1. CFTR – Where It Is Found and What It SHOULD Do Barbara E. Goodman, Ph.D. Respiratory Membrane Transport Physiologist

2. Welsh, M.J. & A.E. Smith. Scientific American pp. 52-59, 1995. CFTR Protein Normal function of CFTR is to allow Cl - transport across cell membranes and to regulate transport of other ions via interactions with their transport proteins

3. Cystic Pancreas In 1936, Dr. G. Fanconi referred to disease as “cystic fibromatosis with bronchiectasis”. In 1938, Dr. Dorothy Anderson named the disease “cystic fibrosis of the pancreas” based on similar tissue.

4. ACINUS: NaCl secretion (with water following) DUCT: NaCl reabsorption (with water following) Junquiera, L.C. & J. Carnero, Basic Histology, McGraw-Hill Companies, 2003. Production of Sweat

5. Reddy, M.M. & P.M. Quinton, American J. Physiology 257:C722-C726, 1989. Ductal Cells in Sweat Glands CELL FROM NORMALCELL FROM CF PATIENT

6. Sweat Chloride Test

7. CF Problems with Sweat Glands Secretion of NaCl in sweat is normal Secretion of NaCl in sweat is normal Reabsorption of Cl - (and accompanying Na + ) out of sweat ducts is low Reabsorption of Cl - (and accompanying Na + ) out of sweat ducts is low Final sweat is high in salt Final sweat is high in salt

8. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Hooper, Celia. Journal of NIH Research 1:76-81, 1989.

9. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

10. Sheppard, D.N. & M.J. Welsh, Physiological Reviews 179:S23-S45, 1999. Model of CFTR

11. Sheppard, D.N. & M.J. Welsh, Physiological Reviews 179:S23-S45, 1999. Model of ATP-dependent Gating of CFTR

12. Regulation of Normal CFTR In absence of R domain phosphorylation – channel is closed In absence of R domain phosphorylation – channel is closed Cyclic AMP stimulates PKA to phosphorylate serines on R domain Cyclic AMP stimulates PKA to phosphorylate serines on R domain CFTR binds ATP at NBFs and conformational change occurs CFTR binds ATP at NBFs and conformational change occurs Open probability and voltage-dependence may depend on tyrosine phosphorylation Open probability and voltage-dependence may depend on tyrosine phosphorylation

13. McCarty, N.A. J. Experimental Biology 203:1947-1962, 2000. 3-D Model of Pore in CFTR

14. Different Classes of Mutations in CF Ashcroft, F.M. Ion Channels and Disease: Channelopathies, Academic Press, 2000.

15. Four Classes of CF Mutations Defective production of CFTR so final product non-functional Defective production of CFTR so final product non-functional Defective processing of CFTR so does not reach correct location in cell (  F508) Defective processing of CFTR so does not reach correct location in cell (  F508) Defective regulation of CFTR (NBF or R domain) Defective regulation of CFTR (NBF or R domain) Defective conduction through CFTR (pore size inhibited) Defective conduction through CFTR (pore size inhibited) First two generally have pancreatic insufficiency and are more serious

16. CFTR is absent or defective Highly salty sweat Highly salty sweat Plugging of bile ducts disrupts liver function Plugging of bile ducts disrupts liver function Occlusion of pancreatic ducts and lack of HCO 3 - causes pancreatic insufficiency Occlusion of pancreatic ducts and lack of HCO 3 - causes pancreatic insufficiency Lack of Cl - secretion leads to intestinal obstruction Lack of Cl - secretion leads to intestinal obstruction Either developmental or physical sterility in males and females Either developmental or physical sterility in males and females

17. Knowles, M.R. & P.R. Durie. New England J of Medicine 347: 439-442, 2002.

18. RECEPTORS AND SECOND MESSENGER SYSTEMS Cl - CFTR CFTR 3Na + 2K + K+K+ Na + 2Cl - PROSTAGLANDINS ACETYLCHOLINE VASOACTIVE INTESTINAL POLYPEPTIDE IONIC MECHANISMS cAMP Ca 2+ Intestinal Crypt Cells W. H. Percy, University of South Dakota

19. CF and Intestinal Crypt Cells Normal stimulation of secretion by various chemicals is blunted Normal stimulation of secretion by various chemicals is blunted Less salt and less water in the intestinal lumen Less salt and less water in the intestinal lumen Constipation or obstruction Constipation or obstruction

20. DUCT LUMEN Pancreatic Duct Cell PLASMA cAMP Cl - CO 2 H2OH2O + HCO 3 - Cl - CFTR SECRETIN W. H. Percy, University of South Dakota

21. CF and Pancreatic Duct Cells Less Cl - secretion into lumen Less Cl - secretion into lumen Cl - buildup inside cell leads to less HCO 3 - secretion into lumen Cl - buildup inside cell leads to less HCO 3 - secretion into lumen Disruption of enzyme production and flow by exocrine pancreas Disruption of enzyme production and flow by exocrine pancreas Pancreatic insufficiency and/or pancreatitis and/or diabetes mellitus Pancreatic insufficiency and/or pancreatitis and/or diabetes mellitus

22. Journal of NIH Research 3: 40-44, 1991. Child with Cystic Fibrosis Receives Chest Physiotherapy

23. Welsh, M.J. & A.E. Smith. Scientific American pp. 52-59, 1995. Absorptive Cells of the Upper Airways

24. Welsh, M.J. & A.E. Smith. Scientific American pp. 52-59, 1995. Secretory Cells of the Upper Airways

25. Kunzelmann, K. News in Physiological Sciences 16:167-170, 2001. Airway Epithelia in Normal and CF Patients– Absorption vs. Secretion Airway Epithelia in Normal and CF Patients – Absorption vs. Secretion

26. RECEPTORS AND SECOND MESSENGER SYSTEMS BasolateralBasolateral Cl - 3Na + 2K + K+K+ Na + 2Cl - IONIC MECHANISMS cAMP Ca 2+ Lumen Cl - Na + H+H+ P2P2 K+K+ Airway Epithelial Cells CFTR CFTR W. H. Percy, University of South Dakota

27. CF and Lung Airways Decreased Cl - secretion into lumen Decreased Cl - secretion into lumen Less water in lumen Less water in lumen Airway mucus less dilute, thick, sticky, tenacious Airway mucus less dilute, thick, sticky, tenacious Culture medium for recurrent respiratory infections and inflammatory response Culture medium for recurrent respiratory infections and inflammatory response Physical and chemical components of airway fluid Physical and chemical components of airway fluid Bronchiectasis and eventual lung failure Bronchiectasis and eventual lung failure

28. Schwiebert, E.M. et al., Physiological Reviews 79: S145-S166, 1999. CFTR Regulates Other Chloride Channels

29. Schwiebert, E.M. et al., Physiological Reviews 79: S145-S166, 1999. Hypotheses for how CFTR Facilitates Transport/Release of ATP out of Cells

30. Hypotheses for how CFTR Regulates Sodium Channels Schwiebert, E.M. et al., Physiological Reviews 79: S145-S166, 1999.

31. Kunzelmann, K. News in Physiological Sciences 16:167-170, 2001. Postulated Role of PDZ Domains in Regulating CFTR/ENaC Activity

32. Schwiebert, E.M. et al., Physiological Reviews 79: S145-S166, 1999. Hypotheses for how CFTR Regulates Potassium Channels

33. Transport in Chloride Secretory Epithelial Cells Schultz, B.D. et al. Physiological Reviews 79:109-144, 1999.

34. Basic Goals of CF Research Fighting chronic lung infections Fighting chronic lung infections Thinning dangerously thick airway mucus Thinning dangerously thick airway mucus Reducing inflammatory response which leads to tissue destruction Reducing inflammatory response which leads to tissue destruction Correct the basic cellular defect Correct the basic cellular defect

35. Therapeutic Approaches to CF for Abnormal Gene Genetic mutation  Aerosolized AAV  Liposomes  Receptor-mediated  Gene transfer  Gentamicin Preston Campbell, Cystic Fibrosis Foundation, 15 th Annual North American CF Conference, 2001.

36. Therapeutic Approaches to CF for Abnormal Protein Protein rescue/activation  Phenylbutyrate  Genistein  7,8-benzoflavones  CPX Preston Campbell, Cystic Fibrosis Foundation, 15th Annual North American CF Conference, 2001.

37. Therapeutic Approaches to CF for Altered Ion Transport and Abnormal Mucus Proper ion transport  INS37217  Moli1901 Preston Campbell, Cystic Fibrosis Foundation, 15th Annual North American CF Conference, 2001.

38. Therapeutic Approaches to CF for Infection & Inflammation Tissue Destruction Anti-infective  TOBI, etc. Anti-inflammatory  Protease inhibitors, etc. Preston Campbell, Cystic Fibrosis Foundation, 15th Annual North American CF Conference, 2001.

39. Therapeutic Approaches to CF for Organ Destruction and Respiratory Failure Transplantation Preston Campbell, Cystic Fibrosis Foundation, 15th Annual North American CF Conference, 2001.

40. Therapeutic Approaches to CF Abnormal gene Abnormal protein Altered ion transport Infection & inflammation Respiratory failure Preston Campbell, Cystic Fibrosis Foundation, 15th Annual North American CF Conference, 2001.

41. Airway Epithelial Cell (ATP or UTP - - P2u agonists) CFTR NUCLEUS RIBOSOME mRNA GOLGI ER P P P (synthetic chaperones) PKA + cAMP PHOSPHATASES ENaC Na Cl Cl (phosphatase inhibitors) (phosphodiesterase inhibitors) (amiloride – sodium channel blocker) (aminoglycosides) CFTR CFTR W. H. Percy, University of South Dakota (Cl - channel activators) (Genetic engineering – restore CFTR)

42. Airway Epithelial Cell CFTR NUCLEUS RIBOSOME mRNA GOLGI ER P P P ENaC Na ClClClCl Cl CFTR CFTR W. H. Percy, University of South Dakota depolymerize DNA in mucus -- rhDNase infection/inflammation: inhaled antibiotics α 1 -antitrypsin secretory leukoprotease inhibitor sever filamentous actin – h plasma gelsolin rehydrate with aerosols of saline

43. Schöni, M.H. & W.H. Nikolaizik. Schweiz Med. Wochenschr. 127: 158-164, 1997. CF Therapies and How They Work

44. Dawson, D.C. et al. Physiological Reviews 79: 47-75, 1999. CFTR Charged Residues