Mechanisms of the inhibition of epithelial Na+ channels by CFTR and purinergic stimulation

Kidney International, Vol. 60 (2001), pp. 455–461

Mechanisms of the inhibition of epithelial Na⫹ channels by CFTR and purinergic stimulation KARL KUNZELMANN, RAINER SCHREIBER, and ANISSA BOUCHEROT Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Brisbane, Australia

Mechanisms of the inhibition of epithelial Naⴙ channels by CFTR and purinergic stimulation. The epithelial Na⫹ channel ENaC is inhibited when the cystic fibrosis transmembrane conductance regulator (CFTR) coexpressed in the same cell is activated by the cyclic adenosine monophosphate (cAMP)-dependent pathway. Regulation of ENaC by CFTR has been studied in detail in epithelial tissues from intestine and trachea and is also detected in renal cells. In the kidney, regulation of other membrane conductances might be the predominant function of CFTR. A similar inhibition of ENaC takes place when luminal purinergic receptors are activated by 5⬘-adenosine triphosphate (ATP) or uridine triphosphate (UTP). Because both stimulation of purinergic receptors and activation of CFTR induce a Cl⫺ conductance, it is likely that Cl⫺ ions control ENaC activity.

purinergic receptors in airway epithelial cells not only leads to activation of a luminal Cl⫺ conductance that is different to CFTR, but it also inhibits ENaC. Because both CFTR and uridine triphosphate (UTP) activate Cl⫺ conductance, it was suggested that changes in the intracellular Cl⫺ concentration during the course of activation of Cl⫺ conductances are correlated to the inhibition of ENaC. Naⴙ ABSORPTION IS CONTROLLED BY CFTR: POSSIBLE IMPACT OF PDZ DOMAIN PROTEINS The mechanisms for interaction of CFTR with ENaC are currently unknown, although recent studies have demonstrated the importance of the first nucleotide binding fold of CFTR for inhibition of ENaC [6]. Several models for the interaction of both proteins have been suggested such as direct protein interaction, interaction via cytoskeletal elements, and the participation of a C-terminal PDZ-binding domain of CFTR. An important role has been attributed to this PDZ-binding domain in protein kinase A (PKA)-dependent regulation of CFTR (Fig. 2). It has been shown that CFTR interacts with the ezrinbinding phosphoprotein 50 (EBP50), the Na⫹/H⫹ exchange regulatory factor (NHERF) and the Na⫹/H⫹ exchanger type 3 kinase A regulatory protein (E3KARP). Since ezrin itself is an A kinase anchoring protein (AKAP), it has been suggested that PKA is translocated to close proximity of CFTR, allowing efficient activation of CFTR [7–9]. In addition, CFTR might be anchored to the luminal membrane via scaffolding proteins and ezrin, which is known to bind to the actin cytoskeleton [8, 10]. Finally, the PDZ domain containing scaffolding proteins would offer a novel way of how CFTR controls the activity of other ion channels such as ENaC (Fig. 2). In that respect, it is noteworthy that EBP50 interacts with the Yes-associated protein YAP65 that recruits both YAP65 and c-Yes to the apical plasma membrane. The nonreceptor tyrosine kinase c-Yes and other Src family kinases are known to regulate ion channel activity [11].

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic adenosine monophosphate (cAMP)-regulated Cl⫺ channel that is defective in cystic fibrosis. A large body of evidence indicates that CFTR is also a regulator of other membrane conductances. There is a growing list of interactions with other ion channels and the control of various cellular processes [1]. A pathophysiologically relevant example is the inhibition of epithelial Na⫹ channel, (ENaC) by CFTR. ENaC is expressed in epithelial tissues such as the kidney collecting duct, colonic epithelium, and airways. It is essential for electrogenic absorption of electrolytes and thus participates in regulation of blood pressure, intestinal function, and mucociliary clearance of the airways. Understanding the regulation of ENaC is therefore essential, allowing some control over tissue functions. The epithelial Na⫹ channel ENaC is inhibited when the CFTR is activated by the cAMP-dependent pathway (Fig. 1). This phenomenon has been studied in detail in epithelial tissues from intestine, trachea, and kidney [2–4], and it explains why in CF an enhanced Na⫹ conductane is detected in parallel with a defect in cAMP-regulated Cl⫺ conductance [5]. Recent studies have found that activation of luminal Key words: ENaC, cystic fibrosis, UTP, ATP, purinergic stimulation, transport proteins.

 2001 by the International Society of Nephrology

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Kunzelmann et al: Inhibition of epithelial Na⫹ channels Fig. 1. Cell models for both (A) cystic fibrosis transmembrane conductance regulator (CFTR)dependent inhibition of epithelial sodium channel (ENaC) and (B) inhibition of ENaC by activation of luminal purinergic P2Y2 receptors by adenosine triphosphate (ATP) or uridine triphosphate (UTP) in airway epithelial cells. (A) Protein kinase A (PKA)-dependent activation of CFTR in a CFTR/ENaC coexpressing epithelial cell probably leads to an initial influx of Cl⫺ and an increase of luminal intracellular Cl⫺ concentration. Activation of CFTR Cl⫺ conductance leads to parallel inhibition of epithelial Na⫹ channels. (B) Stimulation of purinergic receptors located in the luminal membrane of airway epithelial cells activates luminal outwardly rectifying (ORCC) or Ca2⫹-dependent (CACC) Cl⫺ channels probably by an increase in intracellular Ca2⫹. Activation of luminal Cl⫺ channels probably leads to an influx of Cl⫺ into the cell and an increase of intracellular Cl⫺ concentration. Stimulation of luminal Cl⫺ conductance leads to a parallel inhibition of epithelial Na⫹ channels.

Fig. 2. Hypothetical arrangement of cystic fibrosis transmembrane conductance regulator (CFTR), epithelial sodium channel (ENaC), and accessory PDZ domain proteins in a functional membrane microdomain. PDZ domain proteins such as Na⫹/H⫹ exchange regulatory factor (NHERF), ezrin binding phosphoprotein 50 (EBP50), or Na⫹/H⫹ exchanger type 3 kinase A regulatory protein (E3KARP) may contribute to CFTR anchoring and membrane localization, protein kinase A (PKA)-dependent phosphorylation of CFTR, and interaction with other membrane proteins such as ENaC.

So far, little evidence exists that these mechanisms actually take place in the cell. First, ezrin has not been clearly demonstrated to act as an AKAP in the intact cell [8, 9, 11]. Second, preliminary data from our laboratory demonstrate that membrane targeting and activation of CFTR by increase of intracellular cAMP does take place in Xenopus oocytes even in the absence of a functional CFTR–PDZ binding domain (abstract; Boucherot et al, Ion Channel Meeting, Bad Soden, October 30–31, 1999). In contrast, activation of CFTR is only slightly enhanced after coexpression with NHERF and ezrin [8]. Finally, inhibition of ENaC by CFTR does take place even with C-terminal truncated CFTR and is not affected when mu-

tant NHERF1 or NHERF2 is coexpressed in Xenopus oocytes together with CFTR and ENaC (own unpublished data). ACTIVATION OF PURINERGIC RECEPTORS INHIBITS ENaC Epithelial Na⫹ transport is also inhibited by extracellular nucleotides such as adenosine triphosphate (ATP) or UTP by an unknown mechanism (Fig. 3). Purinergic inhibition of ENaC was detected in native and cultured airway epithelial cells and collecting duct epithelial cells [4, 12–14]. Stimulation of luminal purinergic receptors

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Fig. 3. Effects of uridine triphosphate (UTP; 100 ␮mol/L) and amiloride (A; 10 ␮mol/L) on transepithelial voltage (Vte) and transepithelial resistance (Rte) as obtained in a Ussing chamber recording on mouse trachea. Rte was determined from the Vte downward deflections obtained by pulsed current injection. The spontaneous lumen-negative Vte was attenuated by perfusion of the bath with amiloride. Stimulation of the epithelium with luminal UTP induced a transient lumen-negative voltage caused by activation of Cl⫺ secretion. Subsequent application of amiloride in the presence of UTP showed a reduced response, indicating the inhibition of amiloride-sensitive Na⫹ absorption by UTP.

Fig. 4. Model for ion transport in colonic and airway epithelial cells and effects of stimulation with prostaglandin E2 (PGE2) and carbachol. Stimulation with PGE2 activates basolateral K⫹ channels and luminal CFTR Cl⫺ channels, which leads to attenuation of amiloride-sensitive Na⫹ absorption, probably by inhibition of ENaC. Stimulation of basolateral M3 receptors by carbachol increases intracellular Ca2⫹, activates basolateral K⫹ channels, and enhances the driving force for luminal Cl⫺ secretion or Na⫹ absorption. Stimulation with carbachol does not inhibit luminal ENaC conductance.

transiently activates Ca2⫹-dependent Cl⫺ channels of unknown molecular identity (Fig. 1). These Cl⫺ channels may either belong to the class of Ca2⫹-activated CACC channels or may be represented by outwardly rectifying ORCC (ICOR) Cl⫺ channels [1]. In parallel to the activation of a Cl⫺ conductance, UTP inhibits amiloride-sensi-

tive Na⫹ conductance, a phenomenon that takes also place in CF epithelial cells [13, 14]. Inhibition of ENaC during stimulation of purinergic receptors requires Ca2⫹-mediated activation of luminal Cl⫺ channels [13]. Therefore, and in parallel to what has been found for CFTR-dependent inhibition of ENaC [15], activation of Ca2⫹-dependent Cl⫺ channels seems crucial for the inhibition of ENaC by ATP or UTP. Interestingly, stimulation of tracheal epithelial cells by basolateral carbachol also enhances intracellular Ca2⫹ and activates ion transport. However, because no luminal Cl⫺ conductance seems to be activated by carbachol, ENaC is not inhibited (Fig. 4). According to a previously suggested model, ICOR is coactivated with CFTR by an autocrine mechanism based on CFTR-controlled cellular release of ATP [16]. ATP will be secreted to the apical side and bind to purinergic receptors, which couple to ICOR channels that may, in turn, inhibit ENaC. Although appealing, this hypothesis is questionable since (1) CFTR-controlled ATP release is controversial, and (2) CFTR inhibits ENaC in Xenopus oocytes as well as human colonic epithelial cells, although ATP is without effect in these cells [17]. FUNCTION OF PURINERGIC RECEPTORS AND CFTR IN THE KIDNEY Apart from studies on cultured kidney epithelial cells, little is known about the function of purinergic receptors in tubular epithelial cells of the different parts of the nephron [18, 19]. A recent elegant study indicated the presence of luminal P2Y receptors in isolated perfused cortical collecting ducts of mouse but not rabbit [20]. In rat, different

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Fig. 5. Effects of 3-isobutyl-l-methylxanthine (IBMX) and forskolin (100 ␮mol/L and 10 ␮mol/L), amiloride (A; 10 ␮mol/L), and low extracellular bath Cl⫺ concentration on transepithelial voltage (Vte) and transepithelial resistance (Rte) as obtained in a Ussing chamber recording on mouse trachea. Rte was determined from the Vte downward deflections obtained by a pulsed current injection. The spontaneous lumen-negative Vte was attenuated by perfusion of the bath with amiloride. Replacement of most of the extracellular Cl⫺ concentration by gluconate (5 Chloride) led to an increase in transepithelial resistance. Stimulation of the epithelium with IBMX and forskolin in the presence of low extracellular Cl⫺ was without significant effects on Vte. Readdition of Cl⫺ attenuated the amiloride response, suggesting inhibition of ENaC by an increase of intracellular Cl⫺ concentration.

basolateral P2Y receptor subtypes are expressed along the nephron segments [21]. As shown for colonic and airway epithelial cells, large species-specific differences in the expression of purinergic receptors exist. Whether purinergic receptors are present in the luminal membrane of native human nephrons and whether stimulation of these receptors leads to activation of Cl⫺ conductance and inhibition of ENaC are currently unknown. The situation is somehow comparable to expression and function of CFTR in the kidney. CFTR expression was detected all along the kidney tubular system, but evidence for the presence of CFTR Cl⫺ channels is rather limited [22]. In addition, a renal splice variant was detected that probably does not function as an ion channel but might be involved in vesicular trafficking. Also, a basolateral CFTR homologue, EBCR, was found that could participate in absorption of electrolytes in the thick ascending limb of the loop of Henle in conjunction with CLC-Kb [23, 24]. Functional expression of CFTR Cl⫺ channels and activation by an increase of intracellular cAMP has only been demonstrated for cultured collecting duct cells, which do not necessarily resemble the intact collecting duct [4, 25]. In fact, in freshly isolated

rat collecting duct cells, a Cl⫺ conductance could not be detected [26]. Moreover, patients with CF do not develop major renal dysfunctions. Some defects have been identified, like a reduced capacity in both diluting and concentrating urine and a reduced renal NaCl excretion along with enhanced renal drug excretion [27–30]. It is not clear whether these changes in renal function are secondary to an excessive loss of NaCl. However, it is rather likely that a decrease in renal NaCl excretion is caused by malfunction of CFTR, probably because of a lack of ENaC regulation in the collecting duct. This assumption is supported by a recent study demonstrating enhanced amiloridesensitive Na⫹ absorption in CFTR knockout mice during salt restriction [31]. G PROTEINS ARE UNLIKELY TO MEDIATE THE NEGATIVE EFFECTS OF CFTR AND PURINERGIC STIMULATION ON ENaC The finding that inhibition of ENaC by CFTR or purinergic stimulation depends on the presence of Cl⫺ suggests the contribution of Cl⫺-sensitive proteins (Fig. 5). Because Cl⫺-sensitive proteins may participate in the

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Fig. 6. Summaries of experiments examining the contribution of ␣ subunits of trimeric guanosine triphosphate (GTP)-binding proteins to CFTRmediated inhibition of ENaC in Xenopus oocytes. (A) Coexpression of the ␣ subunit of Go (wild-type and dominant-negative form) or ␣Gi2 (wildtype, dominant-negative, and constitutive active forms) had no significant effects on the activity of ENaC when compared with oocytes coinjected with water. (B) The nonhydrolyzable GTP/guanosine diphosphate (GDP) analogues GTP␥s and GDP␤s had no effects on ENaC conductance. (C) Coexpression of ␣Gi2 (wild-type, dominant-negative, and constitutive active forms) together with CFTR and ENaC. In the presence of either of the three types of ␣Gi2, down-regulation of ENaC by stimulation of CFTR with 3-isobutyl-l-methylxanthine (IBMX) and forskolin was abolished. (D) Coexpression of ␣Go (wild-type and constitutive active forms) together with CFTR and ENaC. In the presence of either wild-type ␣Go or dn ␣Go, down-regulation of ENaC by stimulation of CFTR with 3-isobutyl-l-methylxanthine (IBMX) and forskolin was unaffected. (E) Coexpression of regulators of G protein signaling (RGS3, RGS7) or a protein that interferes with RGS function (14-3-3) together with CFTR and ENaC. In the presence of either RGS3 or RGS7 or after coexpression with 14-3-3, down-regulation of ENaC by stimulation of CFTR with IBMX and forskolin was still detectable (number of experiments).

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regulation of ENaC by CFTR, we examined the contribution of G proteins to inhibition of ENaC by CFTR in Xenopus oocytes. G proteins have been demonstrated to participate in the Na⫹ feedback regulation of ENaC [32]. As summarized in Figure 6, coexpression of wild-type and dominant negative ␣ subunits of Go, regulators of G protein signaling (RGS3, RGS7) and 14-3-3, an inhibitor of RGS proteins, did not block ENaC and did not interfere with the inhibition of ENaC by CFTR. Both guanosine triphosphate (GTP) ␥s (N ⫽ 5) and guanosine diphosphate (GDP) ␤s (N ⫽ 9) interfered with activation of CFTR, but otherwise had no direct effects on ENaC. Interestingly, wild-type, dominant-negative, and constitutively active ␣Go, all abolished down-regulation by CFTR. Because these ␣ subunits could function as scavengers for ␤␥ subunits, which otherwise serve as inhibitory signals for ENaC, we cloned the common ␤1 and ␥3 subunits from mouse colon and coexpressed the respective cRNA in oocytes together with CFTR and ENaC. However, ␤1␥3 subunits did not inhibit amiloride-sensitive Na⫹ conductance and did not significantly interfere with the inhibition of ENaC by CFTR. We therefore conclude that G proteins are probably not involved in the regulation of ENaC by CFTR. Thus, future studies are necessary to demonstrate whether other Cl⫺sensitive proteins contribute to this regulatory loop. SUMMARY Taken together, the current studies indicate that ENaC is inhibited by both activation of CFTR and stimulation of luminal purinergic receptors by ATP or UTP. Both CFTR and ATP/UTP have in common the activation of a luminal Cl⫺ conductance that is probably a prerequisite for inhibition of ENaC. Inhibition of ENaC is largely attenuated when the Cl⫺ conductance is suppressed. Although appealing, only a little evidence is currently available indicating that inhibition of ENaC occurs via PDZ domain proteins, probably colocalized in membrane microdomains together with CFTR and ENaC. Furthermore, the Cl⫺ dependence on the inhibition of ENaC by CFTR suggests the impact of Cl⫺sensitive proteins, such as certain subtypes of trimeric G proteins. However, coexpression studies in Xenopus oocytes using wild-type, dominant-negative, and constitutively active forms of ␣Gi2 or ␣Go, nonhydrolyzable analogues of GTP and regulators of G protein signaling (RGS3 and RGS7) did not suggest any contribution of G proteins. Although it is likely that CFTR and UTP control ENaC through changes in intracellular Cl⫺ ion concentration, the basic mechanism remains to be identified. In contrast to the intestinal and airway epithelium where CFTR is the dominating Cl⫺ channel, in epithelial cells of the nephron, CFTR may function as a regulator of other membrane conductances.

ACKNOWLEDGMENTS The study was supported by the Deutsche Forschungs Gemeinschaft grant DFG KU1228/1-1, German Mukoviszidose e.V., and ARC Grant 00/ARCS243. Reprint requests to Karl Kunzelmann, M.D., Department of Physiology & Pharmacology, University of Queensland, St. Lucia, QLD 4072, Brisbane, Australia E-mail: [email protected]

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