Membranes

Fox, Chapter 6, plus some references back to earlier and later chapters and to Freeman

There's enough lipid to make two layers

Fig. 6.13
shows how red blood cells react to hypertonic, isotonic and hypotonic solutions.
Get a good source of membranes:
red blood cells (erythrocytes) from adult human have only plasmalemma.
Gorter and Grendel showed in1925 that there was enough lipid to make two layers.
Put red blood cells into distilled water, they burst from hyposmotic shock and become only "ghosts" - membrane only.
Blood cell counts, and geometry solves for membrane surface.
Extracted lipids on a surface have an increased lateral stability when they reach a monolayer which, when measured is twice the membrane area.
Here is a snapshot I took of oil on a road after rain - when oil is multiple layers, you see color, and layers slip, when oil is one layer, it is black.

Fig. 6.7
(To understand how hypotonic shock burst the erythrocyte, I introduce a fundamental concept, osmosis)
Osmosis - water moves passively from where water is at a higher concentration (for instance pure water) to where water is at a lower concentration (where organic chemicals are dissolved in it)
through a semipermeable membrane (i.e. a membrane which passes water but not the organic molecules).

Membrane structure

(Glucose transport across the cell membrane is so fundamental that it is in the introductory book)

Figure
TRANSPARENCY (Review from an introductory text)
glucose transporter

Fig 6.16
Glucose transporter from your textbook
shows bilayer of lipids with protein in it

Notice that the lipid molecules are drawn in this "cartoon" as a ball with two sticks.
Most membrane lipids are phospholipids with:
(1) a polar (hydrophilic) head group
and
(2) hydrophobic fatty acid (acyl) tails

Another example allows introduction of another fundamental molecule:

TRANSPARENCY (Review from an introductory text)
Here is a famous membrane protein, rhodopsin, the molecule we see with, and how 7 hydrophobic alpha helices of the protein fit into the hydrophobic part of the membrane (the milieu created by the fatty acid tails). For future reference, retinal is the chromophore, the component (chromophore) that makes the protein [proteins are otherwise not colored] into a pigment. Retinal is a derivative of vitamin A. Rhodopsin is in the membranes of rods and cones, visual receptor cells, shown in the diagram. Rhodopsin is the prototypical G protein-coupled receptor (GPCR), and GPCRs are used for hormones, neurotransmitterss, olfaction, taste and others.

Electron microscopy (EM)

Fig. 3.2, Fox
Robertson did work that led to earlier bilayer model.
He saw 2 "electron dense" (dark) lines in EM when stained osmium, an electron dense heavy metal. Davson and Danielli developed a membrane model from Robertson's vistas.
Fluid mosaic Singer and Nicolson the more modern version

Picture I made freeze fracture replicas with this apparatus. Specimen is prepared, frozen to liquid nitrogen temperature, put inside a vacuum, smashed with a razor (membranes break down the middle between the fatty acid tails), blasted from an angle with a platinum gun (to shadow protein with electron dense heavy metal), blasted from above with a carbon gun (to hold replica together), then the tissue is dissolved away.

Here, from my research, is an example of how things look. Picture shows visual membranes in Drosophila. High vitamin A flies have membranes full of protein (the same rhodopsin I mentioned above) while vitamin A deprivation decreases this protein.

Membrane biochemistry

Membrane lipids are composed of:
(1) Phospholipids such as phosphatidylcholine (lecithin)
I did some research on the phospholipids of the Drosophila head. Using radioactively lbeled phosphate, many different phospholipids are visualized after they have been separated on a TLC (thin layer chromatography) plate.
(2) Cholesterol
(3) Glycolipids such as one that accumulates in Tay-Sachs, a hereditary lysosomal storage disease,1/30 American Jews carry, recessive, fatal at 6 mo - 5 yr

Alexa B. Serfis in SLU's Chemistry Department studies membrane lipids and their proteins

Membrane physiology

Relevant to physiology, if the membrane had only lipids, it would have extremely high resistance. This is because the hydrophobic milieu in the center of the membrane does not allow water, a polar solvent, or ions which carry current. The membrane is only permeable because some of the proteins are channels that pass ions. Also, there is high capacitance. The concepts of resistance and capacitance will be dealt with shortly.

Membrane signalling

Lipid makes a barrier to anything polar or big like protein hormone or epinephrine (bind receptor).
(This receptor is the GPCR, mentioned above.)
Steroid hormones can go in

It used to be thought that lipids just sit there. In the 1980's it became clear that they turn over metabolically and that some products of membrane lipid turnover are important mediators of intracellular signalling. This is very fundamental and will come up repeatedly in later.

Fig. 11.9
Hormone -> receptor protein (GPCR) -> G-protein -> cascade makes second messengers (IP3 and DAG [diacyl glycerol, not in your diagram]) from the membrane lipid PIP2 [phosphatidylinositol-4,5-bisphosphate, not in your diagram], note that calcium ion Ca2+) becomes a next messenger in the cascade.

Important points that will come up repeatedly:
Phospholipase C is the enzyme [and I have a research interest in PLC]
IP3 is a "ligand" for a calcium channel.
Ca2+ is sequestered inside endoplasmic reticulum.
Inside a cell's cisterns is tantamount to outside the cell.
Ca2+ is high outside and low inside, like Na+ (sodium ion) unless deliberateluy increased intracellularly.
Ca2+ levels are so important that 3 hormones regulate blood Ca2+, parathormone, calcitonin and vitamin D.

Membrnne channels

Fig. 7.26
Nicotinic Acetylcholine receptor [More on this later])
Acetylcholine is a ligand (neurotransmitter), nicotine is a pharmnacological agonist.
This receptor is a channel (for ions, giving the membrane electrical conductance [g])
Channel is ligand gated.
Sodium (Na+) and potassium (K+) shown going through pore in membrane that can be open or closed.
Sodium, higher outside the cell, is likely to go in.
Potassium, high inside the cell is likely to go out.

The 1991 Nobel prize in physiology and medicine was awarded to prize was awarded jointly to: ERWIN NEHER and BERT SAKMANN; they developed patch clamping that allowed electrical recording from single channels.
In 1963 the Nobel prize was awarded jointly to: SIR JOHN CAREW ECCLES , SIR ALAN LLOYD HODGKIN and SIR ANDREW FIELDING HUXLEY for their discoveries concerning the ionic mechanisms involved in excitation and inhibition in the peripheral and central portions of the nerve cell membrane; Hodgkin and Huxley worked on the voltage gated channels of the axon's action potential and Eccles worked on the neurotransmitter gated channels at synapses.
In summary, the topic of ion channels is pretty fundamental.

Fig. 7.21
Also holes in membranes from one cell to another are important:
Gap junctions - 2 hexamers in register of connexin protein
This is a very big channel.
Important in many places, especially connecting one heart muscle (myocardial) cell to another electrically.

Membrane transport

Fig. 6.19
"sodium pump"
A large fraction of the cell's energy (ATP) goes to pumping ions (active transport)
This creates an ion imbalance, sodium Na+ high outside cell, potassium K+ high inside.
This gives rise to the membrane electrical potential (voltage) important in nerve and muscle cells.

Fig. 3.4
bulk transport:
phagocytosis - cell eating
pinocytosis - cell drinking
Receptor mediated endocytosis - clathrin coated pits turn to vesicles, clathrin is a protein that makes vesicles look fuzzy.
Receptor mediated endocytosis is important in clearing lipoproteins, LDL and HDL, from blood (later), and, of course, a receptor protein in the membrane is important in the transport.

Figure
From my research, a coated pit.

In summary,

Functions of membrane proteins
(1) transport
(2) many enzymes are on the membrane
(3) receptors for hormones, neurotransmitters and developmental signals are on the membrane.
(4) cells are joined by proteins
(5) cells communicate by proteins
(6) cells hook to extracellular proteins by proteins

Reference:

E. Gorter and F. Grendel, On bimolecular layers of lipoids on the chromocytes of the blood, J. Exp. Med. 41, 439-443, 1925

Exam questions from 2004 - 2011 relevant to this outline

The introductory book's transparency indicated that "GLUT-1 facilitates glucose diffusion." Answer either why they selected the words (1) "facilitates," or (2) "diffusion."

(1) glucose needs a path across the membrane, (2) but it is not a pump that requires energy

In the phosphoinositide signal transduction cascade, IP3 gates a channel. Answer either (1) Where (specifically)? Or (2) For what ion?

(1) a smooth endoplasmic reticulum (2) calcium ions

Answer either (1) Why are there rainbow colors on a drop of oil on a rain-wet road? or (2) Why are rhodopsin or hemoglobin colored molecules?

(1) from optics of layers that are different thicknesses (2) the protein has a chromophore that makes the protein colored

"A semipermeable membrane is part of the explanation for osmosis." Permeable to what and not permeable to what?

permeable to water but not to a larger molecule like sugar

"Picture an area where the gravel has been lightly shadowed by a light snow blowing from one direction." Answer either (1) What is this technique called? (2) What do the P-face particles represent? or (3) What heavy metal is used for shadowing?

1 freeze fracture 2 membrane proteins 3 platinum

A slide was shown where all the phospholipids of the Drosophila headwhich had been separated were clearly visualized. Answer either (1) What is the name of this technique? or (2) How come these phospholipids could be clearly visualized?

1 autoradiography (thin layer chromatography) 2 they radioactive and the radioactivity exposes photographic film

Tay Sachs disease: Answer either (1) Why is it called a lysosomal storage disease? (2) What molecule fails to turn over? or (3) Why would the eye and brain be affected first?

1 the substance the lysosomes fail to degrade accumulate in lysosomes 2 a glycolipid 3 there are lots of membranes with lots of membrane lipids

In terms of how they mediate signal transduction, state, in simple terms, the fundamental difference between a G protein coupled receptor and the nicotinic acetylcholine receptor.

GPCR leads to transduction cascade and "second messenger" while NAChR is a channel

In "receptor mediated endocytosis," the pit (that becomes a vesicle) has a receptor (like the receptor for LDL) plus what famous protein that makes these pits and vesicles look the way they do in the electron microscope?

clathrin

"In an isotonic milieu like the blood stream, a red blood cell's shape remains unchanged." To make red blood cell ghosts, Gorter and Grendel Answer one of the following: (1) They moved the cells from an isotonic solution to what kind of solution? (2) What famous membrane transport process mediated the net influx of water into the cell?

hypotonic (distilled water), osmosis

In the third lecture, the importance of G protein coupled receptors (GPCR) was introduced. Where, in the ultrastructural anatomy of either a rod cell or a cell involved in phosphoinositide signaling, would you find a GPCR?

membrane disks in rod, on the plasmalemma (cell membrane


How much energy, delivered by ATP, is used by the GLUT-1 transporter to get glucose into the cell?

none

In contrast with the G protein coupled receptor, why is the nicotinic receptor more direct?

an ion channel is more direct than a signal transduction cascade

In addition to glycolipids and cholesterol, what is the major lipid constituent of the cell membrane?

phospholipids

In addition to the receptor, in receptor-mediated endocytosis, what famous protein is seen in transmission electron micrographs that gives coated pits and coated vesicles their names?

clathrin

How much ATP would a cell use to open and close the nicotinic receptor?

none

"IP3 is a ligand for a calcium channel." Answer one of the following: (1) Where is this Ca2+ channel? (2) Where was the precursor of IP3 (the molecule from which IP3was formed by the action of PLC [phospholipase C])?
 
(1) on a smooth endoplasmic reticulum, a calcium sequestering cistern (2) PIP2 is a membrane phospholipid
 
"Vitamin A deprivation decreases the number of bumps seen in fruit fly photoreceptor membranes (transmission electron microscopy of freeze fracture replicas)." These bumps are a visualization of what molecule?
 
rhodopsin
 
Note that ATP is not used in a channel like the nicotinic receptor that can be closed or, when opened, passes K+ and Na+ (causing depolarization). Where, if at all, is ATP utilized in generating membrane potentials based on K+ and Na+?
 
Working in the background, the Na+ - K+ ATPase establishes Na+ and K+ gradients

Why do you need lead, osmium, uranium or platinum to see aspects of membrane structure in the electron microscope?

heavy metals are electron dense

What did hypotonic shock do to what kind of cells to allow Gorter and Grendel to show that there was enough lipid in the membrane to make two layers?

burst red blood cells to make red blood cell ghosts with a measured membrane surface area

Why do you need a chromophore (such as retinal for rhodopsin and heme for hemoglobin) to make a protein into a pigment?

proteins do not absorb visible light

How does a steroid hormone get into a cell?

that can pass the lipid barrier

How do sodium ions get forced out of the cell?

active transport with a protein that uses ATP

The nicotinic receptor is a cation channel for what two ions?

K+ and Na+

Rhodopsin and neurotransmitter and hormone receptors interact with what downstrean heterotrimeric protein?

the G protein

What do you call electrical junctions from cell to cell with channels composed of hexamers of connexin protein in register?

gap junctions

What must be bound to the G-protein-coupled-receptor protein to make the fully-functional rhodopsin molecule that absorbs light?

retinal

As a result of phospholipase C (PLC) activation, what ion is released into the cytoplasm from smooth endoplasmic reticulum?

Ca2+

In what fundamental way does the location of a steroid hormone receptor differ from that of the receptor for epinephrine?

steroid receptor is in cell, epinephrine receptor is in membrane

In a "cartoon" of a membrane phospholipid, there is a ball with two tails. The ball is the polar (hydrophilic) head group. What are the two tails?

fatty acids (acyl groups)

Which direction does the ATPase pump sodium ions?

out of the cell

Epinephrine binds one G protein-coupled receptor (GPCR). What other GPCR, used for vision, is a pigment that contains a form of vitamin A?

rhodopsin

What is the name of the electrical connection between myocardial cells composed of connexin proteins?

gap junction

Why does an erythrocyte turn into an erythrocyte ghost when placed into distilled water?

because of osmosis, it swells and bursts

When a membrane lipid is drawn as a ball with two sticks in a diagram, what are the ball and sticks respectively?

polar head group, fatty acids

One signal transduction product of phospholipase C (PLC), diacylglycerol (DAG), is in the membrane while the other, IP3, inositol trisphosphate, goes into the cytoplasm. Where is the precursor, PIP2 (phosphatidylinositol-4,5-bisphosphate)?

it is a membrane phospholipid

When the nicotinic acetylcholine receptor channel opens, there is an efflux of K+. Why?

because it allows Na+ and K+, so K+ goes down its chemical gradient

One membrane protein is sometimes called the Na+-K+-ATPase. What is its function?

pumps Na+ out, K+ in

What is the function of a hexamer of connexin proteins in one cell's membrane in register with a similar hexamer on the adjacent cell?

gap junction connects adjacent cells' cytoplasm and passes current

How can it be that a cortisol receptor is intracellular while so many hormone receptors, for instance for epinephrine, are on the membrane?

steroids pass through the membrane

Robertson's pioneering electron microscopy paved the way for Davson and Danielli's bilayer membrane model and Sanger and Nicolson's fluid mosaic model. Why were heavy metals like osmium necessary for that demonstration?

Electron dense

If water and ions are excluded from the center of the membrane, where fatty acids reside, how is it that a G protein-coupled receptor can span the membrane?

There are hydrophobic amino acids

Tay-Sachs disease is a fatal autosomal lysosomal storage disease. What accumulates?

glycolipid

Before I introduced metabotropic receptors (G protein coupled proteins that bind a ligand such as a neurotransmitter), I showed rhodopsin, the prototypical G protein coupled receptor. Why doesn't rhodopsin need to have a ligand bind to it? (i.e. What does it have that a neurotransmitter receptor does not have?)

it has retinal, a vitamin A derivative

Researchers have been able to make artificial membranes out of phospholipids in a hole between two compartments in a water bath. Why would membrane lipids naturally arrange themselves as they are aligned in membranes?

polar heads would orient to water and hydrophobic tails toward eachother

In the phosphoinositide signal cascade, phospholipase C (PLC) makes "second messengers" IP3 (inositol trisphosphate, the polar head group) and what(?) from the membrane lipid PIP2 (phosphatidylinositol-4,5-bisphosphate.) [If you do not remember, I have put enough information into the question that you should be able to figure it out.]

diacyl glycerol

Osmosis was referred to as passive transport. Is the sodium-potassium pump passive? Justify.

no, it is active b/c it uses ATP

After feeding radioactive phosphate, extracted lipids were visualized by audioradiography of a TLC (thin layer chromatography) plate. Why didn't I see the sort of lipids that accumulate in Tay Sachs disease?

it is glycolipids and would not take up phosphate

If you fracture a frozen membrane, proteins are exposed. But you cannot see them in the transmission electron microscope unless you do something. What?

you need to make a replica, shadow it from an angle with an electron dense material (platinum)

Under what circumstances (what do you do?) does a passive process (what process?) let you make a red blood cell ghost from a red blood cell?

Put r.b.c into distilled water, osmosis

Tell me about a famous lysosomal storage disease. Your answer can be biochemical, cell biological, or genetic.

Tay Sachs fails to break down a glycolipid that accumulates in the cell, autosomal recessive carried in Ashkenaze Jews

In the transmission electron microscope, what membrane specialization of receptor mediated endocytosis is visualized?

Clathrin coated pits (vesicles)

"11-cis retinal (a bent form of the aldehyde of vitamin A) is the chromophore of rhodopsin." In terms of the molecule's interaction with visible light, what does this mean.
 
Gives the protein its property of absorbing visible light
 
"The two half leaflets of the membrane, as seen in freeze-fracture, are called P- and E-faces." Answer either (1) why P and E? or (2) describe why these two halves are distinct from each other.
 
1 protoplasmic (on the cell side) exoplasmic (the external side), 2 turns out that the proteins are usually seen on the P-face
 
"Diacyl glycerol." Explain how this name, by itself, would tell you what would have had to happen to a membrane phospholipid to make diacyl glycerol.
 
2 farry acids (acyl groups) attached to a glycerol means that the polar head group had been chopped off
 
Tell me how autoradiography of a TLC (thin layer chromatography) plate can help you identify the numerous phospholipids of the cell membrane.
 
A TLC plate separates lipids, labeling with radioactive phosphate allows exposure of an audorad
 
"IP3 is a ligand for...," answer either (1) What kind of molecule? Or (2) Where is the molecule (that it is a ligand for)?
 
1 channel for calcium ion 2 on a smooth ER that sequesters calcium

Extracts from what kind of cell gave the original demonstration that there was enough lipid in a cell membrane to make two layers?

erythrocytes

What must be true about the amino acids of the alpha helix of rhodopsin that would let it cross the membrane?

they would be hydrophobic

For Tay-Sach's disease, answer either (1) Why is it called a "lysosomal storage disease?" (2) What molecule is missing because of the mutation? Or (3) What type of molecule is not broken down?

(3) glycolipids are not broken down because of (2) an enzyme deficiency leading to (1) accumulation

In signal transduction cascades, what protein binds to either GTP or GDP.

G protein sepcifically the alpha subunit

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