the genes in neuron and study their function Experiment (Used cDNA expression) - Clone cDNA from DRG neurons --> express cDNA in cells --> pool cells that have cDNA expression - To find receptor (find the gene that codes for capsacin receptor) - look for nueron that was not responding to Capsaicin --> THEN when express the cDNA the cell now responds to capsacin (that cDNA codes for the receptor)

Question 51

Step 2 in finding the sensory receptors - Finding the capsacin sensory receptor (Limitation)

Answer 51

Limitation – how do you assay the cells for response to capsaicin (impractical to find clones that respond by measuring the membrane potential for each clone) Solution – use a Calcium dye (because the Capsacin receptor is a sensory receptor so it is permeable to Ca) - Color indicates Calcium release = indicates if sensory receptor chanel is activated

Question 52

Step 2 in finding the sensory receptors - Finding the capsacin sensory receptor (with Ca dye)

Answer 52

Experiment --> add Capcascin to DRG nuerons --> Add the calcium dye to cells --> look at Calcium levels (calcium imaging) - NOT looking at membrane potential INSTEAD looking at color for Ca dye Once find cells that have Calcium release --> take the sub clones that have Ca release to idetify the Capsacin reeptor - Found cDNA that is expressed in the subclones (cDNA had homology with the voltage gated K chanel BUT the capsacin receptor found is not sensitive to voltage)

Question 53

What is the Capsacin receptor sensative to

Answer 53

Found that capcsian receptor is sensative to capsacin AND heat Means that one sensory receotor has chemical sensing and thermocensing End - capsacin receptor (TRPV1) is a heat activated ion chanel in the pain pathway

Question 54

Studying Capsacin receptor in vivo

Answer 54

Looked at pain sensation in mice that lack the TRPV1 chanel to see the physiological importance of the CAP receptor Exepriment 1 - Make a Knockout mouse --> injected capsacin into the paw - WT mice lick the paw ; Mutant TRPV1 KO did not lick the paw (not sensative tp capsacin) Experiment 2 - Added capsacin to water to see how much of the capsacin mice drink - WT mice --> don’t drink the water ; KO mice – drink the water BOTH experiments showed that TRPV1 channel is the capsacin receptor in vivo

Question 55

What happens after the after Capsacin experiments

Answer 55

Did similar screens using menthol to find the sensory receptors that sense cold temperatures Did more expeirments to find even more receptors for dfferent chemicals ALL these epxirments show that pharmocology can inform physiology

Question 56

Where are sensory receptors expressed

Answer 56

Sensory receptors are expressed in sensory cells and in other cells responding to various signals

Question 57

Mechanically Activated ion channels

Answer 57

Mechanically activated ion channels are technically challenging to study Mechanically Activated ion channels are important because important for touch + mechanic force + development How to assaying mechanically activated ion channels --> Need to record the membrane potential using a microelevtrode while the cell is being pushed with a glass pipette - Increase the pushing distance -> get an inwards current

Question 58

How do you design a screen to study mechanically activated ion channels

Answer 58

Hard to screen mechanically activated ion channels because hard to use a micro electrode to study membrane potentials to screen many cells Tried many solution: 1. Used a chemical that they thought would have a mechanical effect BUT that did not work 2. Used mole animals (did a microrray to find genes) 3. People added a cell to a dish --> used a probe to push the membrane and do Calcium image Solution – Microarray and RNAi Screen

Question 59

Microrray to find candiate genes of the mechanosensor

Answer 59

Had 2 cells lines: 1. Cell line that responds to mechanocal stimuli 2. Cell line that does not responds to mechanocal stimuli Did a microaray to see the genes that are different in the repsonders Vs. Non-responders Take the genes that are hits in the microarray --> do siRNA tranfections --> do microelectrodae assay on each of the cells

Question 60

RNAi screen of mammalian mechanosensor

Answer 60

siRNA screen with the microelectrode assay in a non-neuronal cell line that respond to mechanical force and are easier to manipulate Added RNAi guides to cells that normally respond to mechnaical force --> do elerctophysilogy to see if the cell responds - Want cells that normaly have an inward curent when pushed and lose the inward curent when pushed when the RNAi guide is added (guide is KO the gene that codes for the mechanosensor) - Guides = based on the microaray of responders vs. Non-tesponders

Question 61

RNAi screen of mammalian mechanosensor - results

Answer 61

Found 1 gene where there is a decrease in curent (low pA) when the siRNA is added --> means that cell no longer responds to mechanical stiumuli Chart – shows that the cells that have the gene KO have less current END - When add siRNA to cells --> KO the gene that codes for the mechanocesnor rceptor --> have less current

Question 62

Mechancal Force sensing Receptors

Answer 62

Piezo chanels (found in RNAi screen) Structure: Has a blade structure (shows how structure informs protein function) - Trimer + Transmembrane protein How does it work --> when have mechanical force the membrane bends --> the bending of the membrane opens and close the center cavity in the pore - Curved protein --> when flatten the channel is opened

Question 63

Cl- chanels

Answer 63

Cl- is the most abundant free anion in cell Cl- channels are not selective because they don’t have to be (not a lot of any other anion that could go through the channel ; only Cl- is around to go through)

Question 64

Cl- in nuerons

Answer 64

Nuerons express a lot of transporters that remove Cl- from cell (low Cl- inside ; high Cl- outside) - When open Cl- channels --> Cl- goes into cells Example – GABA receptor is a Cl- chanel - Gaba binds to the GABA receotor --> Cl- channel opens --> Cl- goes into cells --> membrane potential is hyperpolarized (basis of nueronal inhibition)

Question 65

Cl- in most cells

Answer 65

MOST Cells expression Cl- loaders that bring Cl- into cells BUT still the conetratin is not as high as the outside of the cell (Cl- Inside is low ; Cl- Outside is high) - BUT when Cl- channel opens --> cl- goes out of the cell Why does Cl- go out if there is more Cl- outside - because the inside of cell is negative --> Cl- is repelled by that negative charge (does not go inside of the cell) - Cl- direction is not about the concetration grdaeint INSTEAD it is about the electric gradient

Question 66

Use/Important of Cl- Channels

Answer 66

1. Fluid secretion 2. Excitability 3. Cell volume regulation 4. Orgenalle pH regulation

Question 67

Cystic fibrosis

Answer 67

IN lung cells – when Cl- chanel opens --> Cl- leaves cell --> water follows salt so the water will also move out of the cell --> keeps the lungs most so cillia can move and clean up the lungs IN Cystic fibrosis – Muated Cl- Chanel --> can’ 'relase Cl- --> water does not go to the surface of the lung --> mucus builds up --> get bacteria infections Mutaions in Cystic fibrosis: 1. Mutaions that causes the chanel to not function - Treat with Potentiator (opens the chanel more frequntely) 2. Mutation that casues the Cl- channel to not go to the plasma membrane - Treat with Drug that helps the protein fold

Question 68

How much does the chaparone drug effect

Answer 68

Drug only corrects a small amount of protein WHY is this ok - Becase only 10% of proetin made in a healthy cell end of u at the plasma mebrane (drug doesn’t have to fix 100% of proteins because 90% of proteins made in a normla healthy cel don’t end up at the plasma membrane) - Only need 10% of the protein made to get to the plasma membrane (because that is the amount in healthy person)

Question 69

How much of proteins are degraded

Answer 69

90% of proteins are degraded - Have costant turnover to be able to maintain the 10% to get enough channels at the plasma membrane Why degrade so much - Because have many copies of proteins it is better to degrade them and make new ones that to correct misfolded proteins (means a lot of proteins are dgeraded) - If always fixing proteins would need fixers for the fixers (no very effcicient) + so that evolution can use positive muttaios - EXCEPTION – DNA 0 you need to have a wya to fix DNA

Question 70

Summary of sensory ion channels and Cl-

Answer 70

Question 71

Summary of expeirments:

Answer 71

Disadavtntge of the expresion clone – assme cDNA is sufficinet to get activity (would not work if have multiple SU in porteins)

Question 72

Summary of disease

Answer 72