Lecture #4 - Membrane Transporters Carriers and Pumps

Question 1

Ways thing can move across the membrane

Answer 1

Passive (No energy): ALL Molecules going down the concentration gradient (High –> LOW concentration)
1. Diffusion - No protein involoved (molecules just go through PM)
2. Channels (facilitated diffusion)
3. Uniporter (facilitated diffusion)

Active: Molecules move Low –> High (uses work)
1. Symporter (Secondary Active)
2. Antiportnter (Secondary Active)
3. Pump (primary Active)
- Makes gradients –> gradient can be used by symporter and antiporter

Question 2

Chanels Vs. Uniprter

Answer 2

Open ion channels have access from both sides of the membrane at one time (BOTH sides of the channel are open at once)

Uniporter - Never open to both sides of the membrane at once (BOTH sides of uniporter are never open at once)
- Binds to molecule on one side –> closes the part of uniporter that was open –> molecule will be occulded (trapped) within the uniporter –> other side of uniporter opens –> molecule can leave
- THIS applies to pumps and Sympoters and antiporters

Channels are uniporters in a thermodynamic sense but their mechanisms and structures are different

Question 3

Symporters and Antiportners

Answer 3

Moves substance down the concertation gradient AND captures the energy of substance going down the gradient to move another substance up its gradinet from low to high (using the energy of the substance going down the concetration gradient to do osmotic work)

Symporter ( co-trasnporter) - both molecules are going in the same direction (Ex. both go into the cell)
- Ex. Na coupled Glucose transporter (Na down ; Glucose up - both into cell)

Antiportnter (exchnagers) - both molecules moves in oppoite directions (One goes into the cell ; one goes out of the cell)
- Ex. Na down ; H+ out (Na in to cell and H+ out) –> Na Gradeint is EXCHANGED for the H+ gradient

Question 4

Secondary Active Vs. Primary active

Answer 4

Primary Active - making the gradients (does not need the grdaeint to exist)
- ALL pumps are primary active

Secondary active = Uses a prexisting gradeint (needs to gradient to already exist)

Question 5

Ions and Solute Pumps (overall)

Answer 5

Requires a direct link to source of energy

Use the most complex mechansim for transport because of energy coupling
- Energy coupling = Captures energy from one source –> coverts energy into a diferent form of energy
- Converts energy to energy of a gradient (form of energy pumps make)

Pumps have high specificty because energy coupling is strict and complex molecularly (Ex. H+ pump ONLY transports H+)
- Ion channels are selective (selective for Na over K BUT they can still transport some K)

Question 6

Energy sources for pumps

Answer 6

Forms of energy that pumps harness:
1. Light - Halobacteria use Rhodopsin to capture light –> light drive H+ or Cl- pumps
2. Redox potential
- Energy used to drive the ETC machinery H+ pumps
-Pumps in the mitocidnrial membrane + bacteria
- Moves H+ and Na
3. Decarboxylation
- Ion-transporting decarboxylase pumps are coupled Decarboxylation
4. Pyrophosphtae
- Used by H+ pyrophosphatase pumps
5. ATP
- Most common source of energy
- ATPase transporters (pumps) use energy of ATP hydrolysis to pump ions and other solutes

Question 7

Prmary pump in mamlian cells

Answer 7

Na/K pump is the primary pump in mammalian cells (use 25% of energy that the cell produces)

Why is it ok that the Na/K pump burns all of the ATP –> because the Na and K gradents that the pump forms is a form of energy that can do work
-Converting the energy released from ATP hydrolysis to make to make energy in the form of gradients

Question 8

Kinds of work ion gradeints can do

Answer 8

1. Chemical work (ATP syntehsis)
2. Chemiosmotic work –> gradeints drives antiporters and symporters (move solutes + metabolizes + nutrients across the membrane)
3. Osmitoc/cell volume regulation –> Cells regulate volume by moving ions across membrane and water following passively
   4, Regulates homeostasis and remove toxic compounds (ex. pH regulation)
4. Mechanical work - Ex. flagella
5. Signal trandiuction
   - Energy used by pumps is involoved insignling because the pumps set up the grdaeints for chanels and chanels are used in signaling

Question 9

dgATP

Answer 9

dG (ATP) = Energy from ATP hydrolysis (work done by ATP)

dG ATP resides in two components:
1. Concertation gradient of ion
2. Membrane potential because ion is charged (When generating concetration gradients –> moving ions that have charge –> creates a charge differnce)

The Concentration and voltage gradients are freely interchangeable

Question 10

Electronuetral pumps

Answer 10

Electronuetral pump = not making a charge gradient

IF a pump is electroneutral (ex. use ATP to drive +1 ion in and +1 ion out) –> dV is 0 (no charge gradient) –> ALL of the energy from ATP is used to make a concetration gradinet across the membrane

In electronuertal transport you can make a BIG concentration gradient (max 100,000) (because the ions won’t be reppeled by a charge gradeint)

Example – H/K ATPAse makes the stomach acidic (need big concentration gradient) - To do this for every ATP it moves 1 H+ into the lumen of stomach K+ leaves lumen –> not worried about electric gradeint –> keep pumping ions

Question 11

What hapens when transport is NOT electronutral

Answer 11

Pump would use the energy of ATP hyolysis –> ALL of the energy from ATP hydrolysis goes inot making a voltage gradient

Question 12

Classes of ATPases Transporters (Major classes of ATP driven primary pumps)

Answer 12

1. F-Type - FoF1 (ATP synthase)
   - Inner mitochondrial membrane, chloroplasts, bacteria membranes
   
   • Transproters H+ and Na (rarely)
2. V-Types - VoV1 ATPase (Lysosomale H+ pump)
   - Similar to F type BUT not synthesizing ATP (uses ATP to make H+ gradeint)
   - Endosomes + lysosme/vaculoe + synpatic vesicles
3. P- Type - cation pumps (3Na/2K ATPase ; 2H/2K ATPase ; 1-2Ca/H ATPase)
   - Sometimes transport lipids or petides
   - PM + ER + golgi
4. ABC pumps (ATP binding cassete)
   - Transports things other than ions (solutes + drugs + proteins)

Question 13

F1Fo ATP Synthatse + ETC

Answer 13

F1Fo ATP Synthase sites next to ETC

ETC is a redox driven pumps that pump H+ from matrix to the inter membrane space (makes H+ gradient) –> H+ flow back down gradient from the intermembrane space into the matrix and go through ATP sythase –> ATP synathses makes ATP

Question 14

Why is F1Fo considered a pump

Answer 14

Considered a pump because can complete the reaction in both ways

1 – If you add ATP to purified F1Fo it will make a H+ gradient
2 – If add H+ gradient with ADP/Pi to F1Fo it will make ATP

In bacteria - IF no O2 to drive ETC –> use ATP from glycosis to dirve F0F1 as ATPase –> F1Fo uses energy from ATP hydrlyisis to make a H+ gradient –> use the gradient to do things

In mitochondria and chloroplast F1Fo are designed for ATP synthsis –> In hypoxia protein inserts into the pump to physicaly block F1Fo fro being ATPase

Question 15

F1 sector

Answer 15

Sticks into the matrix of mitocondria (or into cytoplasm in bacteria)

Contains ring of Alpha and Beta SU (a3b3)
- Beta SU = has ATP binding and catalytic activity ; Alpha SU = has ATP binding (no catalytic activity)

Function – F1 can break or make ATP (ATP synthatse or ATPase )

Question 16

Fo sector

Answer 16

Embedded the membrane

H+ conductions (H+ chanel that conducts H+)

SU structure – a1b2c12
- Has C ring (ring of SU) –> each SU has a negative charge that allows the SU to bind to H+
- A subunit has a positive charge (Pos ion) AND has two hemi chanels that are not connected to each other (ensures there is never a pore through membrane that is open to both sides)

F1 and Fo sectors are connected by the gamma SU (gamma stalk)

Question 17

How are H+ moved through ATP synthase

Answer 17

H+ enters 1 half chanel (by interacting with arginine in A SU) –> H+ binds to the negative charge (E59) in C ring –> SU of the C ring go around in a circle –> C ring bound to the H+ recahes the next hemi chanel –> H+ goes through second hemichanel (top chanel in mage) –> H+ goes to other side of pump (H+ leave)
- Because every SU in the C ring is H+ bound the SU move
around in circle
- Number of SU in C ring chnages depending on the stoichemtry of H+:ATP

Question 18

What is Affinity of the pump/SU for protons based on

Answer 18

Affinity of the pump/SU for protons = based on Pka

Pka – probability of H+ binding or coming off (probability defines if the pump/SU has high or low affinity)

Question 19

Binding Change mechanism - Binding strengths

Answer 19

F1 has 3 catalytic SU = 3 catalytic sites (each can form or break ATP) –> each catalytic SU in F1 has a different binding affinity for ATP at a given time

At a given time one SU will be:
1. Tight (ATP is formed and bound tight ; need energy to remove ATP)
- ADP/Pi in a tight site = gets made into ATP
- Affinity for ATO is high = ATP can’t leave
2. Loose (ADP-Pi is bound lossley)
3. Open
- ATP is not bound (low affinity for ATP)

Question 20

Binding Change mechanism

Answer 20

Energy goes into the system (ADP/Pi enter the open site) –> binding of ADP/Pi opens the Tight site –> NOW ATP can leave site AND what was the loose site becomes tight AND whay was he open site becomes loose (bound ADP-Pi) –> ATP leaves open side –> ADP-Pi binds to new open –> the ADP-Pi becomes ATP in tight site AND loose keeps ADP-Pi

3 catalytic SU that go through alternating catalysis
- System is driven by seqeuntial chnage in binding affinity (changes in binding affinity causes chnage in catalytic activity)

Question 21

What causes the chnages in binding affinity in binding change mechansim

Answer 21

System is driven by seqeuntial chnage in binding affinity

Change in binding affinity occurs because Gamma SU shaft has changes contacts with the catalytic beta SU
- First contacts one SU which gives the SU certain properties THEN gamma SU roattes and contacts the other SU

As gamma SU rotates within F1 (contacts the catlytic SU) –> caises a chnage in binding affinity at each SU for ATP –> chnages in affinity chnages the catalyitic activity of the SU (ATP is made or relased)

Question 22

WHY does the Gamma ring rotate?

Answer 22

As the C sun ring turns in the lipid bilayer it causes strain/torque on the gamma SU –> gamma SU will release that strain in intervals–> release of the strain causes the gamma SU to ratched from one Catlystic SU active site to another to another –> rotation of gamma SU changing the affinity of the calalytic SU for ATP -> causes ATP to be made by
- Torwue causes the rotaion of the gamma SU
- Ratchet movement happens where the Gamma SU contacts the F1 sector

Question 23

F1Fo hydrolyzing ATP

Answer 23

F1Fo can also hydrolyze ATP

Uses the same mechansim BUT the Gamma SU rotates in the opposite directions and H+ will be moved across

Question 24

Experiment that showed the rotation of the gamma SU

Answer 24

Attached F1 to a nickel coated glass using His tags (bind to nickle) –> cross link an actin filament that is fliruenley labled to the F1 hexomer –> Add ATP –> actin filaments rotate (indictaes the gamma SU are rotating in the direction of hydrolysis)

IF slow down the video – can see that the gamma SU pauses at 120 degree angles for each of the 3 steps in catalysis

Question 25

Where are V1Vo Types ATPase Pumps

Answer 25

Endosome compartments inside cell – Lysosomes/vaculous + golgi + clathrin coated vesciles + synaptosomes + secretroy vesciles + endosmes Plasma membrane of specilzied cells that secrete H+ - Macrophages makes regions that they engulf acidic to kill bacteria - Kidney - secrete H+ into urinary filtrate to regulate the pH - Osteoclasts-- Osteaclasts make a tight seal around the bone and secerete H+ using the V1Vo --> acidic pH degrades the bone

Question 26

Function of V1Vo

Answer 26

1. Acidifincation - used for receptor mediated enodcytsosis + targeting of lysosomal vesciles + activation of Acid hydrolases + protein porcessing - Receptor mediated endocytosis --> Acidification is needed for the ligand to fall off the receptor once it is in the endosome 2. Electrochemical H+ gradeint - used for the uptake of solutes via H+-coupled transport (Using the H+ gradient to take up solutes) V1Vo and F-Type ATPase pumps use similar mechasnims

Question 27

What is H+ couple transort is used for

Answer 27

1. Storage of basic amino acids + phosphate + calcium in vaciles 2. Taking up neurotransmitters at synaptosomes --> Nuerotranmitters need to be concetrated in presynaptic vesicles - Done using using the H+ gradinet (Nuerotranimitters go into the vescile and H+ go out) - H+ gradinet made by V1Vo ATPase

Question 28

F-Type (F1Fo) Vs. V-Type (V1Vo)

Answer 28

V-Type function = hydrylozes ATP to make H+ gradient Have similar in structure - V-type has more subunits (more complicated) than F-type When studying F-type Vs. V-Type: - F-Type ATPase pumps are more well behaved --> IF take pump part F1 is always ATPase and Fo is always a H+ channel - V-Type ATPase pumps are less well behaved --> IF take apart the Top and Bottom halves are BOTH non-functional

Question 29

Why are V-type pumps non-functional when apart

Answer 29

V-Type pumps evolved this way because V-Type ATPases are regulated by assembly and dissaly (not active when not assmbled ; active when assembled) Example - To conserve ATP if no glucose in the cell V-ATPase falls apart - NOW Membrane part is not open for H+ (won't lose the energy that was used to make H+ gradient) - V1 is turn off because you don’t want it to hydrolyze ATP

Question 30

Disorders of V-ATPases

Answer 30

1. Distal Renal Tubular Acidosis (dRTA) - Mutation in two V-ATPase subunit that are ONLY expressed in kidney cells and cochlea --> kideny and cochlea can't excrete H+ --> get metabolic acidosis hearing deficit 2. Osteopetrosis - Mutation in V-ATPase subunit expressed in osteoclast cells --> can't remodel bones (get dense bones) --> lethal

Question 31

Answer 31

Answer - B --> There is no Cl as a counter ion = can’t get H+ concetraton gradient = cant get pH gradient - Need to balance the movement of H+ with another ion to be able to move enough ions to get a concetration gradient Answer is NOT C because F1Fo ATPase is in the mitocindria membrane

Question 32

Is V1Vo eltrogenic or electronuetral

Answer 32

V1vo = electrogenic (because only moves H+ = build positive charge) Need to something thing to collaberate with the V1Vo ATPase to get electronutral tranport to get a concentration gradeint (Ex. Need the Cl- and the V1V0 pump to get a concetraion grdaeint – need to work together) - Need counter ion to see a concetration gradeint NOTE - V-Type and F-Type that mostly pump H+

Question 33

Function of P-Type transporters

Answer 33

P-Type pumps transporter different types of ions ((Na, K, Cu, and H) Main P-Type pump = Na/K ATPase pump (Na out and K in) - Generate the Na and K gradients that are needed for ion chanels to have action potential AND for chemiomotoc circuts AND maintains Na and K homeostasis

Question 34

Types of P-Type Pumps

Answer 34

1. Na/K ATPase 2. H/K-ATPAse (acidifies the gastric lumen) 3. Ca/H-ATPase - Function - keep cytoplasmic Ca very low + Set up the Ca gradeint for signaling + restores Ca store in ER + supplies golgi with Ca - Found at ER + plasma membrane + Golgi - Ca pump functions opposite the Ca channel (Ca/H-ATPase pump moves ions out of cytoplasm ; channel brings Ca into the cytoplasm when signlaing) 4. Cu/? ATPAse --> pumps Cu into compartments where copper can be loaded onto enzymes AND removes excess Copper from the body

Question 35

Ca gradient used for signaling

Answer 35

When Ca channels open Ca goes into the cytoplasm -> Increases Ca concetration --> Ca binds to effects that cause downstream effectos Example downstream effects = contraction of muscle or chnages in gene expression or causing cell division

Question 36

Reaction of SR Ca-ATPase (Types of P-Type pump)

Answer 36

Overall - Ca from the cyoplasm into the lumen ; H+ goes from the lumen into the cytoplasm 1. 2Ca form cytoplasm binds to E1 (Ca ATPase pump) in a confirmation that has high Ca binding affinity 2. ATP binds to the E1/Ca complex --> forms E1-Ca-ATP complex 3. ATP is hydrolyzed by E1 pump BUT the phosphate doesn't leave 4. Phosphate is transfered to the E1 protein (Now have E1-P-Ca-ADP ; Aspartatle phosphate reaction covalent intermediate) 5. ADP is released off of Ca --> forms the high energy phosphoenzyme intermediate (E1-P-Ca) 6. Ca site is ripped open and changes so now it faces the opposte side from where it was orignally facing 7. In the low energy confimration (E2) the pump has a milimolar affinity --> Ca will leave AND H+ counter ion binds (have E2-P-H2) 8. Phosphate bond is hydrolyzed and Pi leaves and H+ goes to the cytoplasm (Now have E2H2) --> E2 goes back to E1 --> restart cycle NOTE - pump is electigenic even thought has H+ counter ion

Question 37

E1 Vs, E1 in P-type pumps

Answer 37

E1-P = high energy phospahte (has submicro molar affinity for molecule) Vs. E2-P = lower energy phospahte (has milimolar affinity for molecule) During confirmation change from E1 high energy to E2 low energy --> the Ca binding site is ripped open AND it is moved to face the oppeote site - Ex Ca binding site started facing the cytoplasm and once have the confirmation change the Ca binding site faces the Lumen

Question 38

Key thing that happens in P-type pumps mechanism (ex. Ca-ATPase)

Answer 38

KEY thing that happens --> Keq for hydrolysis of ATP and phosphoenzyme intermediate (E1-P-Ca) is close to 1 - Keq is close to 1 because the energy is not released as free energy INTEAD the energy is captired by the protein (E1) to change the bind affinity for Ca from Sub micromolar (E1) to millimolar (E2) - Diference in affinity for Ca between E1 and E2 is 10^3 THIS IS HOW A P-Type PUMP WORKS

Question 39

Overall how do P-Type pumps work

Answer 39

Overal - Energy from ATP hydrlysis is used to convert ion and solute binding sites from high affinity to low affininty Binds a substarte on one of site of the memebrabe with higher energy --> pump has a confoirmation change --> confimation chnage causes the ion to face the opposite side of the memnrane --> something happens to the binding site (rippped apart) --> affinity of the pump for the ion decreases --> ion comes off

Question 40

What do P-Type pumps look slike

Answer 40

All P-Type pumps look alike (made of 1 polypeptide chain) P-Type pumps can have a second subunit that helps in assembly

Question 41

ATP hydrolysis Vs. Ion binding in P-Type pump

Answer 41

Ion binds in the middle of the membrane Vs. ATP binds and is hydrolyzed on the cytoplasmic side --> ATP hydrolysis is far from where the ion is binding MEANs there has to be energy coupling from ATP hydrlysis part in cytoplasm to membrane part of pump where ion binds in order to change binding affinity - Energy coupling happens by big confirmational changes --> chnages of the pump's binding pocket affinity for ions

Question 42

Diseases releated to P-ATPases

Answer 42

1. Menkes and Wilson disease: Cu2+-ATPases - Menkes - Cu can’t be absorbed Vs. Wilson – Cu can’t be removed (toxic) 2. Brody and Darier disease: ER Ca2+-ATPases - Issue with muscel relaxation AND causes blistering 3. Hailey Hailey disease: Golgi Ca2+-ATPases - Issues with Adhesion molecules that for adherens and desmomes = get blisters on skin

Question 43

Clincal relevnace of P-ATPAses

Answer 43

1. ouabain/digitalis (cardiac glycosides) treatment of heart failure by poisoing cardiac Na+/K+-ATPase - Na/Ca exchangers normally remove Ca using the Na gradient --> when poison the Na/K ATPase pump the Na gradient is off = Ca goes in opposite directions = heart has enough Ca to contract 2. omeprazole (anti-ulcer) drug that inhibits the gastric H+ pump (treat excessive acidity)

Question 44

ABC transporters (PUMPS)

Answer 44

Different because of the substrates that they move (transports proteins + ions + drugs + sugars) Found in all cells - Bacteria have ABC importants and Exporers ; eukryotes only have exporters

Question 45

Structure of ABC Pumps

Answer 45

ALL have a similar structure but have diffrent number of SU that can come to together in different ways to make a proteins with similar structures Overall Structure includes: 1. ATP binding domains (can be 1 or two proteins) 2. Periplasmic bindning domain (binds substrate and brings it to the pump) 4. Transoort pathway (each protein has 6 transmembrane helicies) Tranpsort pathway and ATP binding domains are part of SU code

Question 46

Types of Structure of ABC Pumps

Answer 46

1. Each Subunit is coded by a different gene (each a different protein) --> 2 separate proteins in ATP binding domain + periplasmic binding protein + 2 proteins in transprt pathway 2. Two ATP binding domains are fused (coded for by 1 gene) 3. Transport pathway protein = 1 protein with 12 transmembrane domains 4, MHC molecules (TAP1/TAP2 transporters) - TAp1 and TAP2 each have ATP binding domain and transport pathway (membrane helicies) are fused 6. Example 5/6 - MDR and CFRT --> ALL of the proteins are fused

Question 47

How ABC ATPase transporters work

Answer 47

Example - bacteria cell is taking up B12 Outer membrane has a beta barrel protein that allows B12 to go down its gradient --> after B12 is captured by the periplasmic binding protein on the ABC pump (brings B12 to the ABC pump) --> ABC membrane helicies and ATP binding domains allow B12 to go from the periplasm into the cytoplasm

Question 48

Unconventional ABC ATPase Pump

Answer 48

1. CFTR --> looks like a pump BUT functions like a Cl- chanel - Most pumps ATP hydrolysis is coupled to ion movement (Ex. 1 ATP hydrolyzied : 2 Ions are moved) - in CFTR - ATP hydrolysis is NOT coupled to moving Cl- INSTEAD ATP hydrolysis is linked to gating of the chanel (ATP is hydrolyzed --> chanel opens --> Cl- ions go down hill) - Here ATP hydrolysis is not stoichiometrically coupled to ion movement 2. MDR2 (flippase) --> moves lipids between bilayers (makes the composition of the leaflets different) 3. MDR1 - non-selective --> transports many substrates (Most pumps are very specific)

Question 49

2nd function of ATP transporters

Answer 49

Many ABC pumps (CFTR, MDR, SUR) are NOT transporters but instead regulate ion channels: Regulates - channel activity + sensitivity to drugs

Question 50

ABC transporters involved in disease

Answer 50

1. Overexpression and selection of P-glycoprotein (MDR1) in cancer cells confers resistance to chemotherapy (resistent to drug pateint was on AND other drugs) - Resistnet cells take over tumor - MDR1 will pump drugs out of cell 2. Mutations in CFTR cause cystic fibrosis 3. Sulfonylurea receptor is mutated in patients with persistent hypoglycemia of infancy and is used to treat diabetes 4. Mutations in peroxisomal transporters ALDP and PMP70 --> can't pump long chain fatty acids --> accumulate very long chain fatty acids

Question 51

Pumps Summary

Answer 51

Gradeints can be used to do work (Gradients made by pumps can be used by carriers or ion chanels) Interactions occur far apart (Example – F1 and F0 --> confimration chnages occur over long ranges because F1 is far from the F0 C ring) - Energy transduction between teh ATPase site and the membranes can take place over long distances Energy from ATP hydrlysis is used to convert ion and solute binding sites from high affinity to low affininty

Question 52

Occurrence of transporter families

Answer 52

In genomes of Prokaryotes and Eukryotes secondary transporters (sympoter and antiporter) dominate membrane function - Few genes code for pumps (few types of pumps) - Few genes that code for channels (most channels are voltage gated) - Half of the genes that code for trasnporters code for carriers (Uniporter or antiporter sympoter) WHY so many genes for carriers - because eveyrthing that is moved using a carrier needs a different carrier that is designed to bind it (have many carriers that bind many things ; need different carrier for each thing)

Question 53

Families of transporters

Answer 53

Made faimlies of transporters based on genomic sequence similarities --> showed there are many types of uniporters + symporters + antiporters Have carriers that are specilized to transport anything (Ex. surgars or nitrate or phosphate or drugs) - Eplains why have many genes for carriers --> diffreent gene for each carrier and need a different carier for each thing being transporterd

Question 54

Three types of carriers

Answer 54

1. Uniporter (Ex. GLUT transporters) - Facilitated Diffusion - Substartes move down concetration gradient by itself 2. Antiporter - Substrates EXCHANGE with one another 3. Symporter (co-transport) - Example – Na/Glucose symporter --> use Na gradient to bring glucose into cell (if need get ALL glucose into cell)

Question 55

Complexity of carriers

Answer 55

Stoichometry and the carriers can be complex Carrier transport can be electrogenic (Example – Na/Ca Antiporter (removes the bulk of Ca from the cytoplasm along with a slow pump) - 3Na in : 1 Ca out--> +3 : +2 = electrogenic

Question 56

Example carriers

Answer 56

Carriers includes Symporters + Antiporters GLUT family --> used for sugars Na/H antiporters - found in endosomes + in plasma membrane + in vesciles - In tissues Na/H exchnagers are used for movement of salt and water Band3 – bring CO2 into the lung Na/Ca (antiporter) - used in heart + nerves GAT – moves Nuerotransmitters in the brain

Question 57

Kinetics of RBC Glucose transport - D-Glucose Vs. L-Glucose

Answer 57

Experiment --> put RBCs in solution and Add Glucose - D-Glucose (biologically active form) goes into the cell quickly --> then Reach Equilibrium (Rate of glucose in = rate of glucose out) - L-Glucose (not bilogically active form) goes into cell much slower (does not reach equiliboum quickly) + has a much lower concetration Overall – means that something in the membrane facilitates the movement if D glucose NOT L-glucose

Question 58

Kinetics of RBC Glucose transport - Change concetration of Glucose

Answer 58

Changing the concetration of glucose and look at the change in rate of glucose transport X-Axis = glucose concetration ; Y-Axis = Rate of glucose transport - Increase the concetration of glucose = the rate of D-glucose transporter increased BUT then the rate platuers - The plateu in D-glucose line is paraell to L-Glucose rate (passive movement of glucose/non-specifc leak movement) - D-glucose - L-glucose = Faclitated transport (uses Uniporter) ; L-Glucose = passive movement ('leak') ; D+L glucose = total movement

Question 59

Kinetics of RBC Glucose transport - D-glucose - L-Glucose

Answer 59

D-glucose – L-glucose gives the rate difference Chart - Rate diference looks like the same as it would be for an enzyme --> MEANs whatever is tranporting glucose behaves like an enzyme - When increase the concentration of glucose the rate of transprt (rate difference) increases BUT eventually the Rate difference platues Vmax (occurs when the binding site that becomes saturated when substate concetration is too high) - MEANS there is finite niding sites - Half of the Vmax = Km - This properties of carriers makes carriers different from chanels (don't have finite binding sites)

Question 60

What did RBC experiment show

Answer 60

Shows that in membranes there is a mechanism for transporting solutes and that is specific for biologically active molecules (moves D-glucose not L-Close) AND the thing transporting the solutes has a saturatable rate (reaches Vmax) - Means that the things moving molecules (carriers) = behave like enzymes

Question 61

How do you show that Glucose accumulation Requires Na

Answer 61

Exeriment - looked at glucose going in vs. Glucose going out with and without Na in Intestinal cells Results: Have Na in media --> cells accumulate Glucose (more glucose going in than out) No Na – the glucose goes in slowly AND plateues at a low level (equal amount of glucose in and out) SHOWs Na drives the accumulation of glucose because glucose only goes into cell when have Na THIS shows active transport (symporter ; need Na gradient to drive uptake of glucose)) Vs. RBC exoeirment was a uniporter (passive transport)

Question 62

How do you show something is a symporter (NOT antiporter)

Answer 62

Experiment – looking at the movement of H+ in E.coli using an electrode (measures pH of the media) Results - Add lactose (+substare) - the pH increases --> because the H+ are being removed from the media and going into the cell along with lactose - When add detergent – don't see a change in pH - Shows this is a sympoter (lactose and H+ go in the same direction) + shows that ions can move along with a substrate

Question 63

How can you accumulate something in a cell

Answer 63

If the substrate acucmuluates in the cell (have a ton more of something inside the cell compared to the outside) then it requires active trasnpprt (need energy from an ion grdainet for it to happen)

Question 64

How do uniporters work - carrier hypothesis

Answer 64

1. Carrier faces one direction (Ex. Carrier faces out) and binds to substrate (Sout + Cout --> CS out) 2. When substate binds carrier has a confirmation change that causes the solute to face the opposite side (faces out --> In) - Solute came from outside --> NOW the solute faces the inside - CS out --> CS in 3. Release the Solute (CSin --> Cin + Sin) 4. Carrier has a confirmation chnage that causes the carrier to face the original direction - Have 2nd confirmation when substarte leaves

Question 65

How do Antiporters work

Answer 65

Carrier starts facing in one direction (C1) --> C1 binds to the substrate (CS1) --> carrier changes confirmation to face the other side --> carrier lets go of the substrate --> carrier binds to another substrate (S2) --> carrier can go back to facing the original side --> carrier can release S2 - After carrier releases S1 it can’t go back to orginal confmration to face original side --> carrier needs to bind to nother substarte (S2) to be able to go back (how it can do exchange)

Question 66

How does a symporter work

Answer 66

Carrier starts facing in one direction (C1) --> C1 binds to the substrate (CS1) --> THEN binds to ANOTHER substrate (S’) (NOW have CSS1’) --> carrier bound to both substrates has a confirmation change to face the other side (CSS’2) --> Carrier realese one substrate (Now have CS2) --> Carrier can release the second Substrate (Now gave C2) --> empty carrier can go back to orginal confirmation to face the original side - When bound to one substarte it can’t have a confirmation chnage to face the other side (needs to bind to both substrates) ; needs to release both substrates before can go back to original confirmation

Question 67

Importance of Uniporters

Answer 67

Example - Glucose transporters (bring glucose in/out of cell) Isoforms: 1. Glut1,2,4 --> uniporters - GLUT 1 - Glucose uptake in brain - Mutate GLUT2 - glucose released from glycogen can't leave (glycogen storage disorders) 2. SGLT1 (symporter ; active transport ; coupled to Na) - Used for gut to bring sugar into cell (need to use active mechansim to get ALL glucose) 3. SGLT2 (Symporter l coupled to Na) - Active sugar absorption in from kidney filtrate

Question 68

Topology of carrier

Answer 68

More simple structure compared to pumps (only 1 protein) Often has 12 transmembrane domain Uses the Positive inside rule --> there are more positivley charged amino acids on the cytoplasmic side compared to the peirplasmic side (orientated because of negtaive charge inside of cell) There are few charged amino acids in the trasnmembrane helix - Charge AA in membrane = involoved in binding an ion or binding a solute (ion paired) or important for transport

Question 69

Two halves of the carrier

Answer 69

N and C terminus halves are homologous First 6 transmembrane helicies are duplicate in the second half of the carrier (the two halves are orineted in opposite orinetations) The transmenranes regions are mirror images (a,b,c,d,e,f VS a’,b’,c’,d’,e’,f) --> carrier has Pseudo two fold sympetry In Center have a pathway where transport happens

Question 70

LacY Crystal structures

Answer 70

Shows transporters don’t provide access to both sides of the membrane at once Structure – 2 halves form a V shape (closed at one end) When transporter functions - it goes from V shaped (One side closed ; on side open) --> closed on both sides (Occluded) -> Second side open (NOW the side that was closed before is open and the side that was open is closed ; A shape)

Question 71

Two mechanism of alternating accesisbility in carrier

Answer 71

1. Rocker switch (Major Facilitator Superfamily proteins) - Example - GLUT isoforms 2. Elavator model (Sodium solute symporter proteins) - Example - SGLT1

Question 72

Rocker switch model

Answer 72

Model – two bundles of helices that rock against each other (like LacY) Image - Transporters have been crystilized in different parts of the transport process Steps - Outward open --> substrae bound (outward facing + partially occulded) --> occluded (both sides closed) --> inwrad occuluded --> ligand bound inwards open --> inwards open (ligand can come off)

Question 73

Elevator model

Answer 73

Image – Copper and purple attached to each other to form the core --> Purple and copper slide past each other Ligand binds at top of the pruple when puple is higher (purple is outward facing) --> when the purple slides the ligand bound is closer to the other side (purple is inward facing)--> ligand is relased - When purple slides down the ligand is lowered and released to the inside Substate is ‘going for a ride on the elevator’

Question 74

What sustains a Chemiosimotic circuit

Answer 74

Chemiosimotic circuts are sustained by a few ions pumps + MANY carriers (one for each substane that needs to be transported) + channels - Pump makes gradient --> gradient is used to move a substances across membrane

Question 75

Example transport scheme - epthelial cell

Answer 75

Basal side as Na/K pumps --> generates the Na and K gradeints On apical side - - Na goes back down the gradeint into the cell and brings Glucose in using SGLT symporter - Fructose goes into the apcal suface passivley through GLUT5 Once inside the cell – glucose flow down its gradient through the transporters on the basal surfce --> glcuose goes into the blood

Question 76

How is fluid (salt + water) trasnportered across an epithelum - What is add apical and basal membranes

Answer 76

Apical membrane: CTFR + Aquiporine (water chanel) Basal surface - NA/K ATPase --> makes the Na grdaeint on both sides of the membrane - Na,K/2Cl cotrasnporter --> Uses the Na gradeint to bring Cl in - K chanel

Question 77

How is fluid (salt + water) trasnportered across an epithelum - CFTR

Answer 77

CTFR - Allows Cl to leave cell --> when Cl leaves Have Na and Cl outside of cell = salt --> where salt moves water foolows = water will leave cell through aquproins --> movement of salt and water keep epithelium in airway moist = allows villi to beat = villi can move microbes out Mutate CFTR = Cl can’t leave = Cl builds up and can’t go to the lumen = area gets clogged = microbes get stuck and mucus builds up - Mutation in CFTR = protein folding mutations --> misfolded protein stays in ER - NOW have drigs that act as chaparones to helps CFTR fold = CFTR goes to the epithelium in the airway