Bioscience Reports, Vol. 15, No. 6, 1995
Chloride Channels and Cystic Fibrosis of the
Pancreas
M. A. Gray, l"z J. P. Winpenny, B. Verdon, H. McAlroy, 1
and B. E. Argent 1
Received September 8, 1995
Cystic fibrosis (CF) affects approximately 1 in 2000 people making it one of the commonest fatal,
inherited diseases in the Caucasian population. CF is caused by mutations in a cyclic AMP-regulated
chloride channel known as CFTR, which is found on the apical plasma membrane of many exocrine
epithelial cells. In the CF pancreas, dysfunction of the CFTR reduces the secretory activity of the
tubular duct cells, which leads to blockage of the ductal system and eventual fibrosis of the whole
gland. One possible approach to treating the disease would be to activate an alternative chloride
channel capable of bypassing defective CFTR. A strong candidate for this is a chloride channel
regulated by intracellular calcium, which has recently been shown to protect the pancreas in transgenic
CF mice. Pharmacological intervention directed at activating this calcium-activated C1- conductance
might provide a possible therapy to treat the problems of pancreatic dysfunction in CF.
KEY WORDS: Chloride channels; cystic fibrosis; pancreas; CFTR.
INTRODUCTION
Cystic fibrosis (CF) is a common, often fatal, autosomal recessive disorder
characterised by abnormalities of salt and fluid transport in a variety of exocrine
tissues including the lung, pancreas, liver and intestine. Affected cells lack the
ability to activate a cAMP-regulated C1- conductance, and in the lung, CF
epithelial cells also hyperabsorb sodium (Boucher et al. 1988). Both these factors
contribute to the secretory defect in CF which is thought to underlie the disease.
The major clinical symptoms are chronic obstructive pulmonary disease, pancreatic fibrosis and intestinal malabsorption and obstruction. The gene responsible for the basic cellular defect in CF was cloned in 1989 (Kerem et al. 1989;
Riordan et al. 1989; Rommens et al. 1989) and the protein product was given the
rather awkward name CFTR (for cystic fibrosis transmembrane conductance
1Department of Physiological Sciences, University Medical School Framlington Place, Newcastle
upon Tyne NE2 4HH, U.K.
2 To whom correspondence should be addressed.
531
0144-8463/95/1200-0531507.50/09 1995PlenumPublishingCorporation
532
Gray, Winpenny, Verdon, McAlroyand Argent
Fig. 1. Predicted structure of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel
(adapted from Anderson et aL 1991).
regulator). Using a variety of approaches it was subsequently shown that the CF
gene coded for a cyclic AMP-regulated chloride channel (see Riordan, 1993 for a
summary of this work). Based on the full-length cDNA and hydropathy analysis
of the amino acid sequence, a model for the structure of CFTR was proposed by
Riordan et al. (1989: Fig. 1). CFTR is a 1480 amino acid integral membrane
protein and is a member of a large family of ATP-binding transporters found in
Prokaryotic and Eukaryotic cells. This family includes P-glycoprotein, the
transporter responsible for multi-drug resistance (Higgins, 1992).
From the model shown in Fig. 1 CFTR is predicted to be composed of five
domains. Two of these are membrane spanning domains (MSD), each consisting
of six transmembrane segments, which together are thought to form the ion
channel pore. A number of studies have identified specific residues and peptide
sequences in transmembrane segments 1, 2, 6 and 12 which were suggested to line
the pore, although the recent work of Carroll et al. (1995), which indicated that
segments 1-4 were not essential for channel activity complicates this picture
(see Carroll et al. 1995 for references). The other three domains, which are
cytosolic, have a regulatory function. The R (for regulatory) domain may act as a
plug which closes the channel by blocking the pore formed by the twelve
transmembrane spans. Unplugging the pore, and thus opening the channel,
requires phosphorylation of the R domain which is catalysed by cyclic AMPdependent protein kinase A (PKA: Fig. 1). The R domain contains multiple
consensus sites for phosphorylation by PKA, and four specifi c sites appear to be
vital for expression of full activity (Welsh et al. 1994). Phosphorylation will
increase the net negative charge on the R domain which has been proposed to
lead to electrostatic repulsion of the domain from the internal face of the plasma
membrane (Welsh et al. 1994). In support of this idea replacing serine residues in
the R domain by aspartate residues (which carry a net negative charge), or
removing most of the R domain leads to constitutively open channels in the
absence of PKA phosphorylation (Welsh e t al. 1994). However, in addition to
Chloride Channelsand CysticFibrosis of the Pancreas
533
phosphorylation activation of CFTR requires binding and hydrolysis of ATP. This
occurs at the two nucleotide binding domains (NBD), which contain Walker A
and B motifs found in many other proteins which can bind and hydrolyse ATP.
Recent data (Smit et al. 1993; Baukrowitz et al. 1994; Carson et al. 1995) suggests
that ATP binding and hydrolysis at NBD1 opens the channel (activation) while
the same process at NBD2 closes the channel (deactivation). The two NBD's
therefore have distinct regulatory roles and function to gate the channel.
CFFR CI- CHANNELS AND DEFECTIVE PANCREATIC HCO~
SECRETION
In the pancreas CFTR is located in the apical membrane of the pr0ximal duct
epithelial cells (Marino et al. 1991) and plays a key role in ductal bicarbonate and
fluid secretion (see Fig. 2). The main function of pancreatic duct cells is to secrete
a HCO3-rich isotonic fluid. This secretion flushes digestive enzymes, secreted by
acinar cells located at the termini of the smallest ducts, along the ductal tree and
into the duodenum (Fig. 2). It also neutralises acid chyme entering the duodenum
from the stomach, which is crucial for creating the correct luminal environment
for the digestion of food in the gut (Argent & Case, 1994). When CFTR is either
absent or dysfunctional pancreatic HCO3 and fluid secretion is markedly reduced
(Durie & Forstner, 1989). This causes proteinaceous acinar secretions to become
concentrated in the duct lumen, where they eventually precipitate, causing
blockage of the small ducts and eventual destruction of the gland. This means that
the majority of CF patients are pancreatic insufficient (PI) and require constant
digestive enzyme supplements.
.mtein-~d~ secretion.
)
,~. ~
~
~-~
Pro)dmalDuct
is~"
(NaHCO~ and H20)
~
~
Pancreatic Juice
H=O
Fig. 2. Structure of the proximal part of the exocrine pancreas.
Diagram illustrates the physiologicaland anatomical relationship between the proximalduct cells and acinar cells,and the locationof CFTR
on the apical (luminal) surface of the duct cells. CFTR CI- channels
regulate pancreatic HCO~ and fluid secretion, by controlling the
cyclingrate of the C1-/HCO~ exchanger.
534
Gray, Winpenny, Verdon, McAlroy and Argent
LUMEN
DUCT CELL
BLOOD
C02
~ 002 +
I~O
~C03
HCO~.4~_7~ - HCO3-
e,AN~ Cm'R
r _
LJ
q
~
~AMP
ca~
LJ
~cr
ACh1 Ca~
~ N a+
I
Fig. 3. Cellular model for pancreatic HC03
secretion evoked by secretin and acetylcholine.
The current model is largely based on morphological, fluorescence and electrophysiological studies
on small ducts isolated from rat and pig pancreas.
Secretin uses cyclic AMP as an intracellular messenger whereas acetylcholine (ACh) increases
intracellular calcium concentration. The calcium
pathway is particularly well developed in the duct
cells of rats and mice. For references see Argent &
Case (1994). CA, carbonic anhydrase.
In order to understand how a defect in the C F T R C1- channel is linked to
reduced HCO~- and fluid secretion, requires an understanding of the mechanism
of H C O 3 transport in pancreatic duct cells. Our current model for ductal H C 0 3
secretion evoked by a rise in intracellular cyclic A M P is shown in Fig. 3. The
initial step is diffusion of C 0 2 into the duct cell, and its hydration by carbonic
anhydrase (CA) to carbonic acid. This dissociates to form H § and H C 0 3 , and the
proton is translocated back across the basolateral m e m b r a n e either by an
electrogenic H + - A T P a s e or a N a + / H § exchanger. Effectively, this is the active
transport step for H C O s and it leads to the accumulation of HCO;- inside the
duct cell. H C 0 3 ions are then thought to exit across the apical m e m b r a n e on a
C I - / H C 0 3 exchanger. The rate at which this exchanger cycles will depend on
the availability of luminal C I - , and on the rate at which intracellular chloride
(accumulated by the exchanger) leaks from the cell. Both of these processes are
controlled by the apical C F T R C1- channel which is activated following
secretin-stimulation of the duct cell and the subsequent rise in cAMP. Since
H C O y exit at the apical m e m b r a n e generates a current, there must be equal
current flow across the basolateral m e m b r a n e during secretion. Some of this
current is accounted for by K § efflux through a K § channel, and the remainder by
cycling of the electrogenic pumps, namely H + - A T P a s e and Na § K+-ATPase.
Finally, the negative transepithelial potential, generated by activation of the
apical C1- conductance, draws Na § and a small amount of K § into the lumen via
a cation-selective paracellular pathway. Fluid m o v e m e n t will occur by osmosis.
Hence, any alteration in the normal functioning of the C F T R C 1 - c h a n n e l would
Chloride Channels and CysticFibrosis of the Pancreas
535
be predicted to reduce HCO3 and fluid transport, which has been demonstrated
in CF patients (Durie & Forstner, 1989).
P R O P E R T I E S OF CFFR IN P A N C R E A T I C D U C T CELLS
CFTR C1- channels were first described in rat pancreatic duct cells (Gray et
al. 1988) and then in primary cultures of human fetal cells (Gray et al. 1989),
although these studies were actually performed before the gene was cloned. In
both cases the properties of the channels were found to be very similar and can be
summarised as follows: (i) low conductance (human, 6-7 pS; rat, 3-4 pS); (ii) long
open and closed times (Fig. 4A); (iii) a linear current-voltage plot with
symmetrical C1- gradients; (iv) no marked voltage-dependence of channel
activity; and (v) regulation of channel activity by cAMP. (Gray et al. 1988; Gray
et al. 1989; Gray et al. 1990). The CFTR channel is highly selective for CI- over
Na + and K +, and has a HCO3/C1 permeability ratio of about 0.2, making it
very unlikely that significant amounts of HCO3 could be secreted directly via the
channel (Gray et al. 1990). Extracellular 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) has no effect on the channel; however, 5-nitro-2-(3phenylpropylamino)-benzoic acid (NPPB) exhibits CFTR channel activity (Gray
et al. 1990). The chance of finding CFTR channels on stimulated duct cells is quite
low; less than 1 in 4 patches have channel activity (Gray et al. i988). However,
the channels usually occur in a cluster; most often with two or three, but
sometimes with up to 15 channels per patch (Gray et al. 1988). Figure 4A & B
show that secretion and cAMP increase the activity of single CFTR CI- channels,
and whole cell CI- currents, in pancreatic duct cells. On average 70% of duct cells
have detectable CFTR C1- currents (Fig. 4B); however, the cells are electrically
coupled, and the epithelium probably functions as a syncitium in vivo (Gray et al.
1993). Using data from single channel and whole cell recordings it is possible to
estimate that there are about 1000 active CFTR channels in the apical membrane
of an unstimulated duct cell and that this number increases to about 4000
following exposure to cAMP (Gray et al. 1993). Our data suggests that cAMP
stimulation increases both the activity of individual channels as well as the
number of active channels. Whether this reflects the regulated insertion of
additional channels into the plasma membrane or activation of quiescent channels
that are already present is unclear at the moment. Whatever the explanation, it is
likely that CFTR channels are not evenly distributed over the apical plasma
membrane, but are clustered near the junctional complex (Gray et al. 1993). In
addition to regulation by cAMP, we have recently shown that protein kinase C
can also modulate CFTR function in these cells (Winpenny et al. 1995a), probably
via phosphorylation of the R domain which contain PKC consensus sites. PKC
activation has little effect on current density but does appear to modulate the
stability of the channel in the plasma membrane.
536
Gray, Winpenny, Verdon, McAlroy and Argent
A
control
OSP 0.22
B control
OSP 0.13
C
OSP 0.09
A
secretin 40s
13 secretin 120s
OSP 0.35
E
secretin 200s
OSP 0.50
F
washout240s
OSP 0.24
G washout 390s
OSP 0.15
0.4 p/~
B
Is
stimulants
Secretin 10 rum
!
washout
washout
~
500 pA l .
4os
Fig. 4. Regulation of CFTR chloride channel activity by cAMP. (A) Cellattached patch on the apical membrane of a human pancreatic d u c t cell, held at
a pipette potential of -60mV. Dashed lines indicate the current level when all
channels are closed. Each trace illustrates 10 sec of data under control
conditions or after exposure to 10 nM secretin, OSP is the open state probability
of the channel (from Gray et aL 1989; with permission). (B) Continuous
whole-cell recording showing the effects of cyclic AMP stimulants (top trace)
and secretin on CFTR chloride currents in a cluster of 5 rat pancreatic duct
cells. The membrane potential was held at 0 mV and then alternatively clamped
at -4-60mV for a 1 sec periods. CFTR current densitiy in stimulated cells
averages about 100pA/pF at 60 inV, From such data it can be calculated that
the conductance of the apical plasma membrane is 14,6mS .cm -~, a value
which is consistent with that obtained by circuit analysis of the epithelium
(1lAreS. cm-Z; Novak & Greger, 1991). Thus CFTR CI- channels form the
major conductance pathway in the apical membrane of pancreatic duct cells that
have been exposed to secretin or cyclic AMP. (From Gray el al, 1993; with
permission).
Chloride Channelsand CysticFibrosis of the Pancreas
537
MUTATIONS IN CFFR AND PANCREATIC FUNCTION
Mutations in the CF gene cause CF, but well over four hundred different
mutant alleles have now been described (with many of them being patientspecific). It is therefore not surprising that our understanding of the relationship
between the specific mutation(s) and the clinical phenotype is poor. Indeed, many
tissues, particularly the lung, have a variable phenotype. Tizzano and Buchwald
(1995) have recently hypothesised that the pathogenesis of the disease will be the
result of a combination of three main factors; (1) genotype (2) secretory activity
of CFTR (3) the physiological and anatomical characteristics of the organ in
question. In this respect the pancreas is unusual in that it is the only tissue where
a clear genotype/phenotype relationship has been shown to exist (Kerem et al.
1990; Kristidis et al. 1992). Indeed mutations in CFTR are classified as either
severe or mild based on pancreatic phenotype. Evidence shows (Kristidis et al.
1992) that patients inheriting two severe mutations will be PI, while the presence
of one (or two) mild mutations will produce less pancreatic dysfunction and
clinically the patient will be pancreatic sufficient (PS). This clear-cut difference is
not absolute, and it has been observed that a subset of PS patients do go on to
develop PI after a variable period of time (Couper et al. 1992). As patients are
living longer this problem may in fact get worse. One has to remember that PI
will not be evident until >90% of pancreatic function has been lost.
Welsh and Smith (1993) have classified CF mutations into four groups based
on their effect on the CFTR protein. The most common mutation which occurs in
about 70% of CF patients involves the deletion of a phenylalamine at position 508
(delta F508), which is located within the first nucleotide binding domain (NBD1)
of CFTR (Fig. 1). Patients who are homozygous for this mutation completely lack
a cAMP-stimulated CI- conductance, which we now know is caused, rather
surprisingly, by the protein being misprocessed and not delivered to the plasma
membrane (Cheng et al. 1990; Kartner et al. 1992). In terms of pancreatic
function, delta F508 is a severe mutation and >98% of homozygous patients are
PI (Kerem et al. 1990; Kristidis et al. 1992). In addition to these trafficking
mutations, null mutations (frameshift and nonsense mutations) all give rise to
severe mutations and PI. However, about 15% of CF patients retain some
pancreatic function and are classed as PS. These patients also have less severe
pulmonary disease and survive much better. In order to understand why certain
mutations are associated with PS, a number of groups have investigated the effect
of specific PS-associated mutations on CFTR channel function. Two distinct
groups of mutations have been studied. The first group involve missense
mutations of arginine residues within the pore-forming domain MSD1 (R117H,
R334W and R347P), which collectively account for approximatley 2% of CF
patients. The second group involves residues within NBD1 (A455E and P574H)
which are located close to the walker A (residues 458-464) and B (residues
568-572) motifs, crucial for ATP binding.
When expressed in a heterologous assay system group 1 mutants were
inserted normally into the plasma membrane but the total CI- conductance was
found to be between 4-30% of that generated by the expression of wild-type
538
Gray, Winpenny, Verdon, McAlroyand Argent
CFTR. At the single channel level activation by PKA and ATP appeared normal
but the channel activity (Po) and/or conductance was reduced (Carroll et al. 1993;
sheppard et al. 1993). In contrast the group 2 mutants were incorrectly processed
and a much smaller number of channels got to the plasma membrane (Champigny
et al. 1995; Sheppard et al. 1995). However, these channels were fully functional
with normal conductance and normal, or greater than normal, Po (P574H). It was
also shown in the studies by Champigny et al. (1995) that the functional activity of
the P547H mutant could be further increased by the chloride channel opener
NS004, suggesting a putative form of therapy in these cases. Together these
elegant transfection studies explain why both groups of mutants are associated
with PS, since in all cases a significant residual CI- conductance would be present
in pancreatic cells to prevent failure. They also show that mutations which have
distinct functional effects can produce similar clinical phenotypes.
A L T E R N A T I V E C H A N N E L T H E R A P Y F O R T R E A T I N G THE
P A N C R E A T I C D E F E C T IN CF
Although systemic gene therapy remains a route to treat the pancreatic
disease in CF, we have looked at the possibility that other chloride channels,
already present in the apical plasma membrane of pancreatic duct cells, could be
used to bypass defective CFTR. Strong circumstantial evidence that this approach
could work comes from our recent studies on murine models of CF. Recently,
four groups have produced transgenic mice which are homozygous for a null
mutation of the CF gene (Dorin et al. 1992; Snouweart et al. 1992; Ratcliff et al.
1993; O'Neal et al. 1993). These animals have either no detectable CFTR mRNA
or very reduced levels of the message. Rather surprisingly, these CF mice have
normal pancreatic function, despite evidence of histological changes in their
intestinal and respiratory tracts (Dorin et al. 1992; Snouweart et al. 1992; Ratcliff
et al. 1993; O'Neal et al. 1993), together with defective cyclic AMP-mediated
chloride ion transport in these tissues (Clarke et al. 1992; Dorin et al. 1992;
Ratcliff et al. 1993; O'Neal et al. 1993). In contrast to CF mice, CF patients with
null mutations, have severe pancreatic disease (Kristidis et al. 1992). This
difference may be explained by the mouse pancreatic duct cells possessing an
alternate fluid secretory pathway which is activated by increases in intracellular
calcium ([Ca2+]i). In support of this idea we have shown that an increase in
[Ca2+]i, evoked by either ionomycin or acetylcholine, activates chloride channels
in rat duct cells (Plant et al. 1993; see Fig. 2), and stimulates fluid secretion from
isolated rat pancreatic ducts (Ashton et al. 1993). Calcium-activated chloride
currents (CACC) can also be detected in mouse pancreatic duct cells which have
no detectable CFTR (Fig. 5), clearly indicating that these currents are carried by
an ion channel that is distinct from CFFR. (Gray et al. 1994, Winpenny et al.
1995b). These CACC are of similar magnitude in wild-type and homozygous cf/cf
animals, and about 15-fold larger than the CFTR currents in the wild-type group
Chloride Channels and Cystic Fibrosis of the Pancreas
539
(B) HOMOZYGOTE CFTR
(A) WILD-TYPE CFTR
30.
30.
i
20.
g 20
.
10
o
10
?
-6
o
C PS
Vm (mY} 60
o.
C PS
-60
{C) WILD-TYPE CACC
QC ~ PS c~l~
C PS
Vm (mV) 60
(D}
-60
HOMOZYGOTE CACC
700 -
7O0
<
525
525 9
350
-$
? 1Ts
?
"6
"5
0
Vm (rnVl
C
IO
6O
C IO
-60
175
01
C
I0
Vm (mY} 60
C
I0
-60
Fig. 5. CFTR and CACC current density in wild-type and
Cambridge c f / c f null mouse pancreatic duct cells. (A & B)
CFTR currents can be activated in wild-type (A) but not
homozygous null cells (B). Mean data taken from >15 separate
cells under control (C: open bars) or in the presence of cAMP
stimulants (PS: hatched bars). (C & D) CACC currents are
present in both genotypic groups. Mean data taken from >7
separate cells under control (C: open bars) or in the presence of
1/zM ionomycin (10; hatched bars). Data is expressed as
current density (pA/pF) at Er~+60mV. (Adapted from
Winpenny et al. 1995b).
(Fig. 5). Approximately 70% of wild-type and homozygous cf/cf cells have
CACC.
Although the CF m o u s e is not a good model for the human pancreatic
disease that occurs in CF, it does show that other chloride channels can effectively
substitute for CFTR. This suggests that the alternate channel approach to therapy
might have some chance of success. The obvious question is whether similar
Ca2+-activated CI- channels are present in h u m a n pancreatic duct cells? O u r
recent experiments indicate that the answer is in fact yes, as shown in Fig. 6. The
fact that structural d a m a g e occurs to the pancreas of CF patients implies that
this calcium pathway is either quantitatively less important than the c A M P
pathway (i.e. the secretory activity of C F T R dominates in this tissue), or the
calcium pathway is not physiologically regulated in man. We favour the second
possibility since the size of the C A C C in h u m a n cells (Fig. 6) are comparable to
those of mouse cells (Fig. 5C). Therefore, pharmacological intervention directed
at activating this calcium-activated C1- conductance might provide a possible
therapy to treat the p r o b l e m s of pancreatic dysfunction in CF.
Gray, Winpenny, Verdon, McAlroy and Argent
540
- -
I-~
0.1nM Calcium in pipette [n = 3]
[]
l pM Calcium in pipette (n = 3)
400
200
100
Vm (mY}
60
-60
Fig. 6. CACC currents in human pancreatic duct
cells. Data summary from 3 cells exposed to a pipette
(intracellular) solution containing either 0.1 nM or
1 IxM calcium. Biophysical properties of the currents
activated by the high intracellular calcium were identical to those described for mouse pancreatic duct cells
(Fig. 5) activated by ionomycin. Data is expressed as
current density (pA/pF) at Erev + 60 mV (Gray, Winpenny & Argent; unpublished observations).
ACKNOWLEDGEMENTS
Funded by grants from the Cystic Fibrosis Trust, Medical Research Council
(U.K.), The Nuffield Foundation and the National Institute for Health (USA: DK
43956). HLM is funded by a University of Newcastle upon Tyne Studentship.
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