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 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

3 Na

2 K

ATP

ADP + Pi

Unidirectional Transport Assays

Cells growingin multi-well plates

1. Cells washed in isotonic buffered solution

2. Required transport inhibitor(s) added

3. Flux medium containing radioactive isotope added

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

10

Selectivity filter diversity

11

Ion gradients and membrane potential

Na 117K 3Cl 120Anions 0Total 240

Na 30K 90Cl 4Anions 116Total 240

[+ charge] = [- charge]

0 mV

[+ 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 energy-driven pump to maintain the concentration gradient

13The Goldman Hodgkin Katz Equation

outClinNainK

inCloutNaoutKm [Cl]P[Na]P[K]P

[Cl]P[Na]P[K]Plog60mVV

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.

14Currents During an Action Potential

Time Course of Currents

15

Sodium Channel Gating States

Trypsin – a protease that cleaves after basic residues (R or K).

Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides

Slide courtesy of Andrew Link

Protein of Interest:

Products from Trypsin digest.

Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides

Slide courtesy of Andrew Link

Average length of tryptic peptides = 10 aa residues

Select an Individual Peptide in the Mass Spectrometer

Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides

Slide courtesy of Andrew Link

Performed by adjusting the electrical fields in the mass spectrometer.

Impart energy to the peptide by colliding it with an inert gas (Argon or Helium).

Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides

Slide courtesy of Andrew Link

Measure the masses of the fragment ions.

Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides

Slide courtesy of Andrew Link

Control

Mix Lysates

Identify and Quantify Proteins by Mass Spec

Fractionate Proteins on SDS-PAGEDigest Bands with Trypsin

Treatment 1 Treatment 2

Protein Quantitation with Mass Spectrometry

Bose et al., PNAS 103: 9773-8, 2006

Introduce Stable Isotope by Metabolic Labeling

Studying EGFR Signal Transduction with Quantitative Proteomics

Introduce Stable Isotope by Chemical Labeling

Zhang et al., MCP 4: 1240-50, 2005

Her2/neu and Breast Cancer

• 1987 – Southern blots of genomic DNA from breast cancer patients shows Her2 gene amplification. – 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

Slamon, et al., Science 1987

100%

Time (months)

Patie

nt S

urvi

val

80%

60%

40%

20%

0%

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

HE

R2

EG

FR

HE

R2

HE

R2

Homodimer Heterodimer

Extracellulardomain

Tyrosine-kinasedomains

Monoclonal Antibodies

ATP-mimetic Tyrosine Kinase Inhibitors

The RARα Nuclear Hormone Receptor in Acute Promyelocytic Leukemia (APL)

• 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.

PML Retinoic Acid

Binding

DNA Binding

Retinoic Acid Receptor α

ATRA

GPCR signaling Controls Blood Pressure via the Renin-Angiotensin System

Angiotensinogen

Angiotensin I

Angiotensin II

Renin (kidney)

ACE (lung)(Angiotensin Converting Enzyme)

Angiotensin II Receptor (GPCR)

TACE inhibitors

TAngiotensin Receptor Blockers

Common Blood Pressure Medicines

Erythropoietin (EPO) binds to a Cytokine Receptor

Nucleus

STAT5

STAT5

DNA

JAK2 Tyr Kinase

EPO receptor

Munugalavadla and Kapur, Reviews in Onc-Hem, 2005 

EPO Deficiency causes Anemia of Chronic Kidney Disease

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.

Kidney

BoneMarrow

Increased Red Blood Cell Production

EPO

Good Luck!