Functional Interactions of HCO3

Mike A Gray, Catherine O’Reilly
1
, John Winpenny
2
, Barry Argent
Department of Physiological Sciences, University Medical School. Newcastle upon Tyne, United
Kingdom.
1
Biomedical Imaging Group, Department of Physiology, University of Massachusetts
Medical Centre. Worcester, MA, USA.
2
School of Health Sciences, University of Sunderland.
Sunderland, United Kingdom
Summary
Disruption of normal cystic fibrosis
transmembrane
conductance
regulator-
(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
HCO3
-
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
-
over
HCO3
-
. In addition, extracellular HCO3
-
has a
novel inhibitory effect on cAMP-stimulated
CFTR currents carried by Cl
-
. The block by
HCO3
-
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
-
transport
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
Cl
-
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

Page 2
JOP. J. Pancreas (Online) 2001; 2(4 Suppl):207-211.
JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 Suppl. – July 2001
208
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
-
/HCO3
-
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
HCO3
-
. With this in mind our computer
modeling studies indicate that parallel operation
of CFTR channels and Cl
-
/HCO3
-
exchangers
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
-
content
(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
-
Efflux
through CFTR
During recent anion permeability studies from
native guinea pig PDCs, we observed an
0
0 .3
0 .6
Time (s)
-1
0
1
a
I (nA)
0
0.3
0.6
Time (s)
-1
0
1
b
I (nA)
0
0.3
0.6
Time (s)
-1
0
1
c
I (nA)
Figure 1. Inhibition of cAMP-activated currents by bath
HCO3
-.
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
2
, 5
ethyleneglycol-bis-(beta-aminoethyl
ether)-N,N'-
tetraacetic
acid
(EGTA),
10
N-2-
hydroxyethylpiperazine-N'-2-ethanesulfonic
acid
(HEPES), 1 Na
2
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].

Page 3
JOP. J. Pancreas (Online) 2001; 2(4 Suppl):207-211.
JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 Suppl. – July 2001
209
unexpected and novel effect of extracellular
HCO3
-
on cAMP-activated CFTR Cl
-
currents
[17]. Figure 1 shows that when 140 mM
extracellular Cl
-
is replaced by HCO3
-
this
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
-
concentration,
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
-
is
causing ‘trans’ inhibition of Cl
-
efflux.
This effect of extracellular HCO3
-
was rapid,
fully reversible (Figure 2a) and dose-dependent
over a physiological range of extracellular
HCO3
-
concentrations (Figure 2b).
The data in Figure 2b suggest that a single
binding site is involved in the HCO3
-
induced
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
HCO3
-
containing solutions, pH, HCO3
-
or
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
on
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
1.
(b) Effect of different extracellular HCO3
concentrations
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]).
STIM
HCO3
WASH
a
whole-cell current (pA/pF)
0
25
50
75
100
Vm (Erev +/- 60 mV)
0
20
40
60
80
100
120
140
25
50
75
100
Vmax = 70.7 ± 4.8
Km = 6.8 ± 2.2 mM
External HCO3 Concentration (mM)
% inhibition
b
0
Figure 3. Positively charged residues in the extracellular
loops (EL) of human CFTR.
Abbreviations used. H: Histidine, K: Lysine and R:
Arginine.

Page 4
JOP. J. Pancreas (Online) 2001; 2(4 Suppl):207-211.
JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 Suppl. – July 2001
210
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
-
also
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
HCO3
-
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
-
/HCO3
-
exchange [13, 14]. In
contrast, cAMP-stimulated HCO3
-
secretion is
unaffected by removal of extracellular Cl
-
and
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
normally.
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-
bis-(beta-aminoethyl
ether)-N,N'-tetraacetic
acid; EL: extracellular loops; HEPES: N-2-
hydroxyethylpiperazine-N'-2-ethanesulfonic
acid; PDC: pancreatic duct cell
Acknowledgements Funded by grants from the
Cystic Fibrosis Trust (UK) and the Wellcome
Trust.
Correspondence
Mike A Gray
Department of Physiological Sciences
University Medical School
Framlington Place
Newcastle upon Tyne NE2 4HH
United Kingdom
 
References
1. Argent BE, Gray MA. Regulation and formation of
fluid and electrolyte secretions by pancreatic ductal
epithelium. In: Sirica AE, Longnecker DS, eds. Biliary
and Pancreatic Ductal Epithelia. Pathobiology and
Pathophysiology. New York, NY, USA: Marcel Dekker,
1997: 349-77.
2. Linsdell P, Hanrahan JW. Glutathione permeability
of CFTR. Am J Physiol 1998; 275:C323-6. [98355720]
3. Linsdell P, Hanrahan JW. ATP-dependent
asymmetry of anion permeation in the cystic fibrosis
transmembrane conductance regulator chloride channel. J
Gen Physiol 1998; 111:601-14. [98190100]
4. Smith JJ, Welsh MJ. cAMP stimulates bicarbonate
secretion across normal but not cystic fibrosis airway
epithelia. J Clin Invest 1992;89:1148-53. [92210710]

Page 5
JOP. J. Pancreas (Online) 2001; 2(4 Suppl):207-211.
JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 Suppl. – July 2001
211
5. Ballard ST, Trout L, Bebok Z, Sorscher EJ, Crews
A. CFTR involvement in chloride, bicarbonate, and
liquid secretion by airway submucosal glands. Am J
Physiol 1999; 277:L694-9. [99447278]
6. Hogan DL, Crombie DL, Isenberg JI, Svendsen P,
Schaffalitzky de Muckadell OB, Ainsworth MA. CFTR
mediates cAMP- and Ca2+-activated duodenal epithelial
HCO3
secretion. Am J Physiol 1997; 272:G872-8.
[97287792]
7. Fitz JG, Cohn JA. Biology and pathophysiology of
CFTR and other Clchannels in biliary epithelial cells.
In: Sirica AE, Longnecker DS, eds. Biliary and
Pancreatic Ductal Epithelia. Pathobiology and
Pathophysiology. New York, NY, USA: Marcel Dekker,
1997: 107-25.
8. Curtis CM, Martin LC, Higgins CF, Colledge WH,
Hickman ME, Evans MJ, et al. Restoration by
intratracheal gene transfer of bicarbonate secretion in
cystic fibrosis mouse gallbladder. Am J Physiol 1998;
274:G1053-60. [98359065]
9. Durie PR, Forstner GG. Pathophysiology of the
exocrine pancreas in cystic fibrosis. J R Soc Med 1989;
82 (Suppl. 16):2-10. [89258675]
10. Gray MA, Greenwell JR, Argent BE. Secretin-
regulated chloride channel on the apical plasma
membrane of pancreatic duct cellsJ Membr Biol 1988;
105:131-42. [89110954]
11. Gray MA, Pollard CE, Harris A, Coleman L,
Greenwell JR, Argent BE. Anion selectivity and block of
the small conductance chloride channel on pancreatic
duct cells. Am J Physiol 1990; 259:C752-61. [91051856]
12. Gray MA, Plant S, Argent BE. Cyclic AMP-
regulated whole-cell chloride currents in pancreatic duct
cells. Am J Physiol 1993; 264:C591-602. [93212770]
13. Sohma Y, Gray MA, Imai Y, Argent BE. A
mathematical model of the pancreatic ductal epithelium.
J Membr Biol 1996; 154:53-67. [97045048]
14. Ishiguro H, Steward MC, Wilson RW, Case RM.
Bicarbonate secretion in interlobular ducts from guinea-
pig pancreasJ Physiol 1996; 495:179-91. [97019906]
15. Ishiguro H, Naruse S, Steward MC, Kitagawa M, Ko
SB, Hayakawa T, Case RM. Fluid secretion in
interlobular ducts isolated from guinea-pig pancreasJ
Physiol 1998; 511:407-22. [98372828]
16. Sohma Y, Gray MA, Imai I, Argent BE. 150 mM
HCO3
- How does the pancreas do it? Clues from
computer modelling of the duct cell. JOP. J Pancreas
(Online) 2001; 2(4 Suppl.): 198-202.
17. O'Reilly CM, Winpenny JP, Argent BE, Gray MA.
Cystic fibrosis transmembrane conductance regulator
currents in guinea-pig pancreatic duct cells and their
inhibition by bicarbonate ions. Gastroenterology 2000;
118:1187-96. [20295052

There are no products listed under this category.