MCB Review Exam II

1. MCB Review Exam II Ji Woong Park

2. 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.

3. 3 Na ATP ADP + Pi 2 K Primary Active Transport:Na,K-ATPase • 3 Na outward / 2 K inward / 1 ATP • Km values: Nain ≈ 20 mMKout ≈ 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

4. Cells washed in isotonic buffered solution Required transport inhibitor(s) added Flux medium containing radioactive isotope added At required times flux medium rapidly removed and cells washed (3-4 x) in ice-cold isotonic saline Final wash removed, cells lysed and radioactivity and protein content of samples determined Unidirectional Transport Assays Cells growing in multi-well plates

5. LIMITING JUNCTION/TIGHT JUNCTION

6. CLAUDINS

7. “GATING” OF CONNEXONS

8. Aquaporins- ADH Stimulated Water Permeability

9. SORTING SIGNALS

10. Selectivity filter diversity

11. Ion gradients and membrane potential Na 117 K 3 Cl 120 Anions 0 Total 240 Na 30 K 90 Cl 4 Anions 116 Total 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

13. The Goldman Hodgkin Katz Equation • 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

16. 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

17. 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

18. 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

19. 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

20. Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides Measure the masses of the fragment ions. Slide courtesy of Andrew Link

21. 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, 2006

22. Studying EGFR Signal Transduction with Quantitative Proteomics Introduce Stable Isotope by Chemical Labeling Zhang et al., MCP 4: 1240-50, 2005

23. 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% 80% 60% Patient Survival 40% 20% 0% Time (months)

24. 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

25. Drugs to Target Receptor Tyrosine Kinases Monoclonal Antibodies Extracellular domain HER2 HER2 HER2 EGFR Tyrosine- kinase domains ATP-mimetic Tyrosine Kinase Inhibitors Homodimer Heterodimer

26. 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. Retinoic Acid Receptor α PML Retinoic Acid Binding DNA Binding ATRA

27. GPCR signaling Controls Blood Pressure via the Renin-Angiotensin System Angiotensinogen Renin (kidney) Common Blood Pressure Medicines ACE inhibitors Angiotensin I T ACE (lung) (Angiotensin Converting Enzyme) Angiotensin II Angiotensin Receptor Blockers T Angiotensin II Receptor (GPCR)

28. Erythropoietin (EPO) binds to a Cytokine Receptor EPO receptor JAK2 Tyr Kinase Nucleus STAT5 STAT5 Munugalavadla and Kapur, Reviews in Onc-Hem, 2005  DNA

29. 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.

30. Good Luck!