David Sheppard Flashcards
2 current therapies
Chest physiotherapy - ‘beating’ the chest to clear the air passageway
IV antibiotics - prophylactically - preventative care for infections
Describe the clinical phenotype
Bronchiectasis - end-stage lung failure; permanent dilation of air passageways (can see post-mortem)
Blockage of distal small intestine - 15% of CF babies require surgical repair
Pancreatic insufficiency in 85% (15% have a milder phenotype)
Infertility - more common in males
Diagnostic techniques
Bronchioscopy - image passageway, or sample cells to perform a brionchioalveolar lavage (BAL)
Mucus = high tensile strength - very difficult to remove from CF lungs - due to very high cell count
BAL - can see a greater number of immune cells (fighting bacterial infection)
Cycle between: infection inflammation = cause destruction of the lung tissue
Bacteria
Mucoid pseudomonas auruginosa (mucoid PA) - dominant microorganism, develops in adolescents/adults with CF in later life - lives in lungs for the entire life!
Early on = motile; treated with ABs
Later = develops a biofilm/protective sheath; resistant to ABs
Motile –> colony –> biofilm
Epithelial cells
Line: respiratory tract, reproductive tract, small intestine, liver, pancreas = all blocked by thick, sticky mucus
Polarised due to tight junctions (each cell submits a hemichannel [6x connexin subunits]) - enables basolateral/apical membranes to contain different proteins
Apical membrane - faces the lumen of the duct
Basolateral membrane - faces the interstitium
More basolateral membrane > apical membrane
Epithelial proteins
Basolateral membrane = active pumping of Cl- - accumulate high [Cl-] intracellularly
= NKCC1 (Na+, K+, 2xCl- in)
-Reliant on Na-K-ATPase to set Na+ concentration gradient
Apical membrane = passive Cl- down concentration gradient into the lumber of the ducts
-PKA dependent
Basolateral:
- Na-K-ATPase = set up Na+ concentration gradient (3xNa+ out, 2K+ in)
- K+ channel mediating K+ efflux out of the cell into the interstitium
Cl- movement = TRANScellular
Na+ + H2O movement = PARAcellular - drawn across the epithelium to neutralise Cl- -ve charge (electrochemical gradient)
CF Patients - Paul Quinton self-diagnosed himself;
= loss of passive Cl- channel - apical membrane is impermeable to Cl-!
But - cAMP/PKA signalling pathways are intact!
Identify the defective gene responsible for the disease
CFTR = Cystic Fibrosis Transmembrane Conductance Regulator - function was unclear!
Hypothesis 1 = Cl- channel
BUT - LOF did not explain all the symptoms (ie. hyperabsorption of Na+)
Hypothesis 2 = channel regulator
Whole-cell patch clamp of CFTR in wild-type + F508del = changed IC [Cl-] = shift reversal potential, consistent with Cl- selectivity
THEREFRORE = CFTR has a Cl- current
Experimental CFTR
Fibroblasts in Chinese hamster cells (no CFTR/cAMP-activated Cl- channel)
Transfect: biolistics, lipofection, viral (Sindbus virus)
Transfect: wild-type, F508del
Controls:
Neg - no transfection
Sham - transfection conditions w/ no DNA ie. gold bullet, plasmid with no DNA encoding channel
Whole-cell voltage clamp
IC pipette = EGTA (chelate Ca - keep [Ca] low) + NMDG (large cation, impermeant to K+/Na+ channels - balanced to Cl-)
Bath = NMDG
Activate channels with Forskolin
FOUND:
CFTR = time-independent (does not differ with time) and voltage-independent (the difference between the currents generated by V jumps are all the same)
Is CFTR a channel or a regulator?
Cl- current:
- Isolated CFTR gene, transfected into Chinese hamster fibroblast cells (control, sham control, wild-type, F508del)
- Whole-cell patch clamp at different IC [Ca] = changes in reversal potential generated are consistent with Cl- current
Cl channel
-Site-directed mutagenesis of positive lysine residues outside the MSDs = altered the anion selectivity - therefore a Cl channel!!!!
ATP Binding + Hydrolysis
ATP can only regulate channel opening once the channels has been phosphorylated at the regulatory domain by PKA!
Requires ATP hyrolysis - no current when using ATP non-hydrolysable analogues!
Single-channel currents = looking at cycles of ATP binding + hydrolysing driving channel opening/closing
= the role of ATP binding + hydrolysing acts as a timing mechanism to determine the duration of channel opening + closing
CFTR - ATP binding sites
Used: crystal structure of E. Coli BtuCD = vitamin B12 transporter - led to a refinement of our understanding of ATP binding sites
- Head-to-dimer arrangement
- Amino acid seqeunces from both NBDs make up the ATP binding site
Cannonical binding site = Walker A/B motifs + LSGGQ ABC signature motif
ATP binding site 1 = stable ATP binding
NBD1 = Walker A + B - mutated catalytic base on Walker B
NBD2 = LSHGH - no signature motif
ATP binding site 2 = cannonical; rapid ATP hydrolysis
NBD1 = LSGGQ signature motif
NBD2 = Walker A + B
ATP-driven dimerisation
ATP binding site = ABS
ATP binds tightly to ABS 1
ATP binds to ABS 2 - drives NBD dimerisation (open-to-closed conformation); conformational change is transmitted to the LBDs = whole protein conformational changes drive channel opening
ATP hydrolysed at ABS2 = drives channel closure!
Current thinking - do not have separation at the level of ABS1, just at ABS2 = partial separation!
Molecular models
Crystal structure of E Coli BtuCD protein (vitamin B12 ABC transporter) = knowledge of ATP binding sites
-Head-to-dimer
-Amino acid residues from both NBDs make up each ATP binding site (Walker A/B, LSGGQ)
BUT - more than 12 TMDs - cannot use to build a molecular model of CFTR
Crystal structure of Sav1866 (bacterial multi-drug transporter) - tell us that the 4x IC loops are important in coupling NBDs and MSDs = coupling helices
BtuCD
E Coli protein - vitamin B12 ABC transporter
Knowledge of ATP binding sites
-Head-to-tail dimer
-Amino acid residues from both NBDs make up each ATP binding site (Walker A/B, LSGGQ)
BUT - more than 12 TMDs - cannot use to build a molecular model of CFTR
Sav1866
Used to tell us that the 4x IC loops are important in coupling NBDs + MSDs = coupling helices
