what do carrier proteins do?
how do they work?
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
what are the different classes of carriers?
1) pumps: have intrinsic ATPase activity
2) transporters: do not hydrolyze ATP; instead, exploit energy store in ion gradients, particularly the Na+ gradient
what kind of transport do pumps do?
primary active transport
create initial gradient, by doing ATP hydrolysis - allows pumps to create energy to move solute against gradient
what is primary active transport vs. secondary active transport?
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
what are the different types of pumps?
1) P-class Pumps
2) V/F Class Pumps
3) ABC-Class Pumps
what gets pumped through P-class pumps?
ONLY ions! nothing else
what kind of pump is the Na+/K+ ATPase pump?
what is unique about it?
becomes phosphorylated in course of a cycle
ubiquitous cell membrane protein that consumes up to 40% of cell's ATP
what is the basic schema for an Na+/K+ ATPase pump?
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
describe how the Na+/K+ ATPase pump works
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
what is the energy produced by the Na+/K+ ATPase used for?
what does the Na+/K+ ATPase do to the cell?
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
what does it mean that the Na+/K+ ATPase is electrogenic?
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)
what does SERCA stand for?
what type of pump is it?
what is its basic schema?
sarcoplasmic/endoplasmic reticulum Ca2+ ATPase pump
removes Ca2+ from cytoplasm by sequestering it w/in intracellular storage organelles
describe how the SERCA pump works
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
what is cytoplasmic concentration of Ca2+ in most cells?
what maintains this?
below 1 uM
maintained by SERCA action
what do V/F class pumps pump?
what is the main effect of V-class and F-class pumps' action?
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
what do ABC-class pumps do, broadly?
what do they transport?
what are 2 examples of ABC class pumps?
bidng ATP through ATP-binding cassettes, conserved regions
often transport uncharged, even hydrophobic molecules
eg: multi-drug resistance proteins, cystic fibrosis transmembrane regulator
what kind of pumps are multi-drug resistance proteins?
where are they expressed?
what do they transport?
when are they overexpressed?
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
what kind of pump is the cystic fibrosis transmmebrane regulator (CFTR)?
where is it expressed?
how does it work?
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
how do transporters get their energy to work?
rely on existing gradients to move solutes - do not have ATPase activity, like pumps!
what are uniporters?
what are some examples?
transporters that move a single species of molecule down its gradient
facilitate a thermodynamically favored process
work by facilitated diffusion
what are co-transporters?
what are the different kinds, how do they work?
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)
what is the sodium-glucose linked transported (SGLT) receptor an example of?
where is it located?
how does it work?
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
where are SGLTs re: the renal epithelia?
what is movement of glucose through SGLT --> renal epithelia --> beyond?
apical membrane only
reabsorbed glucose diffuses through the cell, to basolateral membrane, where it's removed to interstitial space by GLUT uniporters
what kind of transporter is the GLUT?
how does it work, and what exposes a binding site?
which binding site is usually exposed, why?
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
what kind of action might GLUTs exhibit, which SGLTs also exhibit?
what does it require?
transcellular transport: movement of, here, glucose, from intracellular to extracellular (or vice versa) space, via the transporter
requires that the cell is polarized
how does the Na+/Ca2+ exchanger (NCX) work?
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
how does digoxin work?
what is it used to treat?
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
why do so many co-transporters us the Na+ gradient as their energy source?
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