2 Journal of the Royal Society of Medicine Supplement No.
19 Volume 85 1992
The biochemical defect in cystic fibrosis
Alan W Cuthbert PhD FRS Department of Pharmacology, University of Cambridge,
Tennis Court Road, Cambridge CB2 1 QJ
Keywords: cystic fibrosis; transmembrane conductance regulator (CFTR); chloride channels; sodium channels; amiloride; adenosine
triphosphate/uridine triphosphate (ATP/UTP)
A cartoon of the protein coded for by the CF gene is
illustrated in Figure 1, and is known as cystic fibrosis
transmembrane conductance regulator (CFTR). It
represents the culmination of a long and difficult
quest to discover the CF gene. This was finally
achieved in September, 19891-3. The gene is large
(some quarter of a million base pairs) and has 24
exons. The protein coded for by the gene (CFTR) has
1480 amino acids-consisting of two halves, each
containing six transmembrane spanning segments
linked to a consensus sequence for a nucleotide
binding fold. A large polar domain, containing
multiple sites for phosphorylation, joins the two
halves of the protein.
In 70% of patients with CF a single codon on the
gene is lost, resulting in the loss of phenylalanine at
position 508 (A 508). This deletion occurs in the first
nucleotide binding fold. Many other mutations (over
70) have now been recorded to account for the other
30% of CF patients, a fact which makes universal
screening for CF an almost impossible task.
Two matters are addressed in this overview. First,
how are the known dysfunctions in epithelial cells
related to abnormal CFTR (ie A 508)? Secondly, are
there pharmacological strategies which can be used
to correct the biochemical defects and relieve the
functional defects of epithelia in CF?
A good starting point is to return to one of the
earliest biochemical defects associated with the
disease, namely the enhanced salt concentration of
the sweat.4 The blind-ended secretory coil of the
sweat gland secretes a near isotonic NaCl solution.
Eventually this fluid moves into the reabsorbing duct
Figure 1. Cartoon ofpossible structure of CFTR, consisting
of two halves each composed of six membrane spanning
sections joined to nucleotide binding folds (NBF). The two
halves are joined by the R-domain containing multiple
phosphorylation sites. The N and C terminal ends of the
protein are shown
COIL DUCT
Na Cl H20 Na Cl H20
NORMAL
Na CI H20 Na NaCI H20
CF w~~~~-i ^6kAA-
v47~~~~~~~~~~~~~~~~~~~~~~~~~~~
Figure 2. Schematic of ion transport processes in the human
sweat duct. Chloride led salt secretion in the coil is
accompanied by an osmotic water flux thus creating an
isotonic primary secretion. In the duct salt is reabsorbed by
a sodium led process but since the duct has a low permeability
to water there is little osmotic drag and hypotonic secretion
appears at the skin surface. In CF glands the primary
secretion is formed as before (there are subtle changes not dealt
with in this review) but salt reabsorption fails because of the
low chloride permeability of the duct giving rise to sweat with
an increased salt content
part of the gland. This section of the gland has a low
water permeability and the ability to reabsorb salt,
so that normally sweat is hypotonic (Figure 2). For
many years it was considered that in CF sweat glands
there was some defect in the handling of sodium ions.
That this was not so was shown in a seminal
observation made by Paul Quinton in 19835. The
mechanism of sodium led salt absorption is given in
Figure 3. Sodium ions enter the apical membrane of
epithelial cells by moving down an electrochemical
gradient through specific membrane channels, which
can be specifically blocked by the drug amiloride. The
sodium ions can then be removed across the baso-
lateral face of the cell by the sodium pump. The
translocation of sodium in this way generates a
transepithelial potential, apical side negative relative
to the basolateral side. The transepithelial potential
provides the appropriate electrical gradient for the
passive movement of the accompanying counterion,
chloride. What Quinton found was that in CF the duct
had a very low chloride permeability, so that salt
could not be absorbed. A further consequence of this
is that the transepithelial potential was greater in CF
than normal ducts.
An important question therefore is whether CFTR
is a chloride channel. There is now a large body of
evidence to indicate that CFTR, a membrane protein,
does indeed function as a chloride conductance. Before
considering this evidence it is important to remember
that chloride channels in epithelia can be activated
by intracellular messengers, cAMP and Ca2+. In CF
activation of chloride channels by cAMP does not
occur. Activation of CF chloride channels by Ca2+ is
 Journal of the Royal Society of Medicine Supplement No. 19 Volume 85 1992 3

Na

Figure 3. Details of electrogenic sodium absorption (a), and electrogenic chloride secretory (b), processes. In (a) sodium ions
enter the cells through the apical membrane through amiloride-sensitive sodium channels. The sodium is expelled through
the basolateral membrane by a sodium pump (p). Chloride ions follow passively down the electrochemical gradient either
through or between the cells. In (b) chloride ions are accumulated in the cells at a concentration higher than expected by passive
diffusion using a Na-K-2 Cl cotransporter, ie an electroneural carrier which moves one Na, one K and two Cl ions during
one translocation. The sodium ions are dealt with by the sodium pump and potassium ions can re-equilibrate across the potassium
perm-selective basolateral membrane. The energy for the Na-K-2 Cl transport process is derived from the gradient ofthe sodium
concentration between outside and inside the cells. Chloride ions in the cell leave by apical chloride channels creating an
electrical gradient for the passive transfer of sodium across the epithelium, either through or between the cells

still a vexed issue and thought to be dependent upon
the tissue examined. In airway epithelial cells which
are clearly of central importance in CF, it has been
shown that cAMP increases the apical membrane
chloride conductance only in normal but not in CF
cells6. Therefore a crucial test of whether or not a
chloride conductance has CF characteristics is its
failure to be activated by cAMP. In molecular terms
this means whether or not the open probability is
increased. It is typical of ion channels that they exist
in open or closed conformations. Increased open state
probability can result from an increase in the
individual duration of open times or an increase in
opening frequency or both.
To return to the question of whether CFTR is a
chloride channel there is a series of recent elegant
studies which make this very probable. The first of
these required the construction of plasmid which
could be used to transfect cells and containing the
cDNA for CFTR. It was shown in vitro that the
recombinant protein generated by the plasmid was
identical to native CFTR. Furthermore the re-
combinant CFTR could be phosphorylated by a
cAMP-dependent protein kinase A, signifying a likely
sensitivity of this protein to cAMP7. How then can
the plasmid be used to show whether CFTR is a
chloride channel? SPQ is a fluorescent dye in which
fluorescence is quenched by halides like chloride or
iodide. Cells loaded with both SPQ and chloride will
have only slight fluorescence. If the bathing solution
is now changed to one containing nitrate then chloride
will leak from the cells, while nitrate will be taken
up. Consequently the fluorescence of the cells will
increase. This is a powerful method since it measures
efflux through all types of chloride channels or
exchange mechanisms and while this does not inform
about which type of translocation mechanism is
involved, it eliminates tedious studies of ion channels
which may subsequently be shown to be irrelevant
to CF. Furthermore by adding cAMP, or substances
such as isoprenaline or forskolin which lead to cAMP
generation, the sensitivity ofthe chloride efflux to the
nucleotide can be assessed.
Transfecting CF airway epithelial cells with
plasmids containing either the cDNA for CFTR or the
cDNA for A 508 gave cells to which isoprenaline could
be added and any fluorescence increase followed using
the method described above. This was greater for cells
transfected with the cDNA for CFTR, than those
transfected with the mutant cDNA. The conclusion
is made that expression of CFTR in CF epithelial cells
corrected the chloride channel defect, ie by-passing
the genetic defect by transfecting with the correct
cDNA corrected the phenotypic defect8.
Using similar technology to that already described
it was found transfection of HeLa cells with the cDNA
for CFTR led to the appearance of a cAMP-dependent
chloride conductance in cells not normally expressing
CFTIR9. More remarkably, site directed mutagenesis
was used to produce mutant forms of CFTR which
were transfected into HeLa cells10. Consequently
mutated amino acids were substituted in the putative
membrane spanning regions. Figure 4 shows a
simplified version ofCFTR with the position of lysines
at position 95 and 335 indicated, as in native or
recombinant CFTR. When these basic amino acids
Figure 4. Another cartoon version of CFTR redrawn from
references 10 and 11. A and B represent the positions of lysines
95 and 335. Changing lysine 95 to aspartic acid and lysine
335 to glutamic acid alters the selectivity of the chloride
conductance fiom chloride> iodide to iodide>chloride When
the R-domain was deleted the CFTR chloride conductance
was increased in the unstimulated state (ie absence ofcAMP)
suggesting that the R-domain is involved in the gating of the
ion channeL A similar mechanism has been proposed for the
gating ofpotassium channels. The dotted outline represents
simply how a conformational change within a protein might
alter a functional responsibility
4 Journal of the Royal Society of Medicine Supplement No. 19 Volume 85 1992
were substituted by aspartic acid (at position 95) or
glutamic acid (at position 335) there was a change in
the anion selectivity of chloride conductance. With
recombinant CFTR anion selectivity was in the order
bromide > chloride > iodide > fluoride, the same as for
endogenous CFTR. Mutations of the lysines (95, 335)
to acidic amino acids converted the sequence to
iodide > bromide > chloride > fluoride.
In yet another study" a plasmid, encoding CFTR
in which amino acids 708 to 835 were deleted (ie the
whole of the R-domain) was transfected into HeLa
cells. Unstimulated cells expressing A R-CFTR
exhibited a high chloride conductance, comparable to
that in stimulated cells expressing native CFTR,
suggesting that the R-domain was intimately involved
in the open-closed kinetics of the channel (Figure 4).
All of the foregoing suggests very strongly that
CFTR is a chloride channel. Other explanations are
just possible; for example HeLa cells might have
nascent chloride channels with CFTR acting as an
activator or regulator. The totally definitive experiment
would be to incorporate CFTR into artificial lipid
bilayers and discover single channel activity with the
correct selectivity and regulated by cAMP. This has
not yet been achieved.
Finally, to return to the sweat gland. Ductal cells
were grown in culture and exposed to an antisense
oligonucleotide complimentary to the first 23 nucleo-
tides of CFTR. Subsequently cells were loaded with
SPQ to report the chloride efflux when the bathing
solution was changed to a gluconate containing one.
An anion impermeability defect was found in the
experimental cells compared to controls, presumably
because of the failure to produce CFTR. The antisense
effect was apparent between 4 and 24 hours suggesting
a rapid turnover of CFTR'12.
Thus the disease phenotype in cells can be both
corrected and introduced by manipulation of the
expression of CFTR. Nevertheless there remains a
difficult paradox in relation to the symptoms of
the disease. In some tissues the chloride channel
appears to be of crucial importance for proper
functioning. For example in the pancreatic duct
chloride secretion is a necessary part ofthe formation
of the bicarbonate-rich pancreatic fluid. However in
the epithelia lining the airways the elaboration of
fluid to maintain the sol phase and mucus hydration
is dependent upon two epithelial transport processes,
vis electrogenic chloride secretion and electrogenic
sodium absorption (Figure 3). As in the sweat gland
CF airway epithelia have an enhanced transepithelial
potential but in this instance it is not due simply to
failure of chloride secretion because of mutant
chloride channels, but also due to the potential
generated by electrogenic sodium absorption. Largely
through the efforts of the North Carolina group the
sodium reabsorptive process in CF epithelia has been
shown to be enhanced13'17. Evidence for this is given
by the greater potential change in CF airway
epithelia caused by amiloride and the increased
transepithelial sodium fluxes. As airway epithelia are
water permeable, water moves osmotically across
them following salt transport. Failure to secrete
chloride and excessive sodium absorption leads to
excessive fluid loss and drying of the airway surface.
The molecular reasons for the excessive sodium
absorption is probably related either to increased
numbers of sodium channels'8, increased channel
open time'9 or both of these. In CF sweat gland cells
sodium channels show an apparent decrease in affinity
for amiloride and an altered relationship between
sodium concentration and transepithelial sodium
transport20.
The ways in which these altered characteristics of
sodium handling are related to the expression of
mutant CFTR are unclear. The evidence reviewed
earlier indicates that CFTR is a chloride channel yet
there is a body of evidence to suggest sodium channels
behave aberrantly. As this sodium transporting defect
is present in cultured epithelia it appears to be
characteristic of the CF phenotype.
Understanding ofthe fundamental biochemical and
functional defects in the CF cell has led to strategies
for a pharmacological approach to treatment. For
example, a drug blocking sodium channels in airway
epithelia will not only reduce sodium absorption but
increase the electrical gradient for any residual
chloride secretion. Knowles and his colleagues were
the first to perform a clinical trial with nebulized
amiloride aerosols. They showed that treatment
reduced the rate of decline of vital capacity compared
to controls and normalized sputum viscosity21. In
another trial amiloride was shown to improve
mucociliary clearance22. As we have seen previously,
CF chloride channels fail to respond to cAMP signals
but the possibility exists that they may be operated
from the apical side by agents not requiring the
agency of the usual regulatory mechanisms. In vitro
studies indicated that ATP and UTP may do this23.
Recently, by measuring changes in nasal epithelial
potentials in vivo not only were changes recorded
consistent with enhanced chloride secretion but the
nucleotides were more effective in CF patients2A.
Nebulized aerosols containing a mixture of both
amiloride and nucleotide is clearly the next step in
the therapeutic battle.
Finally, in a few rare cases a mutation leading to
CF results in the formation of truncated forms of
CFTR or perhaps no protein at all. In this situation
the respiratory symptoms of the disease are mild25.
This has led to the idea that no CFTR is better than
abnormal CFTR. Perhaps this is a further indication
that in airway epithelia the absence of the normal
chloride conductance is of secondary importance.
Clearly this reveals obvious other therapeutic
approaches with antisense oligonucleotides.
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