Functional Interactions of HCO3

Mike A Gray, Catherine O’Reilly
, John Winpenny
, Barry Argent
Department of Physiological Sciences, University Medical School. Newcastle upon Tyne, United
Biomedical Imaging Group, Department of Physiology, University of Massachusetts
Medical Centre. Worcester, MA, USA.
School of Health Sciences, University of Sunderland.
Sunderland, United Kingdom
Disruption of normal cystic fibrosis
(CFTR)-mediated Cl
transport is associated
with cystic fibrosis (CF). CFTR is also required
for HCO3
transport in many tissues such as the
lungs, gastro-intestinal tract, and pancreas,
although the exact role CFTR plays is
uncertain. Given the importance of CFTR in
transport by so many CF-affected organ
systems, it is perhaps surprising that relatively
little is known about the interactions of HCO3
ions with CFTR. We have used patch clamp
recordings from native pancreatic duct cells to
study HCO3
permeation and interaction with
CFTR. Ion selectivity studies shows that CFTR
is between 3-5 times more selective for Cl
. In addition, extracellular HCO3
has a
novel inhibitory effect on cAMP-stimulated
CFTR currents carried by Cl
. The block by
was rapid, relatively independent of
voltage and occurred over the physiological
range of HCO3
concentrations. These data
show that luminal HCO3
acts as a potent
regulator of CFTR, and suggests that inhibition
involves an external anion-binding site on the
channel. This work has implications not only
for elucidating mechanisms of HCO3
in epithelia, but also for approaches used to
treat CF.
It is well established that cystic fibrosis
transmembrane conductance regulator (CFTR)
transports chloride ions in a variety of epithelial
tissues. Disruption of normal CFTR-mediated
transport is associated with a number of
diseases such as cystic fibrosis (CF), certain
types of secretory diarrhoea, and possibly
polycystic kidney disease. CFTR is also
involved in the transport of other
physiologically important anions such as HCO3
[1], glutathione [2] and larger organic anions
[3]. In the case of HCO3
many epithelial
tissues secrete this anion by a mechanism
which is dependent on functional CFTR
channels. This has been observed in the airways
[4], including submucosal glands [5]; the
gastro-intestinal tract [6]; the liver and
gallbladder [7, 8] and the pancreas [9], the
archetypal bicarbonate-transporting gland.
While there is now strong evidence that CFTR
is essential for effective HCO3
secretion the
exact role it plays is still uncertain.
Our studies have focused on the role of CFTR
in the production of an HCO3
rich alkaline
secretion by the exocrine pancreas [1]. We

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JOP. Journal of the Pancreas – – Vol.2, No.4 Suppl. – July 2001
proposed back in 1988 that HCO3
exits across
the apical membrane of pancreatic duct cells
(PDCs) by parallel operation of CFTR Cl
channels and Cl
exchangers [10]. In this
scheme the CFTR channel can be viewed as
having two functions. The first is to provide
luminal Cl
for operation of the anion
exchangers. The second is to act as a leak
pathway to dissipate intracellular Cl
accumulated as the exchanger cycle. Implicit in
this ‘CFTR-anion exchanger model’ is that
CFTR is better at transporting Cl
than HCO3
under normal physiological conditions.
We showed this to be the case in subsequent
patch clamp studies using both single channel
[11] and whole cell current recordings [12], of
CFTR in native rat pancreatic duct cells.
However, it should be noted that in all cases
CFTR did demonstrate a low but measurable
permeability to HCO3
. Therefore, under
conditions where intracellular Cl
is at or near
electrochemical equilibrium then it is possible
that CFTR could act as an exit pathway for
. With this in mind our computer
modeling studies indicate that parallel operation
of CFTR channels and Cl
cannot support the secretion of a pancreatic
juice containing near isotonic NaHCO3, as
occurs in most other species [13]. Secretory
studies on isolated guinea-pig ducts have also
virtual absence of extracellular Cl
which would
not be predicted for the CFTR – anion
exchanger model [14, 15]. The implication of
these findings is that species such as cat, dog,
pig, guinea-pig and human, all of which secrete
a pancreatic juice with a high HCO3
(about 150 mM), employ a different secretory
mechanism to that originally suggested for the
rat, but which is still dependent on CFTR (see
the chapter by Sohma et al. which discusses
this in more detail [16]).
Extracellular HCO3
Blocks Cl
through CFTR
During recent anion permeability studies from
native guinea pig PDCs, we observed an
0 .3
0 .6
Time (s)
I (nA)
Time (s)
I (nA)
Time (s)
I (nA)
Figure 1. Inhibition of cAMP-activated currents by bath
Whole cell currents were recorded at room temperature
under control conditions (a) or after exposure to
stimulants (5 µM forskolin and 100 µM dibutyryl
cAMP) that activate PKA (b and c). Whole cell currents
were obtained by holding the membrane potential (Vm) at
0 mV and clamping Vto ±100 mV in 20 mV steps. The
pipette solution contained (mM): 110 CsCl, 2 MgCl
, 5
(HEPES), 1 Na
ATP, pH 7.2 with CsOH. The bath
solution contained (mM): 145 NaCl, 4.5 KCl, 2 CaCl2, 1
MgCl2, 10 HEPES, 5 Glucose, pH 7.4 or in (c), 140mM
NaCl was replaced with NaHCOand CaClwas omitted
from the solution (pH about 8.0). For further details on
cell preparation and electrophysiology see [17].

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JOP. Journal of the Pancreas – – Vol.2, No.4 Suppl. – July 2001
unexpected and novel effect of extracellular
on cAMP-activated CFTR Cl
[17]. Figure 1 shows that when 140 mM
extracellular Cl
is replaced by HCO3
resulted in a marked inhibition of CFTR
currents. While the reduction in outward
current (anion influx) was expected because of
the decrease in extracellular Cl
the marked block of inward current (anion
efflux) was not predicted as pipette Cl
concentration was unchanged. The reduced
inward current indicates that external HCO3
causing ‘trans’ inhibition of Cl
This effect of extracellular HCO3
was rapid,
fully reversible (Figure 2a) and dose-dependent
over a physiological range of extracellular
concentrations (Figure 2b).
The data in Figure 2b suggest that a single
binding site is involved in the HCO3
inhibition of inward current flow. Since
inhibition was only weakly voltage-dependent
(Figures 1 and 2a), this site is unlikely to
experience the voltage drop across the channel.
We next investigated which component of the
containing solutions, pH, HCO3
pCO2, was responsible for the observed current
inhibition. By varying intra and extracellular
pH over a wide range (6.2-8.0), and changing
pCOfourfold (3-12 kPa) while maintaining a
concentration of HCO3
that caused maximal
inhibition, we were able to conclude that it is
the HCO3
ion itself that inhibits CFTR [17].
Although our experiments have not identified
how HCO3
is able to block CFTR we think that
an external anion-binding site is involved. We
speculate that a positively charged site
(arginine, lysine or possibly histidine) in the
extracellular loops (EL) of CFTR could be
Figure 2. Reversible and concentration-dependent block
of CFTR by extracellular HCO3
(a) Summary of the effect of 140 mM external HCO3
the size of cAMP-activated CFTR Clcurrents. Same
conditions as Figure 1. Current density was calculated by
dividing the total current by cell capacitance. Data
measured at the reversal potential (Erev±60 mV and was
obtained from current/voltage plots of the data in Figure
(b) Effect of different extracellular HCO3
on inward current inhibition. Data measured at Erev –60
mV and fitted to a Michaelis-Menten equation with the
parameters indicated on the figure (diagram adapted
from O'Reilly CM et al., with permission [17]).
whole-cell current (pA/pF)
Vm (Erev +/- 60 mV)
Vmax = 70.7 ± 4.8
Km = 6.8 ± 2.2 mM
External HCO3 Concentration (mM)
% inhibition
Figure 3. Positively charged residues in the extracellular
loops (EL) of human CFTR.
Abbreviations used. H: Histidine, K: Lysine and R:

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JOP. Journal of the Pancreas – – Vol.2, No.4 Suppl. – July 2001
involved (Figure 3). For example in EL1 of
human CFTR residues R104 and R117 are
conserved amongst all species, and R117H is a
known disease causing mutation. Our current
research is aimed at testing this hypothesis. It
should also be noted that HCO3
is not unique
in being able to inhibit Cl
movement through
CFTR, since both extracellular I
and ClO4
cause a significant reduction in inward current,
but with less affinity than HCO3
, and in the
case of iodide, irreversibly [17].
Physiological Implications of HCO3
Inhibition of CFTR
At first sight an inhibitory effect of
extracellular HCO3
on CFTR appears
paradoxical in that it would inhibit HCO3
secretion. At the maximum concentration of
found in guinea-pig pancreatic juice
(about 150 mM) the CFTR conductance would
be more than 70% blocked (Figure 2).
However, it is notable that in guinea pig ducts
basal HCO3
secretion is Cl
dependent and
blocked by 4,4'-diisothiocyanatostilbene-2,2'-
disulphonic acid (DIDS), suggesting that it
occurs via Cl
exchange [13, 14]. In
contrast, cAMP-stimulated HCO3
secretion is
unaffected by removal of extracellular Cl
must therefore involve some other pathway [13,
14]. That pathway is likely to be CFTR.
Inhibiting the CFTR conductance via a negative
feedback mechanism from ‘signals’ in the
lumen of the pancreatic ducts may be
advantageous in that it would limit apical
membrane depolarisation and maintain the
electrical driving force for HCO3
secretion via
the uninhibited fraction of CFTR. Since many
other organ systems (liver, gastro-intestinal
tract and lungs) also secrete HCO3
, this
suggests that HCO3
concentration at the
luminal surface of epithelial cells plays a
general role in the regulation of CFTR, as well
as providing an appropriate physiological
environment for these tissues to operate
Key words Chloride Channels; Cystic Fibrosis;
Ion Transport; Pancreas; Sodium Bicarbonate
Abbreviations CF: cystic fibrosis; CFTR:
cystic fibrosis transmembrane conductance
regulator; DIDS: 4,4'-diisothiocyanatostilbene-
2,2'-disulphonic acid; EGTA: ethyleneglycol-
acid; EL: extracellular loops; HEPES: N-2-
acid; PDC: pancreatic duct cell
Acknowledgements Funded by grants from the
Cystic Fibrosis Trust (UK) and the Wellcome
Mike A Gray
Department of Physiological Sciences
University Medical School
Framlington Place
Newcastle upon Tyne NE2 4HH
United Kingdom
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