MCB Review Exam II
Ji Woong Park
Logistics
• This review covers lectures by Dr. Mercer, Nichols,
and first and third lecture by Dr. Bose.
• It accounts for 60/100 points in the second exam.
• Be sure to not just study but be able to answer
2013 Exam II (at least for my section) – you will
thank me on Tuesday.
• In addition, understand everything on this review
slide as well.
• There will be NO math problem on this exam.
Primary Active Transport: Na,K-ATPase
3 Na
ATP
ADP + Pi
2K
•
•
•
•
•
•
•
3 Na outward / 2 K inward / 1 ATP
Km values: Nain ≈ 20 mM Kout ≈ 2 mM
Inhibited by digitalis and ouabain
Palytoxin “opens” ion channel
2 subunits, beta and alpha (the pump)
Two major conformations E1 & E2
Turnover = 300 Na+ / sec / pump site @ 37 °C
Unidirectional Transport Assays
1. Cells washed in isotonic buffered solution
2. Required transport inhibitor(s) added
3. Flux medium containing radioactive isotope
added
Cells growing
in multi-well plates
4. At required times flux medium rapidly removed
and cells washed (3-4 x) in ice-cold isotonic
saline
5. Final wash removed, cells lysed and
radioactivity and protein content of samples
determined
LIMITING JUNCTION/TIGHT JUNCTION
CLAUDINS
“GATING” OF CONNEXONS
Aquaporins- ADH Stimulated Water
Permeability
SORTING SIGNALS
Selectivity filter diversity
10
Ion gradients and membrane potential
Na 117
K
3
Cl 120
Anions 0
Total 240
[+ charge] = [- charge]
0 mV
11
Na 30
K
90
Cl
4
Anions 116
Total
240
[+ charge] = [- charge]
-89 mV
How does this membrane potential come about?
12
At Electrochemical Equilibrium:



The concentration gradient for the ion is
exactly balanced by the electrical gradient
There is no net flux of the ion
There is no requirement for any energydriven pump to maintain the concentration
gradient
13
The Goldman Hodgkin Katz Equation
 PK [K]out  PNa  [Na]out  PCl  [Cl] in 

Vm  60mV  log 
 PK  [K]in  PNa  [Na]in  PCl  [Cl] out 




Resting Vm depends on the concentration gradients and on the
relative permeabilities to Na, K and Cl. The Nernst Potential
for an ion does not depend on membrane permeability to that
ion.
The GHK equation describes a steady-state condition, not
electrochemical equilibrium.
There is net flux of individual ions, but no net charge
movement.
The cell must supply energy to maintain its ionic gradients.
14
Currents During an Action Potential
Time Course of Currents
15
Sodium Channel Gating States
Identifying a Protein by Mass Spectrometry on
Its Tryptic Peptides
Trypsin – a protease that cleaves after basic residues (R
or K).
Protein of
Interest:
Slide courtesy of Andrew Link
Identifying a Protein by Mass Spectrometry on
Its Tryptic Peptides
Products from Trypsin digest.
Average length of tryptic
peptides = 10 aa residues
Slide courtesy of Andrew Link
Identifying a Protein by Mass Spectrometry on
Its Tryptic Peptides
Select an Individual Peptide in the Mass Spectrometer
Performed by adjusting the electrical fields
in the mass spectrometer.
Slide courtesy of Andrew Link
Identifying a Protein by Mass Spectrometry on
Its Tryptic Peptides
Impart energy to the peptide by colliding it with an inert gas
(Argon or Helium).
Slide courtesy of Andrew Link
Identifying a Protein by Mass Spectrometry on
Its Tryptic Peptides
Measure the masses of the fragment ions.
Slide courtesy of Andrew Link
Protein Quantitation with Mass Spectrometry
Introduce Stable Isotope by Metabolic Labeling
Control
Treatment 1
Treatment 2
Mix Lysates
Fractionate Proteins on SDS-PAGE
Digest Bands with Trypsin
Identify and Quantify Proteins by Mass Spec
Bose et al., PNAS 103: 9773-8,
Studying EGFR Signal Transduction with
Quantitative Proteomics
Introduce Stable Isotope by Chemical Labeling
Zhang et al., MCP 4: 1240-50,
Her2/neu and Breast Cancer
• 1987 – Southern blots of
genomic DNA from breast
cancer patients shows Her2
gene amplification.
–
–
–
–
Patient Survival
100%
Sample 3 & 4: normal level
Sample 1 & 2: 2-5 x normal
Sample 6 & 26: >5 x normal
Sample 18: > 20 x normal
• Correlation between Her2
gene copy number and patient
survival
80%
60%
40%
20%
Slamon, et al., Science 1987
0%
Time (months)
Her2/neu and Breast Cancer
• Transgenic mice bearing the
MMTV-Her2/neu construct
develop breast cancer in all 5
pairs of mouse mammary
glands.
• Tumor formation with Her2 in
this tg model is more rapid
than with the Myc oncogene.
Muller et al., Cell 1988
Drugs to Target Receptor Tyrosine Kinases
Monoclonal Antibodies
Extracellular
domain
EGFR
HER2
HER2
HER2
Tyrosinekinase
domains
ATP-mimetic
Tyrosine Kinase Inhibitors
Homodimer
Heterodimer
The RARα Nuclear Hormone Receptor in Acute
Promyelocytic Leukemia (APL)
Retinoic Acid
Receptor α
Retinoic
Acid
Binding
PML
DNA
Binding
ATRA
• APL has a characteristic
translocation 15;17 that forms
the PML-RARα fusion protein.
• Retinoic Acid (RA) binding
converts PML-RARα from a
transcriptional repressor to a
transcriptional activator.
• All-trans retinoic acid (ATRA)
has made APL the most
treatable and best prognosis
form of adult acute leukemia.
GPCR signaling Controls Blood Pressure via
the Renin-Angiotensin System
Angiotensinogen
Renin
(kidney)
Common Blood Pressure Medicines
Angiotensin I
ACE inhibitors
ACE (lung)
(Angiotensin Converting Enzyme)
Angiotensin II
Angiotensin Receptor Blockers
Angiotensin II Receptor (GPCR)
Erythropoietin (EPO) binds to a Cytokine
Receptor
EPO receptor
JAK2 Tyr
Kinase
Nucleus
STAT5
STAT5
Munugalavadla and Kapur,
Reviews in Onc-Hem, 2005
DNA
EPO Deficiency causes
Anemia of Chronic Kidney Disease
EPO
Bone
Marrow
Kidney
Increased Red Blood
Cell Production
Chronic kidney disease causes a fall in EPO secretion and this results in
decreased red blood cell production (i.e.- anemia). Therefore patients with chronic
kidney disease are given recombinant EPO to prevent anemia.
Good Luck!