Transport across Membranes Lecture Sep 23 Flashcards
What structural component is common amon all transmembrane proteins?
The transmembrane portion of the protein is almost always and alpha helix. It typically takes about 19-21 amino acids to span the whole membrane as a helix and these amino acids will be lipophyllic (such as isoleucine, leucine, valine, phenylalanine, etc.)
What are the 4 ways membrane proteins can move within the membrane?
- Free diffusion: protein is able to move freely anywhere in the membrane
- Anchorage/tethering: this motion is characteristic of proteins that are either directly or indirectly anchored to the cytoskeleton. They are able to spin like a top, but are basically stuck in one location.
- Hop-diffusion: A protein may be temporarily trapped within corrals that are formed by other proteins attached to the cytoskeleton. WHen the corrals disengage temporarily, the proteins can then hop to another domain.
- Confined diffusion: In this situation, the protein is trapped within a corral as in hop-diffusion, but these corrals are permament and the protein is therefore always confined within that one space.
What form of transport does NOT utilize ATP hydrolysis either directly or indirectly?
Passive diffusion and facilitated diffusion do not require ATP hydrolysis for energy - they use the electrochemical gradient alone.
What are the three forms of facilitative diffusion transporters?
Pore
Gated channel
Carrier protein (IF the carrier protein is bringin a molecule down it’s gradient)
How do passive and facilitated diffusion differ in the rate of transport in relationship to concentration?
Simple diffusion is a linear relationship: as the concentration of the target increases, the rate of transport will increase in a linear fashion.
Facilitated diffusion exhibits a hyperbolic relationship. As the concentration of target increases, you do get an increase in transport rate to an extent until the carrier gets maxed out and the rate of transport reaches its maximum.
What does the Km and Vmax mean in terms of facilitate diffusion?
THe Vmax is the maximum rate of transport that can occur (determined by how many transports there are).
The Km is the concentration of transported molecule at which half of the maximum transport rate can be achieved.
What are the two other names for carrier proteins?
What family are they from?
carrier proteins = transporters = permeases
They are from the major facilitator superfamily (MFS)
What three things will mediate opening of a channel?
- membrane potential (depolarization often opens the channel)
- Binding of the ligand
- Phosphorylation
What do aquaporins do?
How were they discovered?
THey allow the mass transport of water across plasma membranes.
They were discovered by Peter Agre, who injected RNA into frog eggs and then placed them in a hypotonic solution. Eggs that had the RNA swelled, and eggs that didn’t have the RNA did not swell. This suggested that the RNA encoded a protein that allowed for water to pass freely through osmosis: aquaporins.
What two types of porins are present in the human aquaporin family? In what tissues are they expressed?
The family includes the aquaporins and the aquaglyceroporins.
Aquaporins are expressed in high concentrations for tissues which have high water content: kidney, GI tract, cornea, brain.
Acquaglyceroporins transport both water AND glycerol, thus they are expressed in high concentrations for tissues with high fat content: liver and adipose tissue
How many transmembrane passes do GLUTs have?
12
How many GLUTs are there? Which class is most importnat for us as medical students?
There are 14 total.
Glut1 thoruhg Glut5 are the most important for us.
Describe GLUT1 transporters.
Km? Tissue? Substrate specificity?
5mM
These are ubiquitous - they are everywhere, but especially in RBCs
They are specific for glucose/galactose
Describe Glut2.
Km?
Tissue?
Special feature?
Substrate specificity?
11 mM (low affinity)
Expressed in the intestine, kidney, liver, and beta-cells
They mediate both uptake and efflux (which is possivle because of the relatively low affinity).
They will transport glucose, galactose, and fructose
Describe Glut 3
Km?
Tissue?
Substrate?
1 mM (High affinity!)
Expressed in neurons
Will move glucose and galactose
Describe Glut 4.
Affinity/Km?
Tissue?
Special feature?
Substrate specificity?
They have 5 mM Km (same at GLUT 1)
THey are expressed in fat, muscle, and heart
This is the GLUT that is regulated by insulin!!!
It will transport glucose, not galactose
Describe Glut 5,
What is the Km?
Tissue?
Special feature?
Substrate specificity?
6 mM
Expressed in the intestine and sperm (check out specificity)
These are found primarily in fructose-metabolizing tissues because they only transport fructose
Describe how insulin brings GLUT 4 to the membrane?
THe GLUT 4 trantsporter will be held in a vesicle just under the surface of the membrane. When insulin bings to its receptor on hte plasma membrane, the vesicle with GLUT 4 will fuse will the membrane, placing the GLUT 4 transports on the outside of the membrane
What equation characterizes active transport?
The deltaG equation:
If the molecule being transported is NOT charged uses a slightly different equation from when the molecule being ttransported IS charged.
What delta G will need ATP hydrolysis?
WHat delta G will NOT need ATP hydrolysis?
A negative delta G means the molecule’s transport will occur spontaneously WITHOUT ATP hydrolysis.
It delta G is positive, it will require ATP hydrolysis.
What is the membrane potential across the typical plasma membrane?
Are the following in higher concentrion on the outside or inside?
NA+
K+
Ca2+
Cl-
Vm = -60 mV
Na+ higher on the outside
K+ higher on the inside
Ca2+ low on both, but higher on the outside
Cl- much higher on the outside.
What establishes the ion gradient across the membrane?
The Na+/K+ ATPase pumps 3 Na+ out into the extracellular space and pumps 2 K+ into the intracellular space, thus making the outside of the membrane more positive and the inside more negative.
That’s what establishes it, but it’s actually the ion channels that really pushed the membrane to how negative it really is - but to do this they require the ATPase pump working.
How does digoxin increase heartrate and stroke volume?
The molecule increases the activity of the Na+/K+ pump, which makes the membrane potential even more negative. THis improves function of the heart.
Describe the intestinal epiethelial cell’s use of secondary ATP transport?
It uses the Na/K pump to pump Na+ out of the epithelial cell into the blood. This creates a higher concentration of Na+ in the intestinal lumen than in the epithelial cell.
It then uses a Na+/glucose symporter that transportsboth glucose and Na+ down Na+’s concentration gradient
It then uses GLUT2 to transport the glucose from the cell into the blood.
Describe the ABC transporters.
THere are 49 members in the Human ATP-Binding Cassette transporter family.
They all use ATP hydrolysis for active transport. They have a structure that easily and specifically binds ATP.
The 49 are split up into 7 different familys: ABCa through ABCg
In class we discussed the ABCa, which has a ATPase that when mutated will cause Tangier’s disease (dysfunctional lipid (and cholesterol) synthesis)
We also discussed the ABCb family which includes the MDR receptos (multidrugs resistance receptors), an example of which is the PGY1
What is the main way we discussed inw hich a cancer cell will develop drug resistance?
Give an example of a receptor involved in this.
Increase expression of efflux pumps, hwich are ATP hydrolysis dependent. They will pump out the drug.
The PGY1 receptor from the MDR family is one that does this.
What receptor is dysfunctional in cystic fibrosis? What specifically is the most common mutation in the gene? What does this mean molecularly?
Describe the structure of the CFTR?
The gene for the CFTR is mutated, most often by deleting a phenylalanine at the 508 position in the nucleotide binding domain #1.
This means the body can’t pump Cl- out of the cell.
In a normal cell, the CFTR will have 12 transmembrane sequences with two nucleotide binding domains and a regulatory domain on the intracellular side. THe regulatory domain will have a series of serine residues, whose hydroxyl groups can be phosphorylated by PKA to become more active. Dephosphorylation by PP2A will deactivate the CFTR.
What are some organs affected by CF and their symptoms?
skin - excessively salty sweat
lungs - bronchiectasis, pneumothorax, hemoptysis
Liver: obstructive biliary tract disease
Pncreas: enzyme insufficiency, IDDM
Small intestine: meconium ileus
Reproductive tract: male infertility
What is the normal purpose of the CFTR?
allow Cl- transport across cell membranes AND to regulate transport of other ions via interactions with their transport proteins.
Why would babies with CF taste salty?
In sweat glands, usually NaCL is secreted in the acinus and water will follow via osmosis.
Then as the water flows upt he sweat gland duct, the NaCl would be reabsorbed with some of the water following and the rest of the water exiting the gland as sweat.
WIth patient who have CF, that NaCl doesn’t get reabsorbed and their sweat tastes excessibly salty.
WHat do CF patients typically die from?
bacterial respiratory infections like pseudomonas.
C diff is also a big issue.
They are mmore susceptible to respiraotyr infections because the inability to pump Cl- out of the respiratory cells means there wont be the necessary layer of water to lubricate the mucous’s excretion thorugh ciliary action. This means the lungs fill up with mucous that can catch bacteria, allowing bacteria to grow in the lungs
How does a CFTR open?
In the CFTR there are bands on either side of the channel that are highly positively charged (lysine residues).
WHen the membrane is polarized like normal, the positive end is attracted INTO the cell because it’s attracted to the negative inside. This interaction stabilizes the channel in a closed configuration.
When the membrane depolarizes, the membrane repell the positively charged region and caus it to move up towards the surface. This conformational change opens the channel and CL- will rush out of the cell.
Note: many voltage sensor channels will act in this way.
Briefly describe the K+ channel.
What molecule will specifically inhibit K+ channels in nerve cells?
The K+ channel is similar to the Ca2+ channel.
It has 6 transmembrane segments that join in to join a tetramer to toal 24 segments.
It has a selectivity filter that controls which ion will br transported. Thus, the selectivity filter is lined with carbonyl oxygen atoms that bear a degree of negative charge and form transient binding sites for the K+ that shed their water shells (since water molecules will cloak the positive K+).
Tetra-ethylammonium ion is a compound that will specifically inhibit K+ channels in nerve cells membranes because the thyl groups will sterically hinder the passage of the cation through the channel.
How are transport mutations somtimes responsible for sudden cardiac arrests?
If a mutation occurs and increases the legnth of the QT interval in heart function, torsades de pointes can results, which leads to syncope, seizures, and sometimes sudden death.
3 transporters that have been implicated in this are SCNSA3, KCNQ1, and KCNH2
The sudden death usually occurs during times of high activity, like a basketball game or swimming.
Collectively, they’re called channelopathies
What are two toxins that will block the Na+/K+ ATPase?
WHere are they found?
Tetrodotoxin from puffer fish
saxitosin from marine dinoflagellates (red tide)
What do ionophores do?
What is an example?
Ionophores are molecules that can bind ions and then passively diffuse across membranes, thus disrupting the ion gradients.
Gramicidin is an example of this and it’s used as an antibacterial agent. If the gramicidin dimerizes, it will actually form a pore in the membrane to allow ions to trasvel down their gradients through the small pore.
Valinomycin is another ionophore which is highly selective for K+- it’s an antiviral.
A23187 is a Ca2+ ionophore
WHat are the 3 classes of ATPases?
P class: located on the plasma membrane; are autophosphorylated during catalysis and include the Na/K ATPase and the Ca ATPase
V class: located in secretory vesicles, they transport H+ into the vesicle and are multimeric
F class: are located in the mitochondria and chloroplasts; physiological ATP is formed through these using the proton gradient as the driving force.
