|Mike A Gray*, Catherine O’Reilly1, John Winpenny2 and Barry Argent
Department of Physiological Sciences, University Medical School. Newcastle upon Tyne, United Kingdom.
1Biomedical Imaging Group, Department of Physiology, University of Massachusetts Medical Centre. Worcester, MA, USA.
2School of Health Sciences, University of Sunderland. Sunderland, United Kingdom
Mike A Gray
Department of Physiological Sciences
University Medical School
Newcastle upon Tyne NE2 4HH
E-mail [email protected]
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.
|Chloride Channels; Cystic Fibrosis; Ion Transport; Pancreas; Sodium Bicarbonate|
|CF: cystic fibrosis; CFTR: cystic fibrosis transmembrane conductance regulator; DIDS: 4,4'-diisothiocyanatostilbene- 2,2'-disulphonic acid; EGTA: ethyleneglycolbis-( beta-aminoethyl ether)-N,N'-tetraacetic acid; EL: extracellular loops; HEPES: N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid; PDC: pancreatic duct cell|
|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- , glutathione  and larger organic anions . 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 , including submucosal glands ; the gastro-intestinal tract ; the liver and gallbladder [7, 8] and the pancreas , 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 . We proposed back in 1988 that HCO3- exits across the apical membrane of pancreatic duct cells (PDCs) by parallel operation of CFTR Clchannels and Cl-/HCO3- exchangers . 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 Claccumulated 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  and whole cell current recordings , 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 . 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 ).|
Extracellular HCO3- Blocks Cl- Efflux through CFTR
|During recent anion permeability studies from native guinea pig PDCs, we observed an unexpected and novel effect of extracellular HCO3- on cAMP-activated CFTR Cl- currents . 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 Clconcentration was unchanged. The reduced inward current indicates that external HCO3- is causing ‘trans’ inhibition of Cl- efflux.|
|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 mM forskolin and 100 mM dibutyryl cAMP) that activate PKA (b and c). Whole cell currents were obtained by holding the membrane potential (Vm) at 0 mV and clamping Vm to ±100 mV in 20 mV steps. The pipette solution contained (mM): 110 CsCl, 2 MgCl2, 5 ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'- tetraacetic acid (EGTA), 10 N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 1 Na2ATP, 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 NaHCO3 and CaCl2 was omitted from the solution (pH about 8.0). For further details on cell preparation and electrophysiology see .|
|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 Cl- currents. 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 ).|
|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 pCO2 fourfold (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 .|
|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 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 .|
|Figure 3: Positively charged residues in the extracellular loops (EL) of human CFTR. Abbreviations used. H: Histidine, K: Lysine and R: Arginine.|
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.|
|Funded by grants from the Cystic Fibrosis Trust (UK) and the Wellcome Trust.|
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