M2M Unit 3 Flashcards
5 properties of malignant cancer cells
- unresponsive to normal signals for proliferation control
- de-differentiated (lack many of the specialized structures/funcs of the tissues in which they grow)
- invasive (capable of outgrowth into neighboring normal tissues to extend the boundaries of the tumor)
- metastatic (capable of shedding cells that can drift through the circ sys and proliferate at other sites in the body)
- clonal in origin (derived from a single cell)
- **can measure transformation in a lab via increased anchorage independence
multi-step process for carcinogenesis
1- susceptibility to cancer is inherited- carcinogenesis is a multi-step process
–accumulation of somatic mutations produced by environmental factors over time
2-an early mutation may be in a DNA repair gene that increases the rate of more mutations
–ex. P53, BRCA1, BRCA2
3- tumor initiation (occurs via mutations in oncogenes and anti-oncogenes)
4- promotion
5- conversion
6-progression
2 types of genes usually mutated in tumor initiation and their effect on cellular proliferation
oncogenes- drive proliferation
- quantitative changes- overexpression (BCR/ABL)
- qualitative changes- create a hyperactive protein
anti-oncogenes- inhibit proliferation or metastasis
-tumor supressors
2 cytogenic abnormalities associated with malignancy
cytogenic analysis to study cancer gave first clues to genetic abnormalities in cancer cells and is used in clinical diagnosis
translocations and gene deletions may activate oncogenes or inactivate tumor supressors
-ex. Chronic myelocytic leukemia (CML) assoc w/ Philadelphia chr
inactivation of tumor supressors may occur by LOH
-ex. retinoblastoma and the APC gene in familial adenomatous polyposis (FAP)
events that can produce LOH
loss of a tumor supressor gene (loss of RB gene)
a tumor supressor gene can be transferred during recombination, leading to a gamete w/o the gene
LOH can occur via:
mutation, mitotic recombination, chromosome loss, environmental factors
Knudson’s theory
2 hits/events needed to acquire a cancer
1 hit-familial individuals already have 1 mutation and just need to lose 1
2 hit-acquired patients need 2 hits to have cancer
how are cancers associated with both dominant and recessive symptoms
susceptibility to cancer is inherited in both fashions
inherited in a dominant fashion: ex. susceptibility to Retinoblastoma
- heterozygotes are non-malignant, but will be cancerous w/ the loss of a single normal RB gene in 1 cell
- thus, RB heterozygotes are likely to develop disease and pass on the defective gene to 1/2 of children, thereby making retinoblastoma a dominant inheritance pattern
in reality- cells must be homozygous RB mutated to be malignant
describe how RB gene was first identified
cytogenic analysis of retinoblastoma cells showed the region around Chromosome 13q14 often had an abnormal structure
- retinoblastoma cells from some patients lack RB completely; both copies of RB have been deleted via PCR analysis
- some patients have partial deletions or other rearrangements of RB
RB protein’s function in the cell cycle and malignancy
RB protein is hyperphosphorylated in rapidly proliferating cells at S or G2 in cell cycle, but is hypophosphorylated in non-proliferating cells in G0 or G1
-hypophosphorylated form of RB protein normally functions to repress the entry of cells into the S phase. When RB becomes hyperphosphorylated, it no longer inhibits this transition and the cells begin a cell division cycle. Thus, when there is no RB protein or it is non-functional, cells cannot downregulate their cell division and grow out of control
RB protein general info
RB is an inhibitor of cell proliferation and is therefore an anti-oncogene or tumor supressor
phosphorylation by CDKs inactivates the RB protein, so cell proceeds from G1 to S phase
RB protein is a target for many animal tumor viruses
- ex. SV40 and HPV
- these viruses drive a quiescent cell into S phase and proliferate by producing a viral protein(s), SV40T antigen (transforming) or HPV E7 protein, that binds to and inactivates the RB protein
hallmark of a tumor supressor gene or anti-oncogene
relates to RB gene
hallmark: LOH and acquired Homozygosity for the susceptible gene
Inherited retinoblastoma: the DNA from the normal tissue of the patient or from another unaffected family member often shows a defect in the RB gene, but has one normal copy per cell.
In these patients it apperas that normal, nonmalignant retinal cells are heterozygotes for RB gene but the tumor cells have descended as a clone from a single cell that has acquired homozygosity for the RB susceptible gene
3 tumor suppressor genes
APC
BRCA1
BRCA2
how does APC function as a tumor supressor
APC is a tumor suppressor in FAP (familial adenomatous polyposis)
FAP is inherited via autosomal dominant- 1 defective APC gene puts you at high risk for colon cancer, but need 2nd mutation (LOH) to develop phenotype
APC gene encodes a cytoplasmic protein that regulates the localization of the beta-catenin in cytoplasm
beta-catenin is bound to E-cadherin at plasma membrane in mormal cells
APC protein cuases degradation of any unbound beta-catenin in cytoplasm
FAP patients lose APC, so beta-catenin goes to nucleus to produce (over) transcription of oncogenes like c-myc
how do BRCA1 and BRCA2 function as tumor suppressor genes
BRCA1 and BRCA2- predisposing genes for breast and ovarian cancer
inherited cases display LOH and have only 1 mutant gene
aquired- these genes haven’t been found in tumors, so it’s believed that mutations in other genes may affect BRCA functions indirectly
homozygous BRCA2 mutations get Fanconi’s anemia
heterozygotes get breast cancer from mammary gland losing the WT allele
describe why p53 was originally incorrectly thought to be an oncogene
initially thought to be an oncogene because certain p53 mutations were dominant to the WT gene in producing cellular transformation; tumors were still heterozygous
the explanation was found by showing that the oncogenic p53 mutations produce a mutant p53 protein that can bind the WT protein and inactivate it
“dominant negative” mutation spoils the WT protein
explain why p53 gene is the “guardian of the genome”
cells missing p53 accumulate mutations at a high rate and have a higher chance of becoming malignant
p53 prevents potentially deleterious mutations though the replication of damaged DNA, and introducing apoptosis in cells with too much damage
cellular function of p53 protein
important for cell response to environmental mutagenesis
acts as a transcription factor important for expression of genes- preventing cells from replicating damaged/foreign DNA
required for apoptosis- when cells commit suicide if their DNA is damaged beyond repair
p53 defective cells replicate DNA and produce mutations leading to cancer (mutant p53 found in ~50% all cancers)
-“hot spots”- common areas for point mutations
oncogenic viruses vs RB and p53
viruses have oncogenes that act by inactivating p53
viruses also inactivate RB protein
destruction of RB and p53 is a major route to cancer
ex of oncogenic virus in humans
HPV Human papilloma virus
HPV E7 binds to RB and deactivates it
HPV E6 binds p53 and causes it to be degraded
how were oncogenes discovered
discovered oncogenes in certain oncogenic retroviruses from animals (chicken)
with 1 particular viral gene segment (v-onc), tumors are rapidly induced in the infected cells
w/o it, integration into host genome occurs w/o activation of oncogenes
method:
put cells in agar, watch for proliferation
normal cells- no anchorage to grow, so no proliferation
infected cells- proliferate regardless of anchorage
define retrovirus
RNA containing membrane-enclosed viruses that bud from cell membrane of infected cells and usually don’t kill infected cell
examples of oncogene discovery
v-src
v-erb
v-abl
v-myc
V-src; oncogene of Rous Sarcoma Virus; caused fibrosarcomas in certain birds
V-erb: oncogene of avian erythroblastosis virus; causes erythroblastosis in chickens
V-abl- oncogene found in Abelson leukemia virus from mice
V-myc- gene usually fused with a portion of the gag gene (in RNA); appears this gene is capable of eliciting neoplastic transformation of cells
protein products of viral oncogenes-
pp60v-src protein
coded by v-src gene is a membrane bound protein kinase that phosphorylates tyrosine residues in several different proteins. The proteins change cell properties by affecting gene expression
protein products of viral oncogenes-
v-erb-B
codes for a protein that is similar in structure to the cell surface receptor for epidermal growth factor (EGFR). This raises the possibility that this protein has growth stimulating properties like EGFR. This receptor is a member of a family of related proteins that exhibit tyrosine-specific protein kinase activity
protein products of viral oncogenes-
v-abl
codes for a protein kinase that phosphorylates tyrosine residues on other proteins. It is similar to the human gene c-ABL that is found on the BCR-ABL translocation in the Philadelphia chromosome and is overexpressed in BRC-ABL CML
protein products of viral oncogenes-
endogenous oncogenes
Endogenous oncogenes are marked c-onc. Notice that c-onc genes are part of normal functioning of human cells, so therapy can only target overexpression, not all of c-onc
Thus- it seems many oncogene products mimic hormones or growth stimulating factors either by resembling natural hormones or by affecting the structure of the cell surface receptors for these hormones.
These altered receptors then send signals to the cell nucleus in an unregulated manner to affect growth
2 changes responsible for oncogene cancerous effects
in the proto-oncogene:
quantitative (too much protein)
qualitative (overactive/unregulated protein)
why are oncogenes useful as molecular markers in prognosis
2 examples:
N-myc
HER2/neu
level of oncogene expression tends to correlate w/ the rapidity of the progress of the cancer
ex. N-myc gene expression used for neurobalstomas
ex. increased HER2/neu gene expression correlates w/ poor breast cancer prognosis
oncogenes vs tumor suppressor genes:
oncogenes- promote cell proliferation
tumor suppressor genes- inhibit cell proliferation
if you have over-activation mutation in an oncogene and an inhibiting mutation in a tumor suppressor gene, then that will lead to cancer
how bioinformatics about a patient’s cancer are being used for targeted therapy and personalized medicine
can design either small molecs or antibodies as therapy
targeting oncogenes:
herceptin- drug antibody therapy against HER2/erb2 oncogene product
small molecs- able to inhibit cancerous proteins, usually by binding to active sties
ex. Gleevac (ATP analog- specific to binding pocket) inhibits ABL tyrosine kinase in patients w/ BRC-ABL translocation on philadelphia chr
targeting tumor suppressor genes:
inject RB directly into RB-negative tumors
use drugs that kill only p53 deficient cells
use drugs that correct mutant conformations of dominant-negative p53 proteins
criteria for classifying a hereditary cancer syndrome such as Li-Fraumeni syndrome
rare inherited cancer susceptibility w/ large range of presentation
must have more than 1 family member w/ cancer (autosomal dominant nature)
usuallly assoc w/ p53 mutation (70%)
diagnostic criterai for Li-Fraumeni Syndrome LFS
a proband w/ a sarcoma diagnosed before 45 AND
a first degree relative w/ any cancer under 45 AND
a first or second degree relative w/ any cancer under 45 or a sarcoma at any age
Knudson two-hit hypothesis
2 hits/mutations must accumulate in a tumor suppressor gene before progressing to cancer
1 inherited state/hit is premalignant
ex. breast cancer= HER2, p53
lung cancer= EGFR, p53
function of p53 in response to UV exposure
p53 is required to protect from UV- a major carcinogen, by protecting skin- sensor of cell stress
CHK1 and CHK2 bind to and activate p53 to act on target genes to help DNA repair and damage prevention
DNA damage, cell cycle abnormalities, hypoxia –>
activate p53 –>
cell cycle arrest; DNA repair and cell cycle restart OR death/elimination of damaged cell –>
cellular and genetic stability
DNA damage can occur/accumulate w/o functioning p53; p53 is a common chemotherapy target and can have personally designed drugs to activate p53 w/o DNA damage
clinical manifestations of Von Hippel-Lindau (VHL) disease
autosomal dominant;
genetic testing can diagnose
high penetrance by age 65
high variability in severity/onset
characterized by formation of cystic and highly vascularized tumors in many organs
major cause of death are metastatic RCC and CNS hemangioblastomas
-cerebellar and spinal cord hemangioblastomas
retinal hemangioblastomas
bilateral kidney cysts clear cell renal carcinomas (RCC)
pheochromocytomas- usually adrenal gland (night sweats; high bp; heart palpitations; panic attacks)
pancreatic cysts and tumors
endolymphatic sac (inner ear) tumors
cystadenomas of genitourinary tract
classification of VHL
based on presence or absence of pheochromocytoma and type of VHL mutation
Type 1: hemangioblastoma + clear cell renal carcinoma
(due to total/partial loos of VHL- impartial folding)
Type 2: pheochromocytoma +/- hemangioblastoma +/- RCC
different combos of Type 2 (due to VHL missence mutation)
molecular basis of VHL
VHL is a tumor suppressor gene located on 3p25-26
VHL protein is part of a complex that targets proteins for proteosomal degradation via ubiquitination
VHL loss/inactivation leads to HIF accumulation, a high rate of aberrant aneuploidy, and disruption of primary cilia maintenance (leads to renal cysts and renal cell carcinoma)
VHL-HIF protein interactions and oxygen condidtions
interactions are dependent on oxygen conditions
Normoxic conditions: HIF is hydroxylated. With WT VHL, HIF is ubiquitinated by VHL protein and undergoes degradation
Hypoxic conditions: HIF doesn’t get hydroxylated and isn’t marked for degradation. HIF protein accumulates and the transcription of downstream genes that are involved in angiogenesis, metabolism, apoptosis, and other cancer-growth promoting processes and survival under low O2 conditions
**Cells w/ mutated VHL act as if they’re hypoxic
pathogenesis of clear cell renal carcinoma
Clear cell renal cell carcinoma (ccRCC) is the most common histologic subtype of RCC
Majority of cases are sporadic (4% inherited)
-VHL loss/mutation is responsible for almost 2/3 of sporadic cases
Knudson’s TWO HIT theory is that the dev of VHL-related tumors requires inactivation of the 2 copies of the VHL gene
VHL disease- patient already inherited 1 VHL gene mutation, so they only need one more hit for tumor
Patients w/ VHL are at risk to develop up to 600 tumors on kidneys
rationale for therapies treating RCC
Management of local renal cell carcinoma typically involves surgical resection w/ either partial or radical nephrectomy
Management for metastatic renal cell carcinoma involves systemic therapy
–vascular endothelial GFR (VEGF-R) tyrosine kinase inhibitors, MTOR inhibitors, and immunotherapies
localized RCC- ongoing surveillance- 20-30% will relapse
metastatic RCC- surgery, radiotherapy, systemic therapy
molecular components of a membrane:
lipid bilayers
composed of lipids, cholesterol, and proteins
~5nm thick
serve as barriers to most water-soluble molecs
dynamic and fluid (depends on composition and temp)
membrane proteins mediate most of membrane func
concept of membrane fluidity:
the degree to which lateral motion is possible among adj phospholipids on a given side of the bilayer.
Some lipids are anchored, some are free-floating within a given membrane.
different compartments of a membrane have different degrees of fluidity.
ex. acyl chains unsaturated makes the membrane more fluid
ex. Cholesterol, when intercalated in membranes, stiffens the membrane and makes it less fluid (Thickens the membrane by straightening out non-polar, hydrophobic groups inside the bilayer)
ex. Membrane composition also determines curvature (size of polar heads and temp)
identify parts of phospholipid
sphingolipid
cholesterol
Phosphoglycerides: have a glycerol-3-phosphate backbone with 2 fatty acyl chains (either saturated or mono- or polyunsaturated) at one end and a polar head group at the other end. Head group determines which phosphatidylglycerol it is
Sphingolipids: have a ‘sphingosine’ (long acyl unit) with either of two polar head groups (which one it is distinguishes which type of sphingolipid it is) and an attached fatty acid chain.
ex. Sphingomyelin: phosphocholine head group.
These are started to be made in the ER and finished in the Golgi apparatus.
Cholesterol: have a characteristic hydroxylated ring structure at one end, and a fatty acid chain on the other.
describe asymmetry of bilayers
particular phospholipids don’t flip from one side to another w/o enzyme activity, but lots of lateral diffusion occurs
Phosphatidylserine, phosphatidylthanolamine, and phosphatiddyinositol are more abundant on internal surface
PC, sphingomyelin, and glycolipids are more abundant on external surface
Cholesterol is thought to be distributed evenly across both membranes
2 ways proteins associate with membranes
Integral proteins (fully or partially embedded in membrane)- have multiple transmembrane domains (can go back and forth through the membrane)
Peripheral membrane proteins- covalently interacting with proteins bound in the membrane, but not themselves embedded in the membrane
obtaining cholesterol- uptake and synthesis
via ingestion and uptake or synthesis by the liver.
Uptake depends on Low-density lipoprotein receptor LDLR
-Negative feedback loop- sufficient cholesterol in the diet decreases synthesis and vice versa
-Sterol regulatory element binding protein (SREBP) regulates both uptake and synthesis of cholesterol
(SREBP- A protein containing a transcription factor that regulates both LDLR and all 30 synthesis proteins)
cholesterol synthesis
depends on approx 30 enzymes
First and rate-limiting enzyme is HMGCoA reductase
- Statins block this step and are used to lower cholesterol
- Low cholesterol- transcription factor is cleaved by SREBP to go to nucleus to activate genes
- -Sensor is in ER membrane, where cholesterol is lowest and changes are easiest to detect
- -Held in ER until cholesterol becomes low, then SREBP moves to Golgi to get cleaved
3 protein complex
- SREBP
- SCAP (SREBP cleavage activating protein) binds both SREBP and sterols like cholesterol
- Insig- binds SCAP when cholesterol is high, blocks SCAP’s signaling
- -SCAP signal domain is recognized by a coat protein COPII for vesicles to move from ER to Golgi
- -As cholesterol conc drops, Insig doesn’t bind SCAP, so SCAP/SREBP moves to Golgi in vesicles
typical volumes of plasma, ECF, and ICF
45 L total plasma- 3 ICF- 27 ECF- 13 + 5 for third space (incl interstitial fluid, plasma, lymph)
ICF and ECF composition- mM and membrane permeability Na+ K+ Cl- (and CO3-) proteins water
Na+
14 ICF; 140 ECF; (-)
K+
145 ICF; 5 ECF; +
Cl-
5 ICF; 145 ECF; +
proteins-
126 ICF; ~0 ECF; -
water
~55,000 ICF = ECF; +
2 most important functional properties of membranes
Lipid-
“impermeable to charge” and strong polarity
Electrically strong; membrane potential -50mV across 5 nm membrane = -100,000 V/cm
Ion channels and transporter proteins
Channels- ions and water
-Selective
-Gated- temperature; mech; chem; electrical
Transporters
-Big molecules
-Pump (move against gradient with an E source- ATP)
3 routes by which a substance can traverse a membrane
Diffusion- passive movement; small and neutral charged
Channels- selective and gated
Transporters- big molecules, sometimes via pumps
4 physical forces that can determine the gating properties of ion channels
temp
mechanical
chemical
electrical
determine direction an uncharged sub will move across a membrane
according to conc gradient
high to low
determine whether a cell will swell or shrink
look at osmotic pressure in and outside of cell
inside the cell:
high osM= cell swells
low osM= cell shrinks
assume ECF is constant and only water can change cell volume; ignore permeable substances
3 mechanisms that different cells have evolved to keep from swelling and bursting
Make a cell impermeable to water
Build a strong wall around the cell to keep it from swelling by brute force
Balance osmotic force osmotically
which equation to determine which direction water will move across a semi-permeable membrane
Pi= sigmaRT*detaC
sigma= reflection coeff R= gas constant deltaC= difference in solute conc across a membrane
osmosis vs diffusion:
osmosis is diffusion of water across a semipermeable membrane
what substance must move to change a cell’s volume
WATER
reflection coefficient and permeabilities
Reflection coefficient of 1: non-permeable
Reflection coefficient of 0: same as water
A different reflection coefficient complicates matters. A molec that crosses half as easily as water will exert half of the ideal osmotic P of a non-permeating solute.
Clinical implications- shock involves having blood go to extremities, and not to brain. You give them an IV of NaCl because it is non-permeable. Clysis- give fluid subcutaneously if you can’t find a vein. Glucose is less permeable, which temporarily sucks water out of cells before it gets into the cells and starts helping them absorb water.
define osmolarity, molarity, and equivalents
Osmolarity- total conc of solute particles
Ex. 1 M soln of CaCl2 gives 3osM soln
Molarity- number of moles of solute per L soln
Equivalents- number of “combining weights” of an ion per liter, calculated by a 2-step process
ex. For each ion, convert to mosM; multiply mosM by the valence of the ion
define tonicity
effect of a soln on a cell; depends on membrane permeability
Hypertonic- soln that makes cell shrink
Hypotonic- soln that makes cell swell and burst
Isotonic- same
importance of sub-cellular protein targeting
- Cell needs to move big proteins without compromising membrane integrity
a. Thus, transport vesicles (contains cargo targeted for specific membrane-bound organelles) - Vesicular transport- basic principle of transporting substances from one intracellular compartment to another
- Incorrect transport frequently results in disease
a. Ex. Cystic fibrosis, due to a failure in chloride ion channel-protein transport
basic principles of membrane fusion
- Membranes don’t automatically merge when they contact e/o; maintain separation
a. 2 membranes coated in water molecs
i. Need to remove water to fuse the lipids
ii. Overcome charge repulsion between lipids in order to allow merging efficiency
iii. Membrane fusion specificity- make sure these should be merging to begin with
func and structure of SNARE proteins
a. 3 shapes:
i. VAMPs: transmembrane domain at one end, with a helical domain in it
ii. Syntaxin: transmembrane domain, one helical domain
iii. SNAP25: no transmembrane domain, 2 helical domains, fatty acid binding region that more or less acts as a membrane-binding domain
1. Notice different types of each protein, and each only binds to specific counterparts
2. Helical domains of all 3 proteins serve to bind with each other- form an aligned bundle or tetramer with coiled coils
3. All vesicles contain VAMPs. All target plasma membranes contain syntaxin and SNAP25
4. When the vesicle nears the target membrane, the helical domains on the vesicle bind to those on the target membrane
a. These effectively press 2 lipid layers together, squeeze out water, overcome charge repulsion, and promote lipid fusion
b. Helical binding needs to be extremely strong to do this (and it is)
regulation of SNARE-based fusion
a. Enzyme that regulates the dissociation of SNARE proteins: NSF protein
i. 6 form a turning barrel around the SNARE complex and twists it apart, using ATP hydrolysis
ii. Alpha snap is a cofactor; binds with NSF
b. After being unwound, SNARE syntaxin becomes unfolded/denatured
i. N-Sec1 refolds it properly and primes it for fusion, but also stays bound and inactivates formation of SNARE complex
ii. N-Sec1 needs to be removed to activate syntaxin
1. Syntaxin w/ n-sec1 can create that same (but inactive) tetramer core complex on its own (2 SNAP25, 1 VAMP, 1 Syntaxin)
2. Ca removes N-Sec1 efficiently (ex. In neurotransmitters)
c. SNARE cycle: VAMP on vesicle, syntaxin and SNAP25 on target membrane, helical binding and lipid fusion, NSF-mediated unwinding, refolding of syntaxin with Sec1
mechanism of viral fusion
a. Enveloped viruses (ex. HIV, ebola, influenza viruses) also need to go through membrane fusion to infect cells
b. Viruses have a fusion machinery similar to SNARE fusion
i. Only 1 protein folded over on itself antiparallel
ii. Still achieves efficiency and specificity (2 objectives of fusion)
iii. (ex. Influenza virus) Achieved by help of special viral fusion protein
1. Protein contains a transmembrane domain at one end (inserted into viral membrane) and a highly hydrophobic fusion domain
a. Normally fusion domain is folded/hidden within viral protein
b. Upon signaling (influenza- pH change), the fusion domain is exposed and inserted in target cell membrane
c. Fusion proteins immediately refold and causes viral and cellular membrane fusion
regulation of viral fusion
- Regulation of viral fusion
a. Signaling- like low pH
i. Influenza is internalized via endocytosis, and is activated in lysosome with drop in pH
b. Binding to CD4- activates HIV
c. HIV also has a pocket domain that can be targeted via drugs
d. Blocking fusion will block transmission
relative strengths of electric and osmotic forces
- Electric forces are MUCH stronger than osmotic forces
a. so relatively few excess ions are needed to counter large conc differences
2 forces acting on an ion moving across a membrane
- Electrochemical gradient
a. Chemical gradient (conc difference)
b. Electrical potential differences (membrane potential from cation/anion imbalance)
define equilibrium potential
- Equil pot relates the conc grad to the electrical force and is the electrical potential diff across the membrane that must exist if the ion is to be at equilibrium at the given conc
a. Specific to ONE ion - If membrane and equil potentials are equal, the ion is at electrochemical equilibrium. Vm=Eion. If not:
a. Membrane is impermeable to that ion or
b. Ion is being pumped across the membrane
equilibrium potential vs recorded membrane potential
- Membrane pot is a measure of the real difference in voltage between the internal environment of the cell and the ECF
- Equilibrium pot is what the voltage diff would be IF a given ion was in equilibrium given a particular ICF/ECF ratio
recognize that each and every ion species has its own, independent equilibrium pot
- Equilibrium pot can be calculated for each individual ion, while the cell will still have only 1 membrane potential
a. Comparison of these 2 indicates whether or not a pump must exist for the ion and which direction it’s pumping
determine if a pump exists for an ion and which way it pumps
- Look at conc in vs conc out- determine which way the ion would naturally move
- Look at charge of ion vs membrane potential- determine which way it would naturally move
- If these are the same direction- must be a pump moving ions across the gradients
- If these are different directions- must compare the equilibrium pot to the membrane potential:
a. If they are equal- there is no pump required
b. If they are different- a pump exists
i. Direction of pump: consider which way the ion movement would move to make Vm closer to E - Pump pushes ions in the opposite direction
number of excess anions vs total number of ions
- Ex. In a typical cell w/ RMP of -80mV: for every 100,000 cations, there are approx. 100,001 anions
a. This gives an indication of just how strong electric force is
bulk solutions are always electrically neutral-
principle of Electric Neutrality
- Principle of Electrical Neutrality- [+]out= [-]out and in=in.
a. Seems contradictory to membrane potentials arising from excess ions
b. Electric force is so powerful that excess number of ions is very small compared to total # ions present in cell
describe cell’s state of equilibrium
- The specific ions don’t have to be equal inside and out
- Cell just must follow Donnan rule: products of ion conc inside must equal product of ion concs outside the cell
a. Only relevant to counter ions (K+ and Cl-) - Nernst equation- at equilibrium when electric and chem gradients are in agreement?
apply osmotic balance, charge neutrality, and Nernst equation to calculate ion conc and membrane potential
- Osmotic balance: mosM inside and out will be equal
- Charge neutrality: can assume equal number of cations and anions inside the cell (and also outside) but not in=out
- Nernst equation: E (mV)=-60/z * log(conc out/conc in)
- If in a steady state with no pumps acting on a cell, Vm=E
describe one cycle of sodium pump
- Each cycle: 3 sodium ions are pumped out from ICF to ECF and 2 potassium ions are pumped into the cell ECF to ICF
a. Pump action requires ATP
equilibrium vs steady state
- Real cells are often in steady state
a. conc’s aren’t changing, but a constant input of E is needed to maintain this (ATP needed to drive Na/K pump)
describe how membrane pot depends on relative, not aboslute, permeabilities to ions
- Dynamic environment
- Depends on RATIO of permeabilities (number of channels)
- Permeability changes to reach new steady states
describe how primary short term determinant of membrane pot is not the Na/K pump, but relative membrane permeabilities to different ions
- a cell with more Na channels than K channels (for ex) will have a different membrane potential than vice versa
- larger cells with more channels are more vulnerable, quicker, to Na/K pump failure than others
define driving force of an ion
- Driving force= at any instant, the difference between Vm and Eion
describe why membrane pot is sensitive to small changes in K+ out, but not Na out
- Membrane pot is already close to E of K+
- Loss of EC Na+ (bringing E of Na+ closer to 0) makes the Vm slightly hyperpolarized (closer to -80mV) but no other significant effect
- Cell is much more permeable to K+ than Na+
- Sensitivity to K conc is higher because its starting conc outside the cell is much smaller than Na+,
a. So it’s much more sensitive to small changes in conc
law of mass action
- Keq= [products]/[reactants]
define pH and pKa
- pH is a measure of the acidity or basicity of a soln
a. pH=-log[H+] - pKa is a measure of the strength of an acid or base by its propensity to donate or accept protons
a. pKa= -logKa
b. Ka= [H+][A-]/[HA]
c. lower pKa= large Ka = more dissociation= stronger acid
Henderson-Hasselbalch Equation
weak acid/base
pH= pKa + log [A-]/[HA]
shows extent of dissociation
pH=pKa when something is 50% dissociated
pH lower than pKa= protonated; pH greater than pKa= deprotoated
