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¹ Division of Maternal and Child Health Sciences, University of Dundee, Dundee DD1 9SY, UK

Abstract

A considerable body of evidence indicates that the intracellular chloride concentration ([Cl^–]_i) is an important regulatory signal in epithelial ion transport. [Cl^–]_i regulates the open channel probability of sodium and chloride channels, the rate of chloride channel recycling to the apical membrane, cell volume homeostasis, the activity of sodium-coupled chloride entry pathways and G-protein activity. Cell volume goes awry in epithelial cells bearing mutant forms of the cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR); however, the pathways that mediate this [Cl^–]_i effect at the apical membrane of polarized epithelia are unknown. Recently, we proposed a mechanism for the transduction of in vitro chloride concentration into a phosphorylation signal to proteins within the apical membrane of respiratory epithelia. Our studies show that an apically enriched plasma membrane fraction from a variety of species, including sheep, human and mouse airway, contains at least two membrane-bound protein kinases which exhibit a number of novel properties. Firstly, the phosphate is located on histidine residues within different families of proteins; one kinase(s) utilizes GTP rather than ATP as a phosphate donor and each kinase has its own unique profile of membrane protein phosphorylation (which itself varies with anion species). Secondly, both kinases mediate Cl^–-dependent phosphorylation of an apical membrane protein around the established physiological values for [Cl^–]_i in airway epithelial cells (40 mM); associated phosphatases also alter the net phosphoprotein profile of the apical membrane. These findings are reviewed and their potential roles explored in relation to the pathogenesis of CF using the control of cell volume as a model for disrupted cellular function in CF-affected epithelia.

(Received 16 September 2005; accepted after revision 6 October 2005; first published online 11 October 2005)
Corresponding author A. Mehta: Division of Maternal and Child Health Sciences, University of Dundee, Dundee DD1 9SY, UK. Email: a.mehta{at}dundee.ac.uk

Cystic fibrosis (CF) diminishes salt and fluid secretion by inducing dysfunction of more than one ion channel protein in mucus-secreting glands and surface epithelial cells (Kerem et al. 1989; Gabriel et al. 1993; Li et al. 1993; Prat et al. 1995; Stutts et al. 1995; Hanrahan et al. 1998). The mechanisms by which mutated or absent CF transmembrane conductance regulator protein (CFTR) causes such defects are both complex and disputed. There is a measure of agreement that the mucus found in CF lung is abnormal, and that fluid secretion is both attenuated and of abnormal composition after cell stimulation via cAMP-dependent pathways. For example, pharmacological inhibition of CFTR can reproduce CF-like lung pathology in a pig mucus gland model (see review Thiagarajah & Verkman, 2003). In such experiments and in CF-affected epithelia as a whole, and especially gland ducts, fluid, chloride and bicarbonate secretion fails when CFTR is mutated and, in some cases, precipitated mucus can obstruct the duct lumen. The complexity arises because CFTR is also controlled by other processes involving the cytoskeleton (Prat et al. 1995), and mutated CFTR generates excessive neutrophil attraction despite a sterile lumen in human fetal CF lung explants engrafted into nude mice (Tirouvanziam et al. 2002). The mechanisms are unknown. Nor is it understood why so many different ion channels are affected by mutated CFTR.

There are two discrete approaches to investigating the malfunction of CFTR in CF: reductive and integrationist. In the reductive approach clones are made of each component (transporter, channel etc.), the regulation of each is examined in turn, and attempts are made to find associated proteins. This is beginning to yield fruit after more than a decade of searching for regulatory factors to explain how [Cl^–]_i controls the entry of chloride through the Na⁺–K⁺–2Cl^– cotransporter (Dowd & Forbush, 2003), but nevertheless requires testable hypotheses derived from the second approach. In the second, integrationist approach, cell physiology techniques are applied to ‘nature-supplied integrators’ to gain insight into some aspect of the process as a whole, looking to generate a molecular pathway (Jentsch et al. 2005). As detailed elsewhere, CF is a good example of a natural integration because so many physiological functions are abnormal (Mehta, 2005). Thus, the present review begins with the second approach, using epithelial cell volume control as a model to illustrate the potential role of the CFTR as an integrator of different cell functions. Thereafter, we describe progress with a parallel protein purification and cloning approach involving the transfer of the terminal () phosphate from ATP and GTP to histidine residues in membrane proteins. Our aim is to familiarize the reader with our working hypothesis about the pathogenesis of CF at the apical membrane of lung epithelium.

The integrative approach: why is cell volume control so critical for an epithelium?

CF is associated with disordered cell volume regulation in small intestinal crypts, as reported by Valverde et al. (1995). This observation has been confirmed by others in the intestine (Seidler et al. 2001), but is not confined to gut because, when CFTR is mutated, cell volume control is also impaired in the airway (Vazquez et al. 2001), nephron (Barriere et al. 2003) and cholangiocyte (Cho et al. 2004). Our unpublished observations (Treharne KJ) show that when CFTR is mutated in bronchial cell lines, CF cells have a different shape compared to wild-type controls. Although the baseline morphology of native airway epithelium is not affected, the above findings may have clinical relevance in that inhaling water without isosmolar salt is predicted to make airway cells swell, and a compensatory regulatory volume decrease will occur by shedding epithelial osmolytes. Further, it is recognized that CF airway cells from patients are swollen (oedematous), a feature usually attributed to coexisting infection. Conversely, should the airway ductal epithelium within glands secrete fluid, ductal cell volume will fall when cellular electrolytes, and so water, enter the lumen. In the face of this threat to cell volume, epithelial cells compensate by recruiting volume from the blood-facing extracellular (basolateral/abluminal) space in order to continue to fill the luminal space. Epithelia can only do this if they can manipulate their intracellular osmolyte composition by activation of inwardly directed ion transport from the external basolateral reservoir. That process is dependent in part on intracellular chloride concentration ([Cl^–]_i; Dowd & Forbush, 2003). In CF, the evidence for dysfunction of the surface apical reservoir has recently been described and is not considered further here (Boucher, 2004). In rat bronchial cells in culture, cell volume change can be very rapid, occurring in seconds to minutes (Shiima-Kinoshita et al. 2004). With the cautionary note that neither a basolateral space nor a bronchial arterial perfusion to maintain fluid pressure in that space are present in vitro, this study found that epithelial cell shrinkage occurs following exposure to terbutaline concentrations in the nanomolar range (Shiima-Kinoshita et al. 2004). Terbutaline is predicted to activate CFTR. These authors also report that the volume-altering effect of this ß₂-agonist could be reproduced in the absence of drug by elevating intracellular cAMP using forskolin, an agent that stimulates enzymatic cAMP synthesis from ATP. Conversely, the effect disappeared when the downstream effector of cAMP, protein kinase A, was blocked. If these effects have a clinical correlate, where such drugs are widely used, their application should reduce epithelial swelling and increase airway lumen diameter.

How does ß-agonist-induced volume decline happen in rat bronchus?

The fact that the exit of intracellular potassium ions plays a critical role in cell volume regulation was shown by the observation that blockade of potassium channels with quinidine reversed terbutaline-induced shrinkage, i.e. the cell volume returned to the preterbutaline level despite the on-going presence of terbutaline (Shiima-Kinoshita et al. 2004). These researchers also found that in the absence of external ß₂-agonist, quinidine alone could also increase resting epithelial cell volume, suggesting that there exists a silent, steady-state potassium loss from the airway cell that is involved in setting basal cell volume. This is relevant firstly because there exists a small basal rate of cAMP production in all cells but its role in cell volume control is unknown and secondly because the regulation of some potassium channels mediating cell volume control fails when CFTR is mutated (Vazquez et al. 2001).

The key roles of intracellular chloride concentration

The role of [Cl^–]_i during volume change is pertinent because significant numbers of potassium ions cannot move out of the cell in isolation because of the retarding effect of the build-up of positive counter-charge across the traversed membrane with each transported cation. To neutralize charge build-up, the intracellular chloride pool is used to supply the neutralizing anion to the other side of the epithelial membrane, thus not only maintaining electroneutrality, but also allowing a quantitatively significant number of osmolytes to move (Willumsen et al. 1994). However, to sustain secretion, this intracellular chloride pool must be replenished (e.g. from the basolateral space, but see Gawenis et al. 2004 for a discussion of control/collapse of that space in vivo after the onset of secretion, and equivalent data from the lung by Willumsen et al. 1994)

[Cl^–]_i is dependent on the balance between ‘accumulative’ chloride influx and ‘dissipative’ efflux across the plasma membrane (Willumsen et al. 1989, 1994). It has to be remembered that chloride in the basolateral space will not enter an epithelial cell by diffusion down its concentration gradient (see below), and ‘accumulative’ implies that energy is needed. That energy is supplied indirectly as the potential energy derived from the inwardly directed sodium ion gradient to drive chloride ‘uphill’ into the cytoplasm, typically using the frusemide-sensitive Na⁺–K⁺–2Cl^– cotransporter (the roles of other sodium-driven transporters are ignored for simplicity). Once again, in the study by Shiima-Kinoshita et al. (2004) the basal role of this cotransporter was shown by the fall in bronchial cell volume in the presence of the Na⁺–K⁺–2Cl^– blocker, frusemide. Conversely, the term ‘dissipative’ refers to energetically favourable or ‘downhill’ chloride movement out of the cell. Critically, [Cl^–]_i itself controls an unusual proline/alanine-rich protein kinase that gates its own entry (Dowd & Forbush, 2003).

Directional control of chloride transport is often called ‘vectorial control’ and depends on the following critical functions. Firstly, influx and exit occur differently in different cells using different proteins that are differently regulated. This prevents signal confusion and is illustrated by comparing the findings of Schwiebert et al. (1994) at the apical membrane with Dowd & Forbush (2003) at the basolateral membrane. Thus the ‘non-blood-facing’ or apical membrane contains different transporters and channels compared to the capillary-facing basolateral membrane. This is elegantly reviewed elsewhere (Jentsch et al. 2005). In fact, further subsegregation occurs because, within an apical membrane, transport proteins congregate further in lipid microdomains called lipid rafts (Fricke et al. 2003), but the predicted consequences for selective fatty acid interactions with the enriched proteins that could facilitate multimer formation (Schillers et al. 2004) are beyond the scope of this review and are discussed elsewhere (Mehta, 2005); an important caveat was revealed in data presented at the Physiology of Anion Transport meeting (Bates et al. 2005), showing that 20% of CFTR resides in clathrin-coated pits and not in lipid rafts. Secondly, these chloride pathways are spatially separated within the different faces of epithelial cells. Thirdly, the direction of chloride flux is principally driven by the magnitude of the (negative-inside) transmembrane potential difference; this is set up by the outward movement of potassium ions and does not necessarily follow the expected direction based on the lower intra- versus extracellular chloride concentrations found in vivo (40–50 and 90–110 mM, respectively); note also the contrast with (intracellular) 10–20 versus (extracellular) 130–140 mM for Na⁺, where concentration difference and potential difference are vectorially additive. This leads to the counter-intuitive result that, despite having more chloride outside than inside, chloride may move out of the epithelial cell provided a gate is open. It is vital to note that across the apical membrane, Cl^– might be at a steady state, with almost balanced opposite voltage and concentration gradients. This means that Cl^– could move in either direction as required. CFTR provides one of a number of such Cl^– gates, but one that may also pass bicarbonate depending on the external chloride concentration (Shcheynikov et al. 2004). Again, the mechanisms are disputed.

Generating vectorial transport

That chloride needs to cross membranes via different transporters and channels itself needs explanation. Why do we need both types of transport? Intracellular potassium accumulates to 120 mM, 30 times above extracellular levels. This elevated potassium concentration requires energy consumption because potassium is driven into the cell by consuming ATP whilst simultaneously pumping out a relative excess of sodium. The net result is that intracellular sodium concentration lies 10-fold below the prevailing potassium concentration (sodium typically around 10 mM). Due to a large number of potassium-selective pore proteins, epithelial cells are inherently leaky to the resulting high concentration of intracellular potassium ions. This steady-state loss maintains the inside of the cell relatively negative, as the positive charge continuously exits the cell through a variety of potassium channels (Ito et al. 2004), each with its own form of regulation (small molecules such as calcium, or voltage etc.), which can in turn trigger release of autocrine/paracrine factors controlling chloride secretion (Ito et al. 2004). The combination affords fine control when coupled to chloride-regulated chloride entry (Dowd & Forbush, 2003). These ideas led us to the notion that a regulator must exist, and the known roles of CFTR made this protein a good choice to study. CFTR is not just one such chloride gatekeeper amongst many, controlling chloride exit, but remains critical for ionic flux through channels unrelated to CFTR. For example, in the airway CFTR also regulates sodium channels (Stutts et al. 1995) and somehow controls bicarbonate flux to alkalinize the secreted fluid (Shcheynikov et al. 2004). How can CFTR perform such diverse roles if it is only a chloride channel?

Chloride controls chloride transport

In epithelia, [Cl^–]_i (typically at 40 mM, three-fold less than intracellular potassium concentration) itself regulates the channels and transporters that accumulate or dissipate chloride. This anion also regulates sodium entry (Robertson & Foskett, 1994). There must be a signal, but the feedback mechanisms which signal [Cl^–]_i to the regulatory pathways for entry/exit are not fully understood. [Cl^–]_i regulates such a diversity of cellular functions (see references in the papers by Robertson & Foskett, 1994, Lytle & McManus, 2002, Devuyst & Guggino, 2002 and Shiima-Kinoshita et al. 2004), including the open channel probability of sodium channels and the CFTR chloride channel, the rate of chloride channel recycling to the apical membrane, cell volume homeostasis and G-protein activity, that there must either be a specific mechanism for each aspect or there exists a latent common regulatory pathway. Here, we argue for the latter hypothesis, suggesting that protein phosphorylation could be involved – albeit using an unusual histidine residue to relay the signal.

Accidents will happen: science and serendipity

In the years immediately after the CFTR was discovered in 1989, there was no molecular explanation for the observation that chloride must be able to signal its concentration to membrane proteins (Robertson & Foskett, 1994), not only those in the apical membrane of the airway epithelium but also those in the basolateral membrane (Lytle & McManus, 2002). To address the problem of how chloride might signal its abundance to the airway membrane, Anil Mehta (A.M.) began by isolating apical membranes from human airway biopsies and exposing them to different chloride concentrations in vitro. Specifically, the fate of the terminal phosphate from ATP within a membrane environment was investigated to determine whether ion species critically determine the destination of this phosphate in vitro. Prior to the cloning of CFTR, A.M. applied conventional approaches, loading intact, beating, ciliated epithelial cells with ³²PO₄^– to fill the cytosolic ATP pool with tracer. The initial answer was not encouraging, with a paucity of labelled membrane proteins (reviewed by Treharne et al. 2001b). But a series of accidents followed when A.M. killed a set of nasal brushings by over-forceful centrifugation and (on the advice of his supervisor, A. W. Segal in London) compensated by adding radiolabelled ATP species to plasma membranes isolated in a cytosol-free manner using sucrose gradients. The critical next step was discovered by chance late one Friday evening, when A.M. added the ‘wrong’ neutralizing alkali when making up membrane buffers. Hitherto, all solutions had used sodium hydroxide to create physiological pH but, on this occasion, potassium hydroxide was used by mistake, being adjacent on the shelf. When chloride concentration was changed in the absence of sodium, the terminal, radiolabelled ³²PO₄^– from ATP moved from free solution to membrane proteins (see Figs 1 and 2 and Treharne et al. 1994). It was noted, however, that exposure of the gels to acid during staining reduced the membrane protein counts considerably. The significance of this finding only became clear much later.

View larger version (88K): [in this window] [in a new window]   Figure 1.  Isolating apical membranes from human nasal epithelium Apical membrane proteins isolated from human nasal brushings are shown in lane 2 of the gel stained with Coomassie Blue (molecular weight markers in kilodaltons in lane 4, marked on the right). Lane 3 shows cytosolic proteins for comparison at the same protein loading. Lane 1 is an autoradiograph of the subset of phosphorylated proteins found in the apical fraction (lane 2) when ATP containing a radiolabel on the terminal phosphate is added in the absence of sodium ions and without staining in acid media (see Treharne et al. 1994). Figure 2 shows the effect of adding chloride or other anions under these circumstances to the apical fraction in vitro.

View larger version (64K): [in this window] [in a new window]   Figure 2.  Phosphorylating apical membrane proteins Autoradiographs of apical membrane phosphoproteins in the presence of different anions (from left to right: 50 mM chloride, sulphate, 10 mM chloride and gluconate). Lane 5 shows the effect of 1 mM chloride (molecular weight markers in kilodaltons on the right).

Further complexity arose from the finding that phosphatases could also play a role, because phosphatase inhibition with phosphothiorate nucleotide analogues also differentially changed the profile of phosphoproteins. Thus, when GTP (or GTPS) was replaced with ATP (or ATPS), not only was a different chloride-dependent profile of phosphorylated membrane proteins generated, but the chloride dependence of these membrane-associated phosphoproteins also differed, and was dependent on the anion chosen to replace the chloride (gluconate, nitrate or sulphate). The pH of the solutions was also critical, with significantly less phosphotransfer below pH 7. Additionally, the rank order of the anion-dependent intensity of labelling disappeared when hydrolysis-resistant ATP (but not GTP) was present, suggesting a complex role for dephosphorylation. This provided a clue to the ‘different signal’ we were looking for, because non-hydrolysable ATP/GTP and hydrolysable ATP/GTP affected different proteins differently. Because the profile of these phosphorylated proteins could not be altered by classical phosphatase inhibitors, such as okadaic acid, this suggested that a novel ‘pathway’ had been discovered.

Further work showed that anion species were independent determinants of the phosphorylation state of multiple apical membrane proteins from purified human airway epithelial membranes in vitro. Thus, anions could differentially alter apical membrane protein structure. The fact that this occurred in the absence of added magnesium was also puzzling because MgATP is normally the preferred substrate for classical protein kinases. Later, a Wellcome Prize PhD student, Lindsay Marshall, confirmed the inhibitory effect of sodium ion concentrations in excess of 10 mM, but noted that this inhibition was confined to the ATP pathway (Marshall et al. 1999). This provided a third way to differentiate the sodium and chloride ‘signals’, but posed a problem because the signals could still theoretically interfere with cyclic-AMP/PKA pathway. This problem was solved when we attempted to identify the phosphorylated residues and found that the ATP- and GTP-donated phosphate on the phosphorylated membrane proteins disappeared completely under acid conditions (Muimo et al. 1998). This was unusual because, following activation of most protein kinases, the terminal ATP phosphate in mammalian cells commonly covalently binds to serine/threonine or tyrosine residues, both of which are stable to acid. This combination of findings provided a further clue to the ‘other signal’, because nitrogen-linked phosphate is acid unstable (Matthews, 1995; Muimo et al. 2000). This suggested that the amino acid histidine, which can accept phosphate at two sites on its nitrogen-containing ring, was a likely candidate for phosphorylation.

Why is apical membrane phosphohistidine present?

Phosphohistidine has four interesting potential properties in relation to epithelial function (Matthews, 1995; Muimo et al. 2000). Firstly, it is very common in mammalian cells and may be even more prevalent than its better-understood cousin, phosphotyrosine (Matthews, 1995). Secondly, the addition and subtraction of phosphate from histidine is close to equilibrium and is therefore both reversible and transferable as high-energy phosphate to other cellular proteins (Matthews, 1995). Most of the 28 kJ mol^–1 of energy available following hydrolysis of ATP to ADP is therefore preserved for use in cellular processes requiring extra energy. In lower evolutionary orders, it is not uncommon for the phosphate to be sent in relay between multiple histidines, alighting on different proteins and transiently altering function as it moves. The simplest analogy is the cascade effect of dominoes as they topple over in relays, each changing the function of the nearest neighbour until a final effector is reached, utilizing the energy of the wave. This relay does not occur for phosphotyrosine or phosphoserine, which each carry one-quarter of the energy of phosphohistidine and remain resolutely resident until removed by local phosphatases. Thirdly, the addition of phosphate to this amino acid can profoundly alter protein function by a different mechanism from phosphoserine and phosphotyrosine. Because the 50% protonation value for histidine residues in intracellular proteins lies very close to physiological intracellular pH (the histidine nitrogen can be protonated or neutral), the addition of phosphate can alter protein function in multiple ways. Thus, for dephosphorylated histidine, small changes in pH can dramatically alter the positive charge on histidine, but the negatively charged phosphohistidine is less affected. Fourthly, although the proteins adding and subtracting phosphohistidine remain poorly characterized (Matthews, 1995), in lower organisms this system of protein histidine phospho-relay is used as a signalling cascade to sense external signals such as osmolar environment in order to mitigate against volume change (Matthews, 1995). The only reason that this system of phosphorylation is so poorly characterized in mammals is that the relatively high-energy status of phosphohistidine (needed to transfer the phosphate) makes it very unstable. To solve this problem, Richmond Muimo (R.M.) joined the group and overcame many of these difficulties between 1998 and 2000, and began the task of cloning the recipient proteins.

The search for function

At the stage of writing, we cannot definitely tie our observations to CF-related function in the airway but, nevertheless, our findings challenge the assumption that apical membranes are inert structures with respect to ion concentration when nucleotides are present (Treharne et al. 1994, 2001b; Muimo et al. 1998, 2000; Marshall et al. 1999). Using an apical fraction from sheep tracheal epithelium that also manifests an equivalent Cl^–-dependent phosphorylation cascade to that found in the human airway, R.M. identified the first of the underlying proteins by purification and protein sequencing (Muimo et al. 1998). The data suggest that transient phosphorylation of nucleoside diphosphate kinase (NDPK), a protein histidine kinase that regulates the cellular secretory pathway and potassium channels, amongst its many different functions (Parks & Agarwal, 1973; Kadrmas et al. 1991; Heidbuchel et al. 1993; Blevins et al. 1994; Maeda et al. 1994; de la Rosa et al. 1995; Wagner & Vu, 1995; Dabernat et al. 1999; Jacobs et al. 1999) precedes the phosphorylation of most other phosphohistidine-containing proteins in this system, such as annexin I (Muimo et al. 2000), a key regulator of inflammation. We note that both proteins manifest ion-sensitive phosphorylation on histidine residues and both regulate processes that are defective in CF. These proteins do not manifest ion-sensitive phosphorylation when purified to homogeneity, but require interactions within the apical membrane environment to become chloride ‘sensors’. The combined data suggest that the apical membrane contains a signal system using phosphohistidine to ‘inform’ membrane proteins about ambient ion concentrations. The most relevant known relationship of NDPK to that notion and cell volume control is its function as a G-protein and potassium channel regulator (Heidbuchel et al. 1993). However, for annexin I our finding of the presence of phosphohistidine was a novel result (Muimo et al. 2000), and the relationship between phosphohistidine, annexin I and inflammation remains unknown. R.M. is now an independent researcher working in this area.

Others immediately challenged our view that the protein histidine kinase NDPK is involved in epithelial ion transport. For example, David Cook (Sydney) had found that [Cl^–]_i can regulate sodium and chloride transport by a process involving G-proteins (Komwatana et al. 1998), but concluded that because classical inhibitors of NDPK fail to alter the effect of chloride on the transport of sodium and chloride, NDPK is unlikely to be involved. Unfortunately, in our hands such inhibitors have no effect on the chloride-dependent cascade (R. Muimo and A. Mehta, unpublished observations), so that matter remains unresolved. One potential explanation is that NDPK in membranes does not act alone and binds to another protein. Our recent data suggest that AMP-activated protein kinase (AMPK; Best et al. 2001; Treharne et al. 2001a; Crawford et al. 2005) can act in this manner and specifically binds to the NDPK-A isoform. This is exciting because Hallows and Foskett have shown that AMPK inhibits CFTR conductance and binds to CFTR near its C terminus (Hallows et al. 2000, 2003a,b). The relationship between that finding and our finding of disrupted phosphorylation of NDPK in CF epithelium (summarized schematically in Fig. 3; Best et al. 2001; Treharne et al. 2001a) are the subject of current investigation.

View larger version (46K): [in this window] [in a new window]   Figure 3.  Cartoon showing metabolic proteins interacting with CFTR ‘A’ represents the adenosine of ATP; ‘P’ represents phosphorylation sites.

We contend that the multiple functions of NDPK as a GTP synthase (from ATP and GDP), dual serine/histidine protein kinase and regulator of AMPK (Crawford et al. 2005) make its regulation a matter of importance for epithelial function. NDPK generates cellular GTP in a membrane-delimited pool that is different from that in the cytosol because it is channelled to G-proteins in a hormone-sensitive manner (de la Rosa et al. 1995). In other tissues, NDPK co-immunoprecipitates with at least one G-protein and may also be located within the secretory pathway from the Golgi apparatus to the apical membrane (Marciniak & Edwardson, 1996). GTP is also essential for cell secretion. Consistent with this theme, NDPK is a regulator of K⁺ channels, which are essential for ionic charge balance during cellular secretion (Heidbuchel et al. 1993). NDPK is also able to bind to cAMP (Anciaux et al. 1997), providing further potential linkage between accumulative and dissipative regulatory signals in ion transport. Thus, our data add a new dimension, because we find that a membrane-bound form of NDPK is present in the apical membrane of the airway epithelium, a site which is critical for epithelial ion transport, volume regulation and cellular secretion, processes which are known to depend on chloride ions and GTP. Understanding the role of this protein kinase on cell volume control and its effect on the phosphohistidine content of the airway membrane remains our target in years to come, hopefully to the ultimate benefit of CF patients.

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Acknowledgements

This review would not have been possible without funding from the Wellcome Trust, the Cystic Fibrosis Trust and the Anonymous Trust.

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