Skip to main content
NCBI home page
The Journal of Physiology logoLink to The Journal of Physiology
. 2007 Feb 1;580(Pt 2):365–372. doi: 10.1113/jphysiol.2006.127449

Binding and direct activation of the epithelial Na+ channel (ENaC) by phosphatidylinositides

Oleh Pochynyuk 1, Qiusheng Tong 1, Alexander Staruschenko 1, James D Stockand 1
PMCID: PMC2075560  PMID: 17272344

Abstract

Several distinct types of ion channels bind and directly respond to phosphatidylinositides, including phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) and phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2). This regulation is physiologically relevant for its dysfunction, in some instances, causes disease. Recent studies identify the epithelial Na+ channel (ENaC) as a channel sensitive to phosphatidylinositides. ENaC appears capable of binding both PI(4,5)P2 and PI(3,4,5)P3 with binding stabilizing channel gating. The binding sites for these molecules within ENaC are likely to be distinct with the former phosphoinositide interacting with elements in the cytosolic NH2-terminus of the β- and γ-ENaC subunits and the latter with cytosolic regions immediately following the second transmembrane domains in these two subunits. PI(4,5)P2 binding to ENaC appears saturated at rest and necessary for channel gating. Thus, decreases in cellular PI(4,5)P2 levels may serve as a convergence point for inhibitory regulation of ENaC by G-protein coupled receptors and receptor tyrosine kinases. In contrast, apparent PI(3,4,5)P3 binding to ENaC is not saturated. This enables the channel to respond with gating changes in a rapid and dynamic manner to signalling input that influences cellular PI(3,4,5)P3 levels.

Ion channels play critical roles in every aspect of physiology. Binding and direct regulation of ion channel proteins by phosphatidylinositides is becoming widely appreciated (Hilgemann, 2004; Hilgemann et al. 2001). This direct regulation plays an important role in physiology for its disruption often leads to abnormal channel activity and in some instances, disease (e.g. Bartter's, Andersen's and long QT syndromes; Schulte et al. 1999; Plaster et al. 2001; Lopes et al. 2002; Donaldson et al. 2003; Park et al. 2005).

Channels responsive to phosphatidylinositides are diverse ranging from those highly selective for Ca2+ (e.g. Cav2 channels; Wu et al. 2002; Gamper et al. 2004), Na+ (e.g. ENaC; Tong et al. 2004; Ma et al. 2002), K+ (e.g. Kv7, Kir6, TWIK channels; Hilgemann & Ball, 1996; Huang et al. 1998; Zhang et al. 1999; Suh & Hille, 2002; Dong et al. 2002; Bian et al. 2004; Lopes et al. 2005) and Cl− (e.g. CFTR; Himmel & Nagel, 2004) to ones selective for cations in general (e.g. CNG and TRP channels; Chuang et al. 2001; Prescott & Julius, 2003). Additional diversity is apparent when considering that these channels are comprised of subunits having very different tertiary structures ranging from those containing only two transmembrane domains to those with six or more. Phosphatidylinositide-sensitive channels, moreover, range from being ligand- to voltage-gated, localized to sensory neurons and epithelial cells, as well as being rectifying and non-rectifying. Despite this apparent diversity, common themes regarding phosphoinositide binding sites and mechanism of regulation are beginning to emerge. Phosphatidylinositides, including PI(4,5)P2 and PI(3,4,5)P3, directly interact with channel subunits at specific sites to modulate gating.

Recent studies are consistent with ENaC being a channel that binds phosphatidylinositides and is sensitive to direct regulation by these signalling molecules (Pochynyuk et al. 2006). ENaC is a Na+-selective, non-voltage gated, non-inactivating ion channel in the ENaC/Deg superfamily (Kellenberger & Schild, 2002; Garty & Palmer, 1997; Alvarez et al. 2000; Benos & Stanton, 1999). ENaC is a heteromeric channel comprised of three similar but distinct subunits: α, β and γ (Canessa et al. 1993, 1994; McNicholas & Canessa, 1997; Fyfe & Canessa, 1998; Fyfe et al. 1998). ENaC subunits share a common tertiary structure having comparatively short amino- and carboxy-terminal cytosolic domains (∼50–100 amino acids) separated by two trans-membrane domains and a large (∼450 amino acids) extracellular domain. All three ENaC subunits contribute to the functional channel and are required for maximal activity (Canessa et al. 1993, 1994; McNicholas & Canessa, 1997; Fyfe & Canessa, 1998; Fyfe et al. 1998).

ENaC is localized to the luminal plasma membrane of Na+-(re)absorbing epithelia, such as that lining the distal renal nephron and colon, and lungs (Garty & Palmer, 1997; Eaton et al. 2004). ENaC is a final effector of corticoid steroids, including glucocorticoids in the lungs and mineralocorticoids, as the end product of the renin–angiotensin–aldosterone cascade, in the kidney (Verrey, 1998; Stockand, 2002). Activity of this channel is limiting for Na+ transport across these epithelial tissues with corticosteroids increasing ENaC activity. Consequently, ENaC plays a critical role in the local movement of electrolytes and water across epithelial barriers, as well as control of total body electrolyte and water homeostasis. This function places ENaC as a central effector modulating blood pressure and epithelia surface hydration. Thus, dysfunction and aberrant regulation of this channel leads to a spectrum of diseases ranging from hyper- and hypo-tension associated with improper renal Na+ conservation and wasting, respectively, to respiratory syndromes linked to overly wet lungs and dry airways (Hummler & Horisberger, 1999; Bonny & Hummler, 2000; Lifton et al. 2001; Rossier et al. 2002; Mall et al. 2004).

Regulation of ENaC by phosphatidylinositides

The first indication that ENaC could be directly regulated by phosphatidylinositides was provided by Ma and colleagues (Ma et al. 2002). This group demonstrated that decay in ENaC activity in plasma membrane patches excised from renal epithelial cells was tightly linked to PI(4,5)P2 metabolism. Addition of PI(4,5)P2 to the intracellular face of ENaC in these excised patches countered spontaneous decreases in channel activity, whereas scavenging and hydrolysis of PI(4,5)P2 accelerated decreases in channel activity. The primary effect of PI(4,5)P2 on ENaC was not related to control of membrane levels of the channel but rather appeared to be an effect on gating. That manipulation of PI(4,5)P2 levels correlated with changes in ENaC activity in excised patches suggested tight spatial coupling between the final effector of this phosphoinositide and the channel.

Several different laboratories have now reconfirmed the relation between changes in membrane PI(4,5)P2 levels and ENaC activity (Yue et al. 2002; Kunzelmann et al. 2005; Tong & Stockand, 2005). Moreover, mounting evidence supports the idea that decreasing membrane PI(4,5)P2 levels serves as an important mechanism through which G-protein coupled receptors (GPCR) and receptor tyrosine kinases (RTK) modulate ENaC activity. For instance, signalling in response to activation of purinergic receptors and other Gq/11-coupled receptors inhibits Na+ absorption and ENaC activity in tracheal and collecting duct epithelial cells and in expression studies (Ma et al. 2002; Kunzelmann et al. 2005; Tong & Stockand, 2005). Gq/11-coupled receptors decrease PI(4,5)P2 levels via activating PLC-β. Scavenging PI(4,5)P2 abolishes purinergic regulation of ENaC (Kunzelmann et al. 2005). Inhibition of PI4-K and DAG kinase, two kinases involved in regeneration of PI(4,5)P2, moreover, slows recovery of ENaC activity from purinergic inhibition in a manner similar to that reported for blockade of PI4-K on recovery of KCNQ activity in response to muscarinic signalling (Kunzelmann et al. 2005). Muscarinic signalling is thought to decrease activity of this latter phosphoinositide-sensitive channel by promoting PI(4,5)P2 metabolism (Suh & Hille, 2002).

Activation of receptor tyrosine kinases, such as the epidermal growth factor (EGF) receptor, which are capable of decreasing PI(4,5)P2 levels via PLC-γ, also decrease ENaC activity. Buffering PI(4,5)P2 to prevent dynamic changes in its levels, uncouples ENaC from inhibition by activation of the EGF receptor implicating this second messenger as the causative agent in changing channel activity (Tong & Stockand, 2005).

Irrespective of how PI(4,5)P2 is manipulated, be it through physiological signalling via membrane receptors or through pharmacological and genetic tools, decreases in the levels of this phosphatidylinositide lead to corresponding decreases in ENaC activity and open probability (Ma et al. 2002; Yue et al. 2002; Tong & Stockand, 2005). Thus, inhibition of ENaC in response to receptor-linked PTKs (RTKs) and Gq/11-coupled receptor signalling has many parallels with regulation of TRP, GIRK, KCNQ and P/Q- and N-type Ca2+ channels by these receptors (Kobrinsky et al. 2000; Chuang et al. 2001; Wu et al. 2002; Prescott & Julius, 2003; Gamper et al. 2004; Li et al. 2005). The generalized conclusion for these other phosphoinositide-sensitive channels is that decreases in PI(4,5)P2 levels in response to RTK and GPCR signalling lead to dissociation of the phosphoinositide from the channel decreasing channel activity.

ENaC gating is also sensitive to the cellular levels of the phosphoinositide products of PI3-K, primary of which is PI(3,4,5)P3 (Ma et al. 2002; Tong et al. 2004; Staruschenko et al. 2004b; Pochynyuk et al. 2005). Direct regulation of ENaC open probability by PI(3,4,5)P3 was first proposed following experiments in a mammalian expression system where changes in channel activity were noted to closely follow changes in the active state of PI3-K and membrane levels of PI(3,4,5)P3 (Tong et al. 2004). In addition, the open probability of ENaC in excised patches increased upon relief of PI3-K from inhibition and in response to addition of exogenous PI(3,4,5)P3. The observation of tight spatiotemporal coupling between changing PI(3,4,5)P3 levels and ENaC activity combined with the observations that PI(3,4,5)P3 affected channel gating and activated the channel in excised patches led to the hypothesis that ENaC or a protein closely associated with the channel contains a functional PI(3,4,5)P3 binding site. With regard to this hypothesis, physical occupancy of this binding site then would affect gating. Testing of this hypothesis has resulted in identification of a putative PI(3,4,5)P3 binding site within the channel that impinges upon ENaC gating for mutation of residues within this site abrogate PI3-K and PI(3,4,5)P3 regulation (Pochynyuk et al. 2005).

The physiological importance of direct regulation of ENaC by PI(3,4,5)P3 signalling in collecting duct principal cells responsible for fine-tuning plasma volume and electrolyte content is exemplified by results such as those shown in Fig. 1. Shown in Fig. 1A are current traces of ENaC before and after inhibition of PI3-K in a cell-attached patch made on the apical membrane of a principal cell in a collecting duct freshly isolated from a rat maintained with a low Na+ diet. In principal cells from such salt-restricted animals, ENaC is active having a high open probability (Palmer & Frindt, 1986; Frindt et al. 1990; Sun et al. 2006). As is clear, ENaC open probability rapidly decreases soon after inhibiting PI3-K. This decrease in channel activity parallels decreases in apical membrane PI(3,4,5)P3 levels following PI3-K inhibition as shown in Fig. 1B. In combination with the emerging understanding of the possible direct effects of PI(3,4,5)P3 on ENaC gating established in heterologous expression systems, the rapidity of simultaneous decreases in ENaC open probability and apical PI(3,4,5)P3 levels argue that this mechanism plays a role in setting the activity of this channel in native principal cells.

Figure 1. Direct modulation of ENaC open probability by PI(3,4,5)P3 in collecting duct principal cells.

Figure 1

Open in a new tab

A, continuous current trace of ENaC before and after inhibition of PI3-K with LY294002 in a cell-attached patch made on a principal cell from a collecting duct freshly isolated from a salt-restricted rat. (Refer to Palmer & Frindt, 1986; Frindt et al. 1990; Sun et al. 2006 for a complete description of ENaC in this preparation.) Traces before and after inhibition of PI3-K are shown below at expanded time scales. This patch was clamped to negative pipette potential (−Vp)=−40 mV with Li+ as the permeant cation in the pippette solution. Inward current is downwards. B, fluorescence micrographs showing emissions from the PI(3,4,5)P3 reporter GFP-AktPH (Haugh et al. 2000; Tong et al. 2004) in the apical membrane of a principal cell (mpkCCDc14; Bens et al. 1999) within a tight monolayer before and after inhibiting PI3-K. Emissions at the apical membrane were optically isolated with total internal reflection fluorescence microscopy (Axelrod, 2001, 2003; Tong et al. 2004). The diary plot below shows the relative decrease in apical PI(3,4,5)P3 levels in this cell over time following inhibition of PI3-K. Some results in this figure re-presented here are also being published in the Journal of the American Society of Nephrology (Staruschenko et al. 2007 in press)

While requiring further study, the predicted physical interaction of phosphoinositides with ENaC stabilizing channel gating has many parallels with PI(4,5)P2 interactions with some types of Kir channels (Rohacs et al. 1999; MacGregor et al. 2002; Rohacs et al. 2003; Enkvetchakul et al. 2005). However, few phosphatidylinositide-sensitive ion channels respond to both PI(3,4,5)P3 and PI(4,5)P2, with most showing preference for the latter (reviewed by Hilgemann et al. 2001). Plasma membrane PI(4,5)P2 levels, moreover, are 100- to 1000-fold greater than the corresponding levels of PI(3,4,5)P3 (Fruman et al. 1998; Blazer-Yost et al. 1999; Nasuhoglu et al. 2002). This mandates that if a common binding site equally capable of accepting both phosphatidylinositides is responsible for regulation, then PI(4,5)P2 must be the more important modulator. Indeed, this is the case for K(ATP) and other types of Kir channels (Rohacs et al. 1999; MacGregor et al. 2002; Rohacs et al. 2003; Enkvetchakul et al. 2005). In contrast, though, this cannot be the case for ENaC, for addition of exogenous PI(3,4,5)P3 and production of endogenous PI(3,4,5)P3 in response to activation of native receptors, increases ENaC open probability even in the presence of relatively high PI(4,5)P2 concentrations (Ma et al. 2002; Tong et al. 2004; Staruschenko et al. 2004b; Pochynyuk et al. 2005). Addition of exogenous PI(4,5)P2 to channels with resting activity and overexpression of kinases involved in PI(4,5)P2 synthesis, however, does not further increase ENaC open probability and Na+ transport (Staruschenko et al. 2004a; Markadieu et al. 2004), though decreasing PI(4,5)P2 levels, as mentioned above, does decrease channel activity. Thus, the effects of PI(4,5)P2 on ENaC are saturated at rest. One interpretation of these findings is that resting levels of PI(4,5)P2 occupy a binding site(s) necessary for channel gating. In contrast, PI(3,4,5)P3 action is not saturated at rest and increasing the levels of this phosphatidylinositide, even in the presence of saturating PI(4,5)P2, correspondingly increases ENaC activity. If ENaC directly interacts with PI(3,4,5)P3, then these observations are most consistent with the PI(3,4,5)P3 binding site(s) not being well occupied at rest and available for dynamic regulation of the channel. In keeping with the idea that phosphoinositides physically interact with ENaC to directly influence channel gating, then the simplest interpretation of these observations is that there are two distinct phosphatidylinositide binding sites within ENaC with one preferring PI(3,4,5)P3 and the other PI(4,5)P2.

Putative PI(3,4,5)P3 and PI(4,5)P2 binding sites within ENaC

Several studies have begun probing putative phosphatidylinositide binding sites within ENaC. However, this area of investigation is just emerging and true understanding of whether ENaC, and for that matter any other phospohoinositide-senstive channel, contains a bona fide binding site and the locale and structures of potential phosphoinositide binding sites within ion channels is currently lacking. Studies investigating putative binding sites within ENaC and other phosphoinositide-sensitive channels most often do so by combining functional electrophysiology measurements of channel activity with mutagenesis. A limitation to using this approach is that it is indirect in its ability to identify actual binding sites. This stands in contrast to some currently available biochemical and structural chemistry approaches that have yet to be applied to this area of investigation.

Two studies point to the cytosolic portion of the amino-terminus of β-ENaC as being important for PI(4,5)P2 regulation (Yue et al. 2002; Kunzelmann et al. 2005). Mutation of this site in ENaC does not affect surface expression of the channel but does decrease resting channel activity and open probability in a manner similar to depleting PI(4,5)P2. While not definitive, one interpretation of these observations is that this portion of the channel contains a binding site that is occupied by saturating concentrations of PI(4,5)P2 at rest with occupancy being necessary/permissive for normal channel gating. It also remains to be determined whether this area of ENaC encompasses a partial or complete binding site, though biochemical results demonstrate it is necessary for PI(4,5)P2 to interact with the channel (Yue et al. 2002).

Our laboratory recently implicated the cytosolic region just following the second transmembrane domain in γ-ENaC as contributing to a PI(3,4,5)P3 binding site functionally linked to control of channel gating (Booth et al. 2003; Tong et al. 2004; Pochynyuk et al. 2005). However, this investigation was also constrained by the limitations noted above. Similar to the amino-terminus of the β-ENaC subunit implicated in PI(4,5)P2 binding, this region contains several well-conserved positive-charged residues. As demonstrated by the results in Fig. 2, which are re-produced from an earlier study, deletion of these residues disrupts the ability of ENaC to physically interact with PI(3,4,5)P3 (Pochynyuk et al. 2005). While our biochemical and electrophysiology studies are consistent with direct interactions between the phosphoinositide and channel, additional study is required prior to fully recognizing this site as a bona fide binding site. Moreover, it is not yet clear whether this domain encompasses a complete or partial binding site.

Figure 2. Deletion of the region just following the second trans-membrane domain in γ-ENaC disrupts ENaC interactions with PI(3,4,5)P3 and PI(3,4)P2.

Figure 2

Open in a new tab

A, Western blots probed with anti-myc antibody. Blots contained whole cell lysate (top) and the PI(3,4,5)P3-(middle) and PI(3,4)P2- (bottom) precipitants of this lysate. Lysates were from CHO cells expressing myc-tagged wild-type α, β, γ-ENaC and tagged channels containing the γΔ573Q-600P and γΔ573Q-583R deletions. Equal amounts of whole cell lysate were used for each precipitation. B, summary graph quantifying disruption of PI(3,4,5)P3 and PI(3,4)P2 binding to ENaC by deletion of 573Q-600P and 573Q-583R in γ-ENaC. *Significant decrease. Results re-presented here were previously published in Pochynyuk et al. (2005).

In contrast to the putative PI(4,5)P2 binding site, though, mutation of charged residues in this putative PI(3,4,5)P3 binding site does not affect basal channel activity but rather prevents the normal increase in channel gating in response to PI3-K and PI(3,4,5)P3 signalling. This is consistent with the hypothesis that this site is not occupied at rest but is available for dynamic regulation.

Results in Fig. 3 typify mutagenesis scans of ENaC subunits attempting to identify potential PI(4,5)P2 and PI(3,4,5)P3 binding sites. Cytosolic regions just following the second transmembrane domains in β- and γ- but not α-ENaC contribute to regulation by PI(3,4,5)P3 but not PI(4,5)P2, whereas the cytosolic domains in the amino-termini of β- and γ-ENaC contribute to PI(4,5)P2 but not PI(3,4,5)P3 regulation. One interpretation of these functional results is that ENaC contains distinct regions important to PI(4,5)P2 and PI(3,4,5)P3 regulation. While still early in our understanding, extrapolation of these observations suggests that binding sites within β- and γ-ENaC subunits bestow phosphatidylinositide sensitivity to the channel.

Figure 3. The NH2- and COOH-termini of β- and γ-ENaC subunits contain potential PI(4,5)P2 and PI(3,4,5)P3 binding sites, respectively.

Figure 3

Open in a new tab

This summary graph shows the relative fold change in activity of wild-type (wt) and mutant mouse ENaC in response to increases and decreases in PI(3,4,5)P3 and PI(4,5)P2 levels, respectively. Activity of recombinant channels assayed in voltage-clamped CHO cells. PI(3,4,5)P3 increased by co-expression of active PI3-K. PI(4,5)P2 decreased by activation of RTK signalling with vanadate. Mutants are: βN, βENaCΔK4-K16; γN, K to A substitutions at residues 6, 8, 10, 12 and 13 in γENaC; αC, αENaCΔR614-R630; βC, βENaCΔK552-R563; and γC, R/K to A substitutions at residues 569, 570, 574, 576, 581, 582 and 583 in γENaC. Some results re-presented here previously published in a different format (Pochynyuk et al. 2005; Tong & Stockand, 2005). The numbers of observations in each group are > 8.

The emerging understanding regarding putative phosphatidylinositide binding sites within ENaC is that they are in cytosolic regions of the channel often close to the gate and that the conserved positive-charged residues within these domains possibly play an important role in phosphoinositide binding. This understanding is similar to that for putative PI(4,5)P2 binding sites in other ion channels, including TRP and Kir, where direct phosphoinositide binding is believed to affect channel gating (Zhang et al. 1999; Shyng et al. 2000; Soom et al. 2001; Dong et al. 2002; Prescott & Julius, 2003).

It is not clear yet what provides selectivity to phosphoinositide binding sites within ion channels; however, alanine substitution of the conserved negative-charged and bulky residues within the putative PI(3,4,5)P3 binding site in γ-ENaC enhances basal activity and responses to PI3-K signalling (Pochynyuk et al. 2005). These findings suggest that these residues, in addition to the conserved positive-charged residues, may influence binding affinity and selectivity. Non-charged and negative-charged residues in the putative binding sites of other phosphatidylinositide-sensitive channels are thought to play a similar role (Zhang et al. 1999; Rohacs et al. 2003).

Functional implications of phosphatidylinositide regulation of EnaC

It is now clear that ENaC is a phosphatidylinositide-sensitive channel responding to both PI(4,5)P2 and PI(3,4,5)P3. The former phosphoinositide is necessary for normal channel gating and may serve as a focal point for convergence of inhibitory signalling from GPCR and RTK. The nature of ENaC modulation by PI(4,5)P2 appears to be amenable to both acute and chronic forms of channel regulation for ENaC activity does not spontaneously recover from PI(4,5)P2 depletion but rather requires re-synthesis of this phosphatidylinositide. In contrast, PI(3,4,5)P3 signalling has the potential to acutely increase the activity of ENaC. The close spatiotemporal coupling between changes in ENaC gating and PI(3,4,5)P3 levels would allow the channel to respond precisely in a dynamic and rapid manner to ever-changing local and systemic cues modulating Na+ movement across epithelial barriers.

References

  1. Alvarez de la Rosa D, Canessa CM, Fyfe GK, Zhang P. Structure and regulation of amiloride-sensitive sodium channels. Annu Rev Physiol. 2000;62:573–594. doi: 10.1146/annurev.physiol.62.1.573. [DOI] [PubMed] [Google Scholar]
  2. Axelrod D. Total internal reflection fluorescence microscopy in cell biology. Traffic. 2001;2:764–774. doi: 10.1034/j.1600-0854.2001.21104.x. [DOI] [PubMed] [Google Scholar]
  3. Axelrod D. Total internal reflection fluorescence microscopy in cell biology. Meth Enzymol. 2003;361:1–33. doi: 10.1016/s0076-6879(03)61003-7. [DOI] [PubMed] [Google Scholar]
  4. Benos DJ, Stanton BA. Functional domains within the degenerin/epithelial sodium channel (Deg/ENaC) superfamily of ion channels. J Physiol. 1999;520:631–644. doi: 10.1111/j.1469-7793.1999.00631.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bens M, Vallet V, Cluzeaud F, Pascual-Letallec L, Kahn A, Rafestin-Oblin ME, Rossier BC, Vandewalle A. Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line. J Am Soc Nephrol. 1999;10:923–934. doi: 10.1681/ASN.V105923. [DOI] [PubMed] [Google Scholar]
  6. Bian J-S, Kagan A, McDonald TV. Molecular analysis of PIP2 regulation of HERG and IKr. Am J Physiol Heart Circ Physiol. 2004;287:H2154–H2163. doi: 10.1152/ajpheart.00120.2004. [DOI] [PubMed] [Google Scholar]
  7. Blazer-Yost BL, Paunescu TG, Helman SI, Lee KD, Vlahos CJ. Phosphoinositide 3-kinase is required for aldosterone-regulated sodium reabsorption. Am J Physiol. 1999;277:C531–C536. doi: 10.1152/ajpcell.1999.277.3.C531. [DOI] [PubMed] [Google Scholar]
  8. Bonny O, Hummler E. Dysfunction of epithelial sodium transport: From human to mouse. Kidney Int. 2000;57:1313–1318. doi: 10.1046/j.1523-1755.2000.00968.x. [DOI] [PubMed] [Google Scholar]
  9. Booth RE, Tong Q, Medina J, Snyder PM, Patel P, Stockand JD. A region directly following the second transmembrane domain in γENaC is required for normal channel gating. J Biol Chem. 2003;278:41367–41379. doi: 10.1074/jbc.M305400200. [DOI] [PubMed] [Google Scholar]
  10. Canessa CM, Horisberger JD, Rossier BC. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature. 1993;361:467–470. doi: 10.1038/361467a0. [DOI] [PubMed] [Google Scholar]
  11. Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, Rossier BC. Amiloride-sensitive epithelial Na channel is made of three homologous subunits. Nature. 1994;367:463–467. doi: 10.1038/367463a0. [DOI] [PubMed] [Google Scholar]
  12. Chuang H, Prescott E, Kong H, Shields S, Jordt S, Basbaum A, Chao M, Julius D. Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns (4,5),P2-mediated inhibition. Nature. 2001;411:957–962. doi: 10.1038/35082088. [DOI] [PubMed] [Google Scholar]
  13. Donaldson MR, Jensen JL, Tristani-Firouzi M, Tawil R, Bendahhou S, Suarez WA, Cobo AM, Poza JJ, Behr E, Wagstaff J, Szepetowski P, Pereira S, Mozaffar T, Escolar DM, Fu YH, Ptacek LJ. PIP2 binding residues of Kir2.1 are common targets of mutations causing Andersen syndrome. Neurology. 2003;60:1811–1816. doi: 10.1212/01.wnl.0000072261.14060.47. [DOI] [PubMed] [Google Scholar]
  14. Dong K, Tang L, Macgregor GG, Hebert SC. Localization of the ATP/phosphatidylinositol 4,5 diphosphate-binding site to a 39-amino acid region of the carboxyl terminus of the ATP-regulated K+ channel Kir1.1. J Biol Chem. 2002;277:49366–49373. doi: 10.1074/jbc.M208679200. [DOI] [PubMed] [Google Scholar]
  15. Eaton DC, Chen J, Ramosevac S, Matalon S, Jain L. Regulation of Na+ channels in lung alveolar type II epithelial cells. Proc Am Thorac Soc. 2004;1:10–16. doi: 10.1513/pats.2306008. [DOI] [PubMed] [Google Scholar]
  16. Enkvetchakul D, Jeliazkova I, Nichols CG. Direct modulation of Kir channel gating by membrane phosphatidylinositol 4,5-bisphosphate. J Biol Chem. 2005;280:35785–35788. doi: 10.1074/jbc.C500355200. [DOI] [PubMed] [Google Scholar]
  17. Frindt G, Sackin H, Palmer LG. Whole-cell currents in rat cortical collecting tubule: Low-Na diet increases amiloride-sensitive conductance. Am J Physiol. 1990;258:F562–F567. doi: 10.1152/ajprenal.1990.258.3.F562. [DOI] [PubMed] [Google Scholar]
  18. Fruman D, Meyers R, Cantley L. Phosphoinositide kinases. Annu Rev Biochem. 1998;67:481–507. doi: 10.1146/annurev.biochem.67.1.481. [DOI] [PubMed] [Google Scholar]
  19. Fyfe GK, Canessa CM. Subunit composition determines the single channel kinetics of the epithelial sodium channel. J Gen Physiol. 1998;112:423–432. doi: 10.1085/jgp.112.4.423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fyfe GK, Quinn A, Canessa CM. Structure and function of the Mec-ENaC family of ion channels. Semin Nephrol. 1998;18:138–151. [PubMed] [Google Scholar]
  21. Gamper N, Reznikov V, Yamada Y, Yang J, Shapiro MS. Phosphatidylinositol 4,5-bisphosphate signals underlie receptor-specific Gq/11-mediated modulation of N-type Ca2+ channels. J Neurosci. 2004;24:10980–10992. doi: 10.1523/JNEUROSCI.3869-04.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Garty H, Palmer LG. Epithelial sodium channels: function, structure, and regulation. Physiol Rev. 1997;77:359–396. doi: 10.1152/physrev.1997.77.2.359. [DOI] [PubMed] [Google Scholar]
  23. Haugh J, Codazzi F, Teruel M, Meyer T. Spatial sensing in fibroblasts mediated by 3′ phosphoinositides. J Cell Biol. 2000;151:1269–1280. doi: 10.1083/jcb.151.6.1269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hilgemann DW. Biochemistry. Oily barbarians breach ion channel gates. Science. 2004;304:223–224. doi: 10.1126/science.1097439. [DOI] [PubMed] [Google Scholar]
  25. Hilgemann D, Ball R. Regulation of cardiac Na+,Ca2+ exchange and KATP potassium channels by PIP2. Science. 1996;273:956–959. doi: 10.1126/science.273.5277.956. [DOI] [PubMed] [Google Scholar]
  26. Hilgemann D, Feng S, Nasuhoglu C. The complex and intriguing lives of PIP2 with ion channels and transporters. Sci STKE. 2001;111:RE19. doi: 10.1126/stke.2001.111.re19. [DOI] [PubMed] [Google Scholar]
  27. Himmel B, Nagel G. Protein kinase-independent activation of CFTR by phosphatidylinositol phosphates. EMBO J. 2004;5:85–90. doi: 10.1038/sj.embor.7400034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Huang C-L, Feng S, Hilgemann DW. Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gβγ. Nature. 1998;391:803–806. doi: 10.1038/35882. [DOI] [PubMed] [Google Scholar]
  29. Hummler E, Horisberger JD. Genetic disorders of membrane transport. V. The epithelial sodium channel and its implication in human diseases. Am J Physiol. 1999;276:G567–G571. doi: 10.1152/ajpgi.1999.276.3.G567. [DOI] [PubMed] [Google Scholar]
  30. Kellenberger S, Schild L. Epithelial sodium channel/degenerin family of ion channels: a variety of functions for a shared structure. Physiol Rev. 2002;82:735–767. doi: 10.1152/physrev.00007.2002. [DOI] [PubMed] [Google Scholar]
  31. Kobrinsky E, Mirshahi T, Zhang H, Jin T, Logothetis D. Receptor-mediated hydrolysis of plasma membrane messenger PIP2 leads to K+-current desensitization. Nature Cell Biol. 2000;2:507–514. doi: 10.1038/35019544. [DOI] [PubMed] [Google Scholar]
  32. Kunzelmann K, Bachhuber T, Regeer R, Markovich D, Sun J, Schreiber R. Purinergic inhibition of the epithelial Na+ transport via hydrolysis of PIP2. FASEB J. 2005;19:142–143. doi: 10.1096/fj.04-2314fje. [DOI] [PubMed] [Google Scholar]
  33. Li Y, Gamper N, Hilgemann DW, Shapiro MS. Regulation of Kv7 (KCNQ) K+ channel open probability by phosphatidylinositol 4,5-bisphosphate. J Neurosci. 2005;25:9825–9835. doi: 10.1523/JNEUROSCI.2597-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lifton RP, Gharavi AG, Geller DS. Molecular mechanisms of human hypertension. Cell. 2001;104:545–556. doi: 10.1016/s0092-8674(01)00241-0. [DOI] [PubMed] [Google Scholar]
  35. Lopes CM, Rohacs T, Czirjak G, Balla T, Enyedi P, Logothetis DE. PIP2 hydrolysis underlies agonist-induced inhibition and regulates voltage gating of two-pore domain K+ channels. J Physiol. 2005;564:117–159. doi: 10.1113/jphysiol.2004.081935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lopes C, Zhang H, Rohacs T, Jin T, Logothetis D. Alterations in conserved Kir channel–PIP2 interactions underlie channelopathies. Neuron. 2002;34:933–944. doi: 10.1016/s0896-6273(02)00725-0. [DOI] [PubMed] [Google Scholar]
  37. Ma HP, Saxena S, Warnock DG. Anionic phosphoinositides regulate native and expressed epithelial sodium channel (ENaC) J Biol Chem. 2002;277:7641–7644. doi: 10.1074/jbc.C100737200. [DOI] [PubMed] [Google Scholar]
  38. MacGregor GG, Dong K, Vanoye CG, Tang LQ, Giebisch G, Hebert SC. Nucleotides and phospholipids compete for binding to the C terminus of KATP channels. Proc Natl Acad Sci U S A. 2002;99:2726–2731. doi: 10.1073/pnas.042688899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. McNicholas CM, Canessa CM. Diversity of channels generated by different combinations of epithelial sodium channel subunits. J Gen Physiol. 1997;109:681–692. doi: 10.1085/jgp.109.6.681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mall M, Grubb B, Harkema J, O'Neal W, Boucher R. Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice. Nat Med. 2004;10:487–493. doi: 10.1038/nm1028. [DOI] [PubMed] [Google Scholar]
  41. Markadieu N, Blero D, Boom A, Erneux C, Beauwens R. Phosphatidylinositol 3,4,5-trisphosphate: an early mediator of insulin-stimulated sodium transport in A6 cells. Am J Physiol Renal Physiol. 2004;287:F319–F328. doi: 10.1152/ajprenal.00314.2003. [DOI] [PubMed] [Google Scholar]
  42. Nasuhoglu C, Feng S, Mao J, Yamamoto M, Yin HL, Earnest S, Barylko B, Albanesi JP, Hilgemann DW. Nonradioactive analysis of phosphatidylinositides and other anionic phosphoinositides by anion-exchange high-performance liquid chromatography with suppressed conductivity detection. Anal Biochem. 2002;301:243–254. doi: 10.1006/abio.2001.5489. [DOI] [PubMed] [Google Scholar]
  43. Palmer LG, Frindt G. Amiloride-sensitive Na channels from the apical membrane of the rat cortical collecting tubule. Proc Natl Acad Sci U S A. 1986;83:2767–2770. doi: 10.1073/pnas.83.8.2767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Park K-H, Piron J, Dahimene S, Merot J, Baro I, Escande D, Lousssuoarn G. Impaired KCNQ1-KCNE1 and phosphatidylinositol-4,5-bisphosphate interaction underlies the long QT syndrome. Circ Res. 2005;96:730–739. doi: 10.1161/01.RES.0000161451.04649.a8. [DOI] [PubMed] [Google Scholar]
  45. Plaster NM, Tawil R, Tristani-Firouzi M, Canun S, Bendahhou S, Tsunoda A, et al. Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome. Cell. 2001;105:511–519. doi: 10.1016/s0092-8674(01)00342-7. [DOI] [PubMed] [Google Scholar]
  46. Pochynyuk O, Staruschenko A, Tong Q, Medina J, Stockand JD. Identification of a functional phosphatidylinositol 3,4,5-trisphosphate binding site in the epithelial Na+ channel. J Biol Chem. 2005;280:37565–37571. doi: 10.1074/jbc.M509071200. [DOI] [PubMed] [Google Scholar]
  47. Pochynyuk O, Tong Q, Staruschenko A, Ma H-P, Stockand JD. Regulation of the epithelial Na+ channel (ENaC) by phosphatidylinositides. Am J Physiol Renal Physiol. 2006;290:F949–F957. doi: 10.1152/ajprenal.00386.2005. [DOI] [PubMed] [Google Scholar]
  48. Prescott E, Julius D. A modular PIP2 binding site as a determinant of capsaicin receptor sensititivity. Science. 2003;300:1284–1288. doi: 10.1126/science.1083646. [DOI] [PubMed] [Google Scholar]
  49. Rohacs T, Chen J, Prestwich GD, Logothetis DE. Distinct specificities of inwardly rectifying K+ channels for phosphoinositides. J Biol Chem. 1999;274:36065–36072. doi: 10.1074/jbc.274.51.36065. [DOI] [PubMed] [Google Scholar]
  50. Rohacs T, Lopes C, Jin T, Ramdya P, Molnar Z, Logothetis D. Specificity of activation by phosphoinositides determines lipid regulation of Kir channels. Proc Natl Acad Sci U S A. 2003;100:745–750. doi: 10.1073/pnas.0236364100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Rossier BC, Pradervand S, Schild L, Hummler E. Epithelial sodium channel and the control of sodium balance: interaction between genetic and environmental factors. Annu Rev Physiol. 2002;64:877–897. doi: 10.1146/annurev.physiol.64.082101.143243. [DOI] [PubMed] [Google Scholar]
  52. Schulte U, Hahn H, Konrad M, Jeck N, Derst C, Wild K, Weidemann S, Ruppersberg JP, Fakler B, Ludwig J. pH gating of ROMK (Kir1.1) channels: control by an Arg-Lys-Arg triad disrupted in antenatal Bartter syndrome. Proc Natl Acad Sci U S A. 1999;96:15298–15303. doi: 10.1073/pnas.96.26.15298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Shyng SL, Cukras CA, Harwood J, Nichols CG. Structural determinants of PIP2 regulation of inward rectifier KATP channels. J Genl Physiol. 2000;116:599–608. doi: 10.1085/jgp.116.5.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Soom M, Schonherr R, Kubo Y, Kirsch C, Klinger R, Heinemann SH. Multiple PIP2 binding sites in Kir2.1 inwardly rectifying potassium channels. FEBS Lett. 2001;490:49–53. doi: 10.1016/s0014-5793(01)02136-6. [DOI] [PubMed] [Google Scholar]
  55. Staruschenko A, Nichols A, Medina JL, Camacho P, Zheleznova NN, Stockand JD. Rho small GTPases activate the epithelial Na+ channel. J Biol Chem. 2004a;279:49989–49994. doi: 10.1074/jbc.M409812200. [DOI] [PubMed] [Google Scholar]
  56. Staruschenko A, Patel P, Tong Q, Medina JL, Stockand JD. Ras activates the epithelial Na+ channel through phosphoinositide 3-OH kinase signaling. J Biol Chem. 2004b;279:37771–37778. doi: 10.1074/jbc.M402176200. [DOI] [PubMed] [Google Scholar]
  57. Stockand JD. New ideas about aldosterone signaling in epithelia. Am J Physiol Renal Physiol. 2002;282:F559–F576. doi: 10.1152/ajprenal.00320.2001. [DOI] [PubMed] [Google Scholar]
  58. Suh BC, Hille B. Recovery from muscarinic modulation of M current channels requires phosphatidylinositol 4,5-bisphosphate synthesis. Neuron. 2002;35:507–520. doi: 10.1016/s0896-6273(02)00790-0. [DOI] [PubMed] [Google Scholar]
  59. Sun P, Lin DH, Wang T, Babilonia E, Wang Z, Jin Y, Kemp R, Nasjletti A, Wang WH. Low Na intake suppresses expression of CYP2C23 and arachidonic acid-induced inhibition of ENaC. Am J Physiol Renal Physiol. 2006;291:F1192–F1200. doi: 10.1152/ajprenal.00112.2006. [DOI] [PubMed] [Google Scholar]
  60. Tong Q, Gamper N, Medina JL, Shapiro MS, Stockand JD. Direct activation of the epithelial Na+ channel by phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate produced by phosphoinositide 3-OH kinase. J Biol Chem. 2004;279:22654–22663. doi: 10.1074/jbc.M401004200. [DOI] [PubMed] [Google Scholar]
  61. Tong Q, Stockand JD. Receptor tyrosine kinases mediate epithelial Na+ channel inhibition by epidermal growth factor. Am J Physiol Renal Physiol. 2005;288:F150–F161. doi: 10.1152/ajprenal.00261.2004. [DOI] [PubMed] [Google Scholar]
  62. Verrey F. Early aldosterone effects. Exp Nephrol. 1998;6:294–301. doi: 10.1159/000020536. [DOI] [PubMed] [Google Scholar]
  63. Wu L, Bauer CS, Zhen XG, Xie C, Yang J. Dual regulation of voltage-gated calcium channels by PtdIns(4,5),P2. Nature. 2002;419:947–952. doi: 10.1038/nature01118. [DOI] [PubMed] [Google Scholar]
  64. Yue G, Malik B, Yue G, Eaton DC. Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates epithelial sodium channel activity in A6 cells. J Biol Chem. 2002;277:11965–11969. doi: 10.1074/jbc.M108951200. [DOI] [PubMed] [Google Scholar]
  65. Zhang H, He C, Yan X, Mirshahj T, Logothetis D. Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5),P2 interactions. Nature Cell Biol. 1999;1:193–188. doi: 10.1038/11103. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES