LEC 49 Transporters & Pumps Flashcards
What are transporters?
catalysts that work in repeated cycles with the protein alternating between inward-facing and outward facing conformations
Slide 4
Transporters function very similarly with respect to kinetics to what other class of molecules?
Enzymes
Slide 5
Direction of net transport through a facilitated diffusion transporter is always in what direction?
Down the gradient
High to low concentration
Slide 7
In facilitated diffusion, is ATP required?
No
Slide 3
Which glucose transporters are insulin-dependent?
GLUT4
Slide 8
What are the insulin-independent glucose transporters?
GLUT1, GLUT2 (bidirectional), GLUT3, & GLUT5 (fructose)
BRICKLIPS
Brain, RBCs, Intestine, Cornea, Kidney, Liver, Islet, Placenta, Sperms
Slide 8
How does GLUT1 also function bidirectionally?
think blood-brain barrier
GLUT1 is on both sides of the endothelial cell. On the blood side, transport is inward; on the brain side, transport is outward. In both cases transport is from higher concentration to lower concentration
Slide 8
What are some of the main points regarding facilitated diffusion?
- Requires a membrane protein that can be in either inward-facing or outward-facing conformation
- One substrate at a time; no ATP hydrolysis
- Substrate saturation
- Competition between substrates
- Direction of transport can be either inward or outward, depending on the substrate gradient; transport is always downhill, from high to low concentration.
- Both empty and substrate-bound forms of the transporter
can reorient between inward- and outward- facing states
Slide 9
What is primary active transport?
Energy of ATP hydrolysis drives uphill transport of one or more substrates, including cations, drugs, lipids (flippase), but not anions
Slide 11
What is the most important example of primary active transport?
Na, K - ATPase
Sodium/Potassium Pump
Slide 11
How does the sodium-potassium pump work?
What does it pump and where? What’s required?
Pumps 3 Na+ out and 2 K+ in hydrolyzes ATP in the process
Slide 12
Why is the ATP required in the Na/K ATPase?
the energy of the ATP is required to overcome the energetically unfavorable conformational changes in order to bind/release the sodium and potassium
Slide 12
Explain the phosphorylation/dephosphorylation of the sodium-potassium pump.
2 K+ go into the cell (pump dephosphorylated)
3 Na+ go out of the cell (pump phosphorylated)
Slide 13
Explain the Na/K-ATPase catalytic cycle?
- 3 cytosolic Na+ bind to E1 conformation (high Na affinity)
- E1 phosphorylated by ATP forming E1-P
- Conformational change to E2-P which releases the Na+ (low Na affinity) and binds 2 extracellular K+ (high K affinity)
- E2-P dephosphorylated and conformational change to E2 which releases to K into the cytosol and returns it to E1
Slide 15
How do ATP-Binding Cassettes work?
- two membrane domains with 6 TM segments each and two nucleotide binding domains
- Drug, from either cytosol or inner half of lipid bilayer, binds to pocket between the two membrane domains
- ATP binds to Nucleotide Binding Domains and induces conformational change that expels drug into extracellular medium
Slide 17
Whats another name for a coupled cotransporter?
Symporter
Symporter = SAME DIRECTION
Slide 18
What is an example of a coupled cotransporter?
2 Na-Glucose (SGLT)
Slide 19
How does SGLT (sodium-glucose cotransporter) work?
- In glucose-absorbing epithelial cells, the inward Na+ gradient across the luminal (apical) membrane drives downhill Na+ influx via SGLT.
- SGLT couples Na+ influx to glucose influx, and the Na+ gradient drives glucose influx against a concentration gradient, with no ATP hydrolysis.
Slide 19
Which cotransporter is a target of Furosemide (Lasix)?
Na-K-2Cl Cotransporter
Slide 21
How does the Na-K-2Cl cotransporter work?
- Couples the transport of 1 Na+, 1 K+, and 2 Cl- ions in the same direction
- No ATP hydrolysis, but regulation by phosphorylation
- Driving force is normally inward because the inward Na+ and Cl- gradients overcome the outward K+ gradient
Slide 21
How does Na-K-2Cl- Cotransporter couple the ion fluxes?
By constraining the translocation event to all sites filled or all sites empty (same idea as 2 Na-Glucose cotransporter, but now there are 4 substrates)
Slide 21
What is another name for coupled exchangers?
Antiporters
Slide 22
What are coupled exchangers?
Substrates are required to move in opposite directions
Slide 22
What makes antiporters different from symporters in terms of their conformational changes (translocation event)?
translocation event requires bound substrate; empty transporter cannot re-orient between inward-facing and outward-facing
Slide 22
How does the Na-H exchanger work?
Inward Na+ gradient drives efflux of H+; this is a mechanism for extruding acid from cells
Slide 23
How does the 3Na-Ca exchanger work?
Inward Na+ gradient drives Ca2+ efflux. Inward Na+ gradient is only ~ 10x, but the 3:1 exchange (with one net positive charge going inward) makes the energetics favorable for Ca2+ efflux, even against a large gradient
Slide 23
How does Cl-HCO3 exchange work as a part of CO2 transport in tissue capillaries?
Cl- influx, HCO3- efflux across basolateral membrane of acid-secreting epithelia and in red blood cells
Slide 23
What are the functions of ion gradient?
- Resting membrane potential, action potential.
- Ca2+ signaling in muscle, nerve, secretion.
- Coupled transport of nutrients (glucose, amino acids).
- Reuptake of neurotransmitters (targets of many drugs).
- Transepithelial absorption, secretion.
- Cell volume regulation.
- Cytosolic pH regulation (Na+-H+ exchange).
Slide 25
What is the only transporter in humans that mediates Na efflux?
Na/K ATPase
Slide 27
What drug inhibits the Na/K ATPase and is related to the stimulation of cardiac contractility?
Digoxin
Slide 28
Where in the cell is Ca2+ stored?
ER or SR
Slide 29
Why is cytosolic concentration of Ca2+ kept at low levels?
This allows small increases in free [Ca2+]to act as a signal for muscle contraction, secretion, and other functions
Slide 29
What transporter pumps Ca2+ into the SR/ER?
SR/ER Ca2+ - ATPase
Slide 29
How is Ca2+ pumped out of the cell?
- a related P-type ATPase pumps Ca2+ across plasma membrane
- In heart and some neurons, 3Na-Ca exchanger pumps Ca out of the cell
Slide 29
How does calcium enter the cell?
Calcium ion channels
Slide 29
How does digoxin elevate cardiac cytosolic Ca2+?
- Digoxin level sufficient to block about 10% of Na pumps
- Intracellular Na increases slightly
- Increased [Na] causes decreased driving force for Na-Ca exchange
- less Ca2+ extrusion by exchanger
- Increased cytosolic Ca2+ and increased force of contraction
Slide 30
What are the clinical uses of Digoxin?
- Heart failure (+contractility)
- Atrial fibrilation (-conduction @ AV node & depression of SA node
Slide 31
What are the adverse effects of Digoxin?
- Cholinergic effects (N/V/D)
- Blurry yellow vision
- arrhythmias
- AV block
- Hyperkalemia (digoxin upsets pump-leak balance for K+ and increased extracellular [K]
Slide 31
How does malignant hyperthermia occur?
What is the mechanism?
- inhaled anesthetics or succinylcholine induce severe muscle contractions & hyperthermia
- mutations in ryanodine receptor (RYR1 gene) cause Ca2+ release from SR
- increased PM & SERCA Ca-ATPase activities d/t increased intracellular Ca2+ results in lower ATP which in turn causes an increase in Oxidative Phosphorylation
- Causes an increase in body tempurature
Slide 32
How does the MDR1 pump work?
- MDR1 pumps drugs out of cells, but hydrophobic drugs diffuse back in.
- This creates a pump-leak steady state, in which cellular drug concentration depends on MDR1 expression.
Low MDR1 expression = higher cytosolic drug concentration
Slide 33
