I) Permeability through membranes is generally proportional to solubility in lipids (an important example is that steriod hormones diffuse directly into nuclei) (evidence that membranes were lipids)
(Another interesting side-issue, NOT mentioned in textbooks is that
anesthetics block conduction of nerve impulses, & general anaesthetics
are lipid soluble chemicals, but it isn't really known how they work!!)
(local anaesthetics much better understood; block ion channels)
II) ratios of concentrations of ions in cytosol versus outside cell.
Na+ about 10 to 30-fold higher outside cell (low inside)
K+ about 30-fold higher inside cells (causes voltage!)
Ca++ kept VERY low inside cytosol (pumped out & into ER)
H+ kept a little higher inside, usually (messed up in cancer cells)
Mg++ kept a little lower inside
III) Passive transport and ionophore chemicals
Gramicidin (for monovalent cations = Na+ and K+ mostly)
many artificial ionophores can be bought: A23187 a calcium ionophore
Graph of rate of diffusion through membrane vs concentration
IV) symports and antiports (borrowed energy)
(these are membrane proteins that let one molecule leak across the membrane down its gradient, & also pump some other molecule)
Also the concept of transcellullar transport through epithelia
(pump something in one end; let it leak out the other end, etc.)
V)* the Sodium-Potassium pump (ATPase) "sodium pump"
(this is an integral membrane protein, which uses the energy of ATP
to force ions of sodium outward through the plasma membrane.
(& also to force potassium ions inward)
(as much as half or more of a cell's ATP may be used for this!)
Remember: the outside is more positive than the inside,
because potassium ions tend to leak out, and they are positive!
Voltage is electrical pressure; each 10-fold difference in concentration
is equivalent to 1.4 kcal/mole of ions, and that equals ~ 60 millivolt
(forces are energy-change per displacement of something)
QUESTIONS: If the ratio of K concentrations were 100 to 1
then what would the resting voltage be? What if 1000 to 1?
About how much energy does it take to pump Avogadro's number
of ions up a transmembrane gradient of 10 to 1? 100 to 1, etc.
& How does 80 millivolts relate to about a 30 to 1 gradient?
NOTE: Nearly all elementary textbooks fudge the issue of how the resting voltage (positive outside) can be caused by a positive ion that is more concentrated inside that outside! So make sure you understand.
(Textbooks tend to claim it's due to negative proteins, etc. which is b.s.)
If membranes were permeable to sodium, but not to potassium, then
the resting potential would be positive inside.
The voltage is generated by leakage; and creates the excess charge on the side TOWARD WHICH the leakage occurs. That's the point.
If membranes leaked both ions, then both would simply equilibrate;
the voltage results from only one being able to leak!
VII) nerve impulses (the action potential)
temporary opening of voltage-gated sodium channels
("threshold voltage")
(and subsequent opening of voltage-gated potassium channels)
Waves of depolarization also spread across muscle cell membranes.
& longer lasting depolarization waves spread on egg cell membranes!
(the latter is part of a set of mechanisms that block more than one sperm from being able to fertilize eggs)
These sodium channels open only for about 1/1000th second
(and then close: "refractory period"
A little more about nerve impulses:
Local anaesthetics (novocain, etc.) block sodium channels.
Some insecticides (DDT) work by slowing repolarization;
so that nerves don't get back beyond the threshold voltage
by the time that the refractory period is over.
(& I think newt poisons work that way, also! Don't eat newts!)
VIII) myelin sheath: plasma membranes of special cells get wrapped around nerve fibers; speed up nerve impulses about 10-fold
(but if 1.4 kilocalories is involved in this, I don't know how!!!)
Apart from that, the way to speed up nerve impulses is to increase the diameter of the axon. Squids evolved giant axons (mm diameter)
which have been useful to experimenters.
IX) How to send messages from one cell to another?
two methods: 1) direct electrical conduction (not used so much)
2) One cell secretes a certain chemical whenever it gets depolarized
& this chemical then stimulates opening of sodium channels
in other cells' plasma membranes, initiating action potentials
synapses neurotransmitters (neurotransmitter substances)
For example, acetyl-choline is the neurotransmitter substance
for the synapses between nerves and muscle cells.
But adrenaline is the neurotransmitter for many other synapses.
Glutamic acid, glycine are among neurotransmitters used inside the brain
(about 20 different neurotransmitters are used in humans:
some open sodium channels (depolarize), and others open Cl- etc.
and cause hyper-polarization (=make the resting potential bigger!)
Many drugs act by stimulating or inhibiting synaptic transport.
Amphetamines mimic adrenaline;
Nicotine affects some receptors for acetyl choline
Nerve gas covalently binds to serine at the active site of the
enzyme that splits acetyl choline into choline & acetic acid
(so muscle stimulation becomes permanent!)
X) ABC transporter proteins (ATPases) (pump molecules)
malaria (protozoa pump out quinine, etc.)
evolution (natural selection) of cancer cells with multidrug resistance
cystic fibrosis
XI) patch clamping method; for detecting opening of ion channels in tiny bits of membrane at tips of micropipettes.
Review questions to check your understanding about resting potentials, etc.
1) What is an electrical pressure? And what units is this pressure measured in?
2) In resting nerve and muscle cells, which side of their plasma membranes has how much charge relative to the other side?
3) Do any other kinds of differentiated cells have this same voltage difference (comparing the electrical potential in the cytosol versus the space outside the cell)?
4) The resting potential is cause by diffusion of which kind of ion?
5) When a diffusing positive ion causes a voltage difference, then does this create an excess positive charge in the place where this ion has the highest concentration, or somewhere else?
6) What causes sodium and potassium ions to have such different concentrations inside versus outside the plasma membrane of living cells?
7) If plasma membranes could somehow be magically changed so that they were more permeable to sodium than to potassium, then would this change the voltage difference?
8) Does such a change in relative permeabilities ever occur?
9) Which of these processes are caused by integral membrane proteins?
10) What is meant by saying that a given ion channel is "gated" by voltage, or by temperature, etc.
11) If a molecular biologist wanted to cause some ordinary cell to be able to propagate electrical impulses like nerves do, how could that be done?
12) If a dentist needed to block the propagation of impulses in pain-sensing nerves, how could this be done at the level of chemicals?
13) Could such a mechanism also be used to block messages sent to muscles from the brain, thereby causing paralysis?
14) Why does depolarization of one location on a nerve's surface immediately cause surrounding areas of this cell's membrane to become depolarized?
15) What would happen if a nerve's sodium channels opened permanently.
16) What would happen if these sodium channels closed permanently, after once having opened for their thousandth of a second of glory?
17) What if a certain kind of animal had nerves with sodium channels that could be stimulated to open by magnetic fields?
18) What would be the evolutionary advantage to a plant if it could make a certain chemical that would stimulate opening of the temperature-gated sodium channels on the nerve fibers that sense heat?
19) How do nerves stimulate muscles to depolarize?
20) Bears or other dangerous large animals can be "knocked out" by darts that inject the chemical succinyl-choline. This chemical has succinic acid, instead of acetic acid, bound to choline. can you figure out whether it really a "tranquilizer", as it is usually said on the news?
How might synapses be used to store memories? Why should some cells crawl toward negative electrodes, but macrophages crawl toward positive electrodes, and structural cells line up perpendicular to external electric fields? What if you had a stack of flat muscle cells or nerve endings, all of which have voltage gated sodium channels only on one side, but not the other?
How can fish sense weak external electric fields? Do embryos ever generate electric fields?
Do these electric fields serve any useful function? How could you hope to find out?
Answers:
1) Voltage; measured in volts; millivolts are thousandths of a volt.
2) The resting potential (=voltage) is about 70 millivolts positive outside relative to negative inside.
3) Nearly all the cells of the body have resting potentials, too; but textbooks only mention nerves and muscle cells having them.
4) Diffusion of potassium ions, from high to low concentrations is the cause of resting potentials.
5) Although it seems paradoxical, diffusion of an ion creates an excess of that ion's charge in the place toward which it is diffusing! (even though that it where it has the lowest concentrations!)
6) The sodium pump is an ATPase enzyme, in plasma membranes, that pulls sodium ions outward and potassium ions inward.
7) Diffusion of sodium in the inward direction would create a positive charge in the cytoplasm, because that it the place toward which positive sodium ions would diffuse.
8) Action potentials (such as nerve impulses) are caused by temporary opening (for a thousandth of a second) of special sodium channel proteins.
9) The sodium channels, the potassium channels, and the sodium pump ATPase, all are examples of integral membrane proteins?
10) When a certain ion channel protein has the property of being stimulated to open in response to a certain variable, then the channel is said to be "gated" by that variable.
11) If you could put voltage gated sodium channels into any given cell's plasma membrane, that should be sufficient to allow it to propagate action potentials.
12) Blocking the sodium channels (with novacaine, etc.) will prevent propagation of action potentials.
13) Sure; it will block any nerve impulse. It might block egg cells from preventing fertilization by extra sperm, for all I know; or prevent Paramecia from backing up, maybe!
14) Because the sodium channels are voltage gated in the sense that they become open wherever and whenever the membrane voltage becomes less than some particular amount? If, instead, these channels became open when the voltage was more than a certain amount, then that property wouldn't allow the positive feedback of depolarization, so it wouldn't allow you to send signals.
15) Permanent opening would let sodium ions continue to diffuse inward until the concentrations had become the same; in fact, that would also let the potassium concentration equilibrate. So there wouldn't be any voltage differences any more, not even the resting potential!
16) If the sodium channels closed permanently, then that nerve would only be able to propagate one action potential. (At least until it had synthesized some more voltage-gated sodium channel proteins)
17) That kind of animal would thereby be able to "feel" magnetic fields!
18) Such a plant would taste "hot" to any animal that ate it! Like hot peppers, etc.
19) Nerve endings secrete vesicles of certain chemicals, called synaptic transmitters, or neurotransmitters, such as acetyl-choline. These stimulate depolarization of the muscle, because the plasma membrane has sodium channels that are gated by acetyl choline.
20) It works by blocking synaptic transmission of motor nerves, thereby paralyzing the animal.
Look up the structure of succinic acid: you can see that this is a good example of a chemical analog.
The majority of this final list of questions are "unsolved problems". But the answer to the one about cells that have action potentials only on one side is that that's how electric eels work!
I) Different "compartments" of eucaryotic cells
IV) The main mechanism... Signal peptides, etc.
"signal sequences" & "signal patches"
"put me in the ER!" "put me in the inner part of mitochondria"
Remember the joke about "Sleeping Giant State Park" in Connecticut; and the High School student in the Yale sweatshirt!
V) How did Gunther Blobel discover signal sequences?
Studying mutant lines of SV-40, a cancer-causing virus
Found mutants in which a certain viral protein, that "normally"
goes into the nucleus, instead went to cytoplasm
pro-pro-lys-lys-lys-arg-lys-val pro-pro-lys-thr-lys-arg-lys-val
"nuclear localization signal"
Incidentally, for most organelles, once the protein gets put there, the signal peptide gets cut off BUT NOT in nucleus (guess why!)
VI) Nuclear pores "nuclear pore complex" octagonal structure
nucleoporins, etc. octagonal control big molecule entrance
"gated transport"
VII) What makes rough ER binding of ribosomes
stuck to ER membranes by nascent proteins & receptor proteins
VIII) Some directionality of protein transport uses GTP hydrolysis
RAN GTPase movement through nuclear pores
IX) In other cases, (mitochondria) heat shock proteins are used to carry unfolded proteins from place to place.
X) Phospholipid exchange proteins
(carry [or stabilize, solubilize] phospholipids from ER
to mitochondria, peroxisomes, or wherever..
XI) Sometimes membrane are moved around by budding
and then fusion of vesicles; either carrying contents of lumen
and/or carrying the pieces of membrane themselves.
XII) The general concept of topological equivalence
of position and orientation.
XIII) an interesting kind of organelle called a peroxisome
today's amino acid: Histidine (& relation to histamine)
I) phagocytosis, pinocytosis etc. endocytosis & exocytosis
and also vesicular transport from one organelle to another.
II) Several kinds of pinocytotic vesicles have darkly-staining sides = called "coated pits", based on their appearance in TEM
These turn out to be 3 different kinds; each with a different mechanism; the best understood kind folds because of a special protein called "clathrin" which forms " trisklions"(little 3-armed swastikas)
"clathrin-coated vesicles" "adaptin" "dynamin"
2 other kinds of coated vesicles:
COPI coated vesicles and COPII "cope-one" & "cope-two" ?
III) monomeric GTPases control assembly of coated pit vesicles
ARF SAR1
How small GTPases can control vesicle formation (endocytosis) and also control vesicle fusion to other membranes.
Researchers were studying mutations that caused cytoplasmic vesicles to behave abnormally; & discovered that the mutated genes coded for GTPases & give directionality to vesicle behavior.
When ARF binds to GTP, it stimulates coatomer subunit aggregation on membrane surface, so that membrane folds inward and buds off to
form a vesicle. Later, when this vesicle touches its target membrane
(i.e. appropriate for it to bind to) this stimulates hydrolysis of the ARF-GTP to form ARF-GDP This allows the coat to disassemble, and the vesicle to fuse to the target membrane.
Without ARF, or something like it, vesicles might bud off the target membrane and fuse with the source membrane!
Similar functions for Rab1, 2, 3A, 4, 5, 6, 7, 9 &Sec4
RAB GTPase "checks the fit" between v-SNARE and t-snare.
By localizing the exchange factors to one set of membranes,
and stimulating GTPase hydrolysis to locations near other membranes
cells can control which membranes bud, and get fused with !
IV) SNARE proteins
needed to cause merger of the hydrophobic parts of the membranes (of the vesicle & of the other membrane)
Several important kinds of viruses have membrane "envelopes"
so their entry into cells is much like fusion of a vesicle with a membrane; including HIV, and also the flu virus & the ones used to fuse cells. (this similarity motivates lots of research)
HIV specificity for CD4 cell surface protein (which is only on the surface of a kind of lymphocyte; which HIV cells therefore selectively bind to and enter)
NSF protein pry snare proteins apart, after fusion
V) Rab GTPases "ensure specificity" of vesicle docking
Rab1 etc. Rab effectors
VI) "The Golgi apparatus" (around the centrosome, next to the nucleus)
packages proteins made in rough ER
cis face (entry face) trans face (exit face) to cell surface
vesicles bud off one layer of Golgi & then fuse with next
"Transport vesicles" carry membrane (& vesicle contents) to next membrane
Golgi is biggest/ best developed in cells specialized for secretion.
Cells that secrete polysaccharides usually make them in Golgi
Glycosaminoglycans (cartilage [=gristle] in animals) is made there
Pectin & hemicellulose in plants (made there BUT NOT cellulose)
leave ER via the COPII type of coated vesicles
VII) mechanisms that selectively allow only properly-folded proteins to leave the ER, to cis Golgi via "vesicular tubular clusters"
Active propulsion of membrane vesicles along microtubules
If they have no special signal, then secreted by fusion with plasma membrane
But, certain signal peptides cause delivery to other places
Any sugar chains are usually added in cis golgi
ER retention signal: KDEL; checkpoint for misfolded or tangled proteins
Lysine, aspartate, glutamate, leucine
BiP (Binding Protein) ~related to heat shock proteins: in ER & is an ATPase
VIII) Lysosomes (acid hydrolases in animal cells)
full of acid hydrolase enzymes: at ph 5 (cytosol 7+)
proteases, nucleases, lipases, etc, dozens of enzymes for destroying everything
"are heterogeneous" = come in many different kinds, even within one cell: different kinds
Have their own signal peptide: KFERQ = "deliver this to lysosomes"
Lysine, phenylalanine, glutamate, arginine, glutamine
Phagocytized waste, etc. in vesicles, which fuse with lysosomes,
VIII 1/2) large vacuoles in higher plants and some fungi
XI) There are a few special cases in which certain kinds of lysosomal structures get secreted out of cells.
melanocytes melanosomes in mammals & birds (including humans)
XII) Special cell types whose jobs are to phagocytize bacteria and other cells, etc.
macrophages
neutrophils Mast cells histamine
XIII) Cholesterol taken up by cells in receptor-mediated endocytosis
LDL low density lipoproteins act as carriers for cholesterol in blood
cells needing cholesterol, just make LDL receptors ! bingo!
An analogous carrier molecule is transferrin, used to carry iron ions
XIV) Synaptic vesicles: are a special form of exocytosis
polarized patterns of secretion: axon is analogous to apical surface.
XV) lipid rafts, and all that...
Energy is used to pump hydrogen ions across (the inner-most) membranes of mitochondria and chloroplasts
(in mitochondria, the H+ is pumped outward through the inner membrane; but in chloroplasts, the H+ ions are pumped inward through the thylakoid membranes)
this causes pH differences of about 1 unit in mitochondria,
and 2 1/2 units in chloroplasts
The H+ ions then diffuse from high to low concentrations, driving ATP synthase enzymes in these membranes
(& bacterial have the ATP synthase in their plasma membranes)
[this idea, called chemiosmosis, was invented by Mitchell ~1961 and was accepted only grudgingly by the experts,
a very few of whom still don't accept it]
The evidence for chemiosmosis included
More recently (but I don't know why the book doesn't say more about this) ATP synthase was shown to rotate!!
{this rotation is analogous to bacterial flagella motor}
II) A chain of enzymes carries electrons (& energy)
from NADH to O2 cytochromes Fe+++ {analogous to hemoglobin}
other metalloproteins with FeS Cu+
(cyanide poisons cytochromes)
III) Photosynthesis
chlorophyll (which is fluorescent! and why it should be!)
captures light energy; uses this energy to reduce NAD
and also to make ATP (apart from making it by chemiosmosis)
IV) photosystem I and photosystem II
are believed to have evolved separately, in different bacteria
(green sulfur bacteria; that used light energy to split H2S into elemental sulfur and H+ analogous to release of O2 from water.
V) In photosynthesis, the capture (fixing) of carbon dioxide
to make sugars is entirely separate from the capturing of the energy.
ATP energy is used to make sugar
(thus, for example, if you gave them ATP from some other source, they could fix carbon in the dark)
The distinction between "dark reactions" which don't need light
as opposed to "light reactions" which directly need light.
Sugar synthesis (and carbon fixation) are dark reactions.
5C sugar + CO2 -> 6C sugar --> two 3C sugars -> etc.
- and which 5 carbon sugar is used? which would you guess?
V) Rubisco ~ 3 reactions /sec (very slow)
and also it is a very big protein (~ 1/2 million atomic weight)
therefore ~1/2 the weight of chloroplasts
In dry conditions, this 4C method allows less loss of water.
(but if you seal an air-tight container containing both 3-C and 4C plants, then the 4C plants will get almost all the CO2
VII) DNA genomes in both mitochondria & chloroplasts
(that only code for part of their proteins)
and they do have their own, bacteria-like, ribosomes;
and their DNA codes for t-RNA & some proteins.
Various interesting things about genomes
{human mitochondrial DNA sequenced way back in 1981}
{human m-DNA transcribed into 2 complete loops of RNA
which are then processed to make all the t-RNAs, rRNA and messenger RNA}
higher mutation rates useful for figuring out evolution
slightly different code(s)
not nearly enough different t-RNAs for all the codons (only 22)
(thus, they really do "wobble")
it is a puzzle why all of the genes haven't transferred to nucleus
(maybe because the change in codes prevented further transfer?)
Even when most of their DNA is lost, mitochondria still get made!! "petit" mutants in yeast!; showing that even if all the DNA had been lost, such an organelle could persist
(note: because of signal peptides, etc. with the receptor proteins that grab these signal peptides also having the same signal peptide: so self-perpetuation would be possible for an organelle, even without having its own genes (maybe peroxisomes;
and maybe basal bodies of cilia & flagella.
Review questions:
a) What expected change in acidity in fluid around mitochondria
when making ATP
b) why the change in the opposite direction in fluid around chloroplasts?
c) If you started with mitochondria that had no pH gradient,
then could you help them make more DNA by putting them into a solution with a different acidity? (yes)
d) Why do H+ ionophores poison mitochondria and chloroplasts?
e) How might weak ionophores be used to help people lose weight? (only occasionally killing a few such people)
f) How direct (hint, not very) is the connection between the energy absorbed from light and the energy used to capture carbon from carbon dioxide, in order to make sugar.
g) Do C4 plants really use a different sequence of reactions to make sugars from CO2 (if not for making sugars, then what good is the C4 fixation route?)
h) What is photoxidation?
i) Compare rates of mutation in mitochondria versus nuclear DNA.
j) What are some odd peculiarities of mitochondrial t-RNA?