LEC15: Carrier Proteins

Question 1

what do carrier proteins do?

how do they work?

Answer 1

operate in cycles, ferry substrate across the membrane

move solutes against their electrochemical gradient - which no channel can do

requries source of energy, usu via hydrolysis of ATP to ADP

Question 2

what are the different classes of carriers?

Answer 2

1) pumps: have intrinsic ATPase activity
2) transporters: do not hydrolyze ATP; instead, exploit energy store in ion gradients, particularly the Na⁺ gradient

Question 3

what kind of transport do pumps do?

Answer 3

primary active transport

create initial gradient, by doing ATP hydrolysis - allows pumps to create energy to **move solute against gradient **

Question 4

what is primary active transport vs. secondary active transport?

Answer 4

both are movements done by pumps to move a solute against the chemical gradient

primary: if solute has a binding site on a pump, which directly carries it through the membrane

secondary: pump doesn’t participate directly in moving solute; instead est. a large gradient for another solute (often Na⁺) across membrane, and use this gradient to move solute

Question 5

what are the different types of pumps?

Answer 5

1) P-class Pumps
2) V/F Class Pumps
3) ABC-Class Pumps

Question 6

what gets pumped through P-class pumps?

Answer 6

ONLY ions! nothing else

Question 7

what kind of pump is the Na+/K+ ATPase pump?

what is unique about it?

Answer 7

P-class pump

becomes phosphorylated in course of a cycle

ubiquitous cell membrane protein that consumes up to 40% of cell’s ATP

Question 8

what is the basic schema for an Na+/K+ ATPase pump?

Answer 8

removes 3 Na+ ions from cell, brings in 2 K+ ions, at expense of 1 molecule of ATP

works by presenting Na+ and K+ binding sites w/ different affinities at cytoplasmic and extracellular faces of membrane

Question 9

describe how the Na+/K+ ATPase pump works

Answer 9

1) initial pump conformation, E1, 3 Na+ ions occupy high-affinity sites on cytoplasm side; 2 low affinity K+ sites are unoccupied
2) ATP binds pump, is hydrolyzed by pump’s ATPase activity

a high-energy phosphate bond is formed w/ an aspartate residue on cytoplasmic side of pump by the ATPase

3) phosphate binding energy causes protein’s conformation to change to E2. = a power stroke.
4) Na+ ions move to low-affinity sites in extracellular space; 2 high affinity K+ binding sites exposed on extracellular side
5) 3 Na+ ions diffuse away from their low-affinity sites, into extracellular space. 2 K+ ions from outside bind their high-affinity sites.

Hydrolysis of aspartyl-phosphate bond: P is dropped from E2

6) Phosphate loss returns pump to E1 state, transfers K+ ions to their low-affinity binding sites facing inside of cell, they dissociate into cytoplasm

Question 10

what is the energy produced by the Na+/K+ ATPase used for?

what does the Na+/K+ ATPase do to the cell?

Answer 10

1) steep gradients for Na+ and K+ across membrane can be exploited to do work for cell
2) intracellular concentration of Na+ in most cells is kept low; intracellular K+ kept high, fxn of this pump

Question 11

what does it mean that the Na+/K+ ATPase is electrogenic?

Answer 11

it expels 3 Na+ for every 2 K+ that enter the cell

this electrical current produces small voltage across membrane

makes inside of membrane negative to outside (by a few mV)

Question 12

what does SERCA stand for?

what type of pump is it?

what is its basic schema?

Answer 12

sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump

P-class pump

removes Ca²+ from cytoplasm by sequestering it w/in intracellular storage organelles

Question 13

describe how the SERCA pump works

Answer 13

1) in E1 conformation, has high affinity binding site for Ca²⁺ in cytoplasmic side
2) when binds Ca²⁺, ATP is hydrolyzed, & SERCA **phosphorylates **via high energy bond w/ an aspartate on cytoplasmic side
3) Per phosphorylation, SERCA undergoes conformational change to E2. Closes off Ca²⁺ pocket from cytoplasmic side, traps Ca²⁺ in the protein.
4) In E2, exposes a **low affinity Ca site **to the sarcoplasmic reticulum
5) Ca²⁺ diffuses from this site, accumulates inside of sarcoplasmic reticulum
6) SERCA returns to E1 conformation

Question 14

what is cytoplasmic concentration of Ca²⁺ in most cells?

what maintains this?

Answer 14

below 1 uM

maintained by SERCA action

Question 15

what do V/F class pumps pump?

what is the main effect of V-class and F-class pumps’ action?

Answer 15

ONLY PROTONS!

establish proton gradients, or work in reverse of gradients, and generate ATP by moving protons across membrane in reverse

V-class pumps: acidification of organelles (i.e. lysosomes) by pumping protons from cytoplasm to lumen of organelle, use ATP

F-class pumps: highly expressed in mitochondria; move protons down their gradient; make ATP from ADP + Pi

Question 16

what do ABC-class pumps do, broadly?

what do they transport?

what are 2 examples of ABC class pumps?

Answer 16

bidng ATP through ATP-binding cassettes, conserved regions

often transport **uncharged, even hydrophobic **molecules

eg: multi-drug resistance proteins, cystic fibrosis transmembrane regulator

Question 17

what kind of pumps are multi-drug resistance proteins?

where are they expressed?

what do they transport?

when are they overexpressed?

Answer 17

ABC-class pumps

MDR proteins - highly expressed in epithelial cells

transport small, polar molecules, including metabolism products

can pump wide variety of drugs out of cell

**tumors **overexpress MDR proteins & are resistant to ttmnt by multiple & unreleated cancer drugs

Question 18

what kind of pump is the cystic fibrosis transmmebrane regulator (CFTR)?

where is it expressed?

how does it work?

Answer 18

expressed in lung & other organs

ABC-class pump, but has no “pumping fxn”- just has an ABC-binding cassette

has a Cl^- channel, regulated by PKA

if lose fxn of CFTR, reduce Cl^- transport across pulmonary epithelial cells, result in mucus secretion by epithelial cells to be very viscous, compromises gas exchange & predisposes to lung infection

Question 19

how do transporters get their energy to work?

Answer 19

rely on existing gradients to move solutes - do not have ATPase activity, like pumps!

Question 20

what are uniporters?

what are some examples?

Answer 20

transporters that move a single species of molecule down its gradient

facilitate a thermodynamically favored process

work by** facilitated diffusion**

eg: GLUT1

Question 21

what are co-transporters?

what are the different kinds, how do they work?

Answer 21

transporters that couple thermodynamically favorable mvmnt of 1 type of molecule (down its gradient) to **unfavorable mvmnt of another **(secondary active trasnport)

usually use potential energy of Na+ gradient

have symporters (move diff solutes in same direction across membrane) and **antiporters/exchangers **(move diff solutes in opposite directions)

Question 22

what is the sodium-glucose linked transported (SGLT) receptor an example of?

where is it located?

how does it work?

Answer 22

**symporter **in kidney tubule

in early part of tubule near glomerulus, [glucose] in urine is high, so SGLT-2 transporter doesn’t need to work against large concentration gradient to take up glucose into cell

**SGLT-2 takes 1Na+:1 glucose **

however, further down in tubule, most of glucose gone from urine; reabsorption becomes challenge b/c glucose gradient resists movements from lumen into cell

SGLT-1 symporter now expressed, b/c SGLT-1 is 2Na+:1 glucose

SGLT-1 can now work against glucose gradient

result: no glucose excreted in urine

Question 23

where are SGLTs re: the renal epithelia?

what is movement of glucose through SGLT –> renal epithelia –> beyond?

Answer 23

**apical **membrane only

reabsorbed glucose diffuses through the cell, to basolateral membrane, where it’s removed to interstitial space by GLUT uniporters

Question 24

what kind of transporter is the GLUT?

how does it work, and what exposes a binding site?

which binding site is usually exposed, why?

Answer 24

glucose uniporters: bidng inslge molecule of glucose at a time

conformational change occurs, exposing glucose-binding site alternatively to extra- and intracellular sides

rate of cycling is accelerated by occupation of binding site in either conformation

b/c [glucose] is higher outside than inside the cell, extracellular binding site is more likely to become occupied by glucose

this causes conformational change; glucose exposed to low concentration inside cell, & it diffuses away from binding site

GLUT spontaneously returns to orig. conformation; another molecule of glucose binds from outside

Question 25

what kind of action might GLUTs exhibit, which SGLTs also exhibit?

what does it require?

Answer 25

transcellular transport: movement of, here, glucose, from intracellular to extracellular (or vice versa) space, via the transporter

requires that the cell is polarized

Question 26

how does the **Na⁺/Ca²⁺ exchanger (NCX) **work?

Answer 26

antiporter, 3 Na⁺ in, 1 Ca²⁺ out per cycle

accumulates lots of Ca outside the cell

makes the cell a bit more positive w/ each cycle, so it’s an **electrogenic **event

Question 27

how does digoxin work?

what is it used to treat?

Answer 27

drug that inhibits Na⁺/K⁺ APase

this depletes the Na⁺ gradient; reduces activity of Na+/Ca2+ exchanger

so **interferes w/ secondary active transport of Ca²⁺ out of the cell **

consequently accumulates intracellular Ca²+ level

this enhances muscle contractility

so used for congestive heart failure, when heart pumping activity is reduced

Question 28

why do so many co-transporters us the Na⁺ gradient as their energy source?

Answer 28

at resting membrane potential, the driving force for Na+ (= 195 mV) is much greater than that of K⁺ ( = 137 mV)

thus much more energy provided by allowing 1 Na⁺ ion to enter cell than by allowing 1 K⁺ to leave

Ca²⁺ is not helpful b/c it’s maintained at very low concentration inside cell, and is an important second messenger; would also activate many enzymes w/in cell if used to drive transporters