1“Woe to the child which when kissed on the foreheadtastes salty. He is bewitched and soon will die” - old proverbCystic Fibrosis- lethal autosomal recessive disease- incidence: 1 in 2000-3000; predominantly Caucausian populations (carrier frequency 1 in 22-28)- disease gene CFTR (cystic fibrosis transmembrane conductance regulator) is a regulated epithelial Cl- channel; influences other ion channelsClinical Features of CFaffects epithelia in multiple organs• chronic lung infections and inflammatory destruction of lungs• nutritional abnormalities due to gastrointestinal obstruction and lack of fluid secretion by intestinal cells• pancreatic insufficiency• males infertile due to congenital malformation of vas deferens• excessive salt loss from the sweat glands• ion imbalance, dehydration, cardiac arrhythmiasthick, viscous mucus2none50-100none10-49congenital absence of vas deferens< 10positive sweat test and symptoms below< 5pulmonary infection and below< 4.5pancreatic insufficiency and below< 1clinical features% normal CFTR functionRelationship of clinical features to residual CFTR functionave. life expectancy - male: 31 female: 28- 34% of patients reachadulthood- 10% live past age of 30Lung Physiology - the lung and other airways (nasal, tracheal) contain several different cell types (all play a role in innate immunity against pathogens)Epithelia: polarized cells that form the lining of the airway tissues; they contain cilia that beat in a single, coordinated directionGoblet cells: mucus producing cells; principal component in mucus is mucins, highly glycosylated and negatively charged molecules that help trap pathogensSubmucosal glands: contains both mucus (mucoid) and fluid (serous)-producing cells and ducts that mix and carry the two components to the surface epithelial layer3Overview of the organization of lung cells and their functions* the epithelial lining of tissues in the digestive system share a similar physiologyElectron Micrograph of Lung Epithelial Surface4human airway lining contains two, distinct aqueous layers:1. mucus layer - traps inhaled bacteria and foreign particles2. airway surface liquid (ASL) - provides microenvironmentfor beating cilia to clear mucus layer with assistance of coughingLung Defenses(ASL: rich broth of proteases/antiproteases, antibiotics, antibodies, and oxidant/antioxidants backed by cellular immune mechanisms)Defensins: small peptide molecules (12-50 amino acids, contain positively charged and hydrophobic residues) that kill microbes in a salt-sensitive manner (mechanism unclear; might involve membrane disruption or neutralization of cytosolic factors in pathogens)Non-specific action of defensins makes it difficult for microbes to acquire resistancehigh salt normally inactivates defensinsCF lung disease- lungs are plagued by persistent bacterial infections main culprits: P. aeruginosa, B. cepacia, S. aureus- colonization occurs early and is nearly impossible to eradicate; lung tissue eventually destroyed by onslaught of immune cells(i.e. neutrophils) that respond to the infections5Transmembrane Topology of CFTR and Charge SelectivityArg-352positive charge of Arg = electrostatic barrierCl- to Na+ permeability ratio = 150 (without Arg352 = 15)Domains in CFTR protein- membrane-spanning domains (MSD) - two set of six membrane- spanning segments that anchor protein to the plasma membrane and form the ion channel (represent 19% of the total protein)- nucleotide-binding domains (NBD-1, NBD-2) - responsible for binding and hydrolyzing ATP, control ion channel gating (opening and closing- regulatory domain (R) - responsible for activation of CFTR6Classes of CFTR mutationsClass I - premature termination codons (truncated proteins) and splicingabnormalities (unstable mRNAs) leads to severe reduction in CFTR production; roughly 5% of total mutations fall into Class IClass II - these mutations including the common DF508, deletion of a Phe codon, results in CFTRs that are not folded properly and are degraded in the endoplasmic reticulum; represent large majority of total mutations (75%)Class III - mutations in the nucleotide-binding regions and regulatory domain lead to defective regulation and gating (protein reaches surface of cell but channel remains closed); severe disease seen in patients with these mutations Class IV - mutations in the membrane-spanning regions result in CFTR molecules that are correctly processed but exhibit altered channel functions (unable to move chloride ions through efficiently); result in less severe disease with no pancreaticdysfunctionOnly 50% of the newly-synthesized CFTR is correctly folded and trafficked to the cell surface (large size, etc.)nature of mutations in CFTR correlate well with severity of pancreatic disease and degree of sweat Cl- abnormality (genotype <--> phenotype)relationship between genotype and pulmonary phenotype less robust- genetic modifiers (stronger immunity, etc)- environmental factors7genetic defects leading to reduced CFTR protein expression or chloride transport capacity lead to cystic fibrosisconversely…overstimulation of CFTR in intestinal epithelial cells by bacterial toxins leads to secretory diarrhea (having less CFTR may provide a selective advantage) - toxins (such as cholera) activate the protein kinases responsible for “priming” the CFTR channel- much larger world health problem: 3 million deaths per year of children under the age of 5Why is there such a high frequency of CFTR heterozygotes?Function of ion channels in epithelium- besides the general function of regulating osmolarity, ion channels suchas CFTR provide more specific functions1) by pumping Cl- ions out of the cell, CFTR can regulate water secretionin some epithelial cells (pancreas and intestine)2) by pumping Cl- ions into the cell, CFTR can regulate absorption of ions inexcess of water, thereby creating hypotonic water outside the cell (lung)8CFTR is the main chloride channel in epithelia of various tissues- ENaC - epithelial sodium channel- CFTR - chloride channel- AQP - aquaporin (water channel)Na+/K+Cl- apicalbasolateralbasal fluid compartmenttight junctionCFTRCl-ENaCNa+AQPH20CFTR is the main chloride channel in epithelia of various tissuesepithelia perform diverse functions: 1) water or volume-absorbing (airways and intestinal tract) 2) salt-absorbing (sweat duct, lung) 3) water or volume-secretory (pancreas, lung)all processes involve