Identification of a Functional Phosphatidylinositol 3,4,5-Trisphosphate Binding Site in the Epithelial Na+ Channel

Abstract

Membrane phospholipids, such as phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ), are signaling molecules that can directly modulate the activity of ion channels, including the epithelial Na ؉ channel (ENaC). Whereas PI(3,4,5)P 3 directly activates ENaC, its binding site within the channel has not been identified. We identify here a region of ␥-mENaC just following the second trans-membrane domain (residues 569 -583) important to PI(3, Direct regulation of ion channel activity by phospholipid signaling molecules is becoming widely appreciated (reviewed in Ref. 1). This mechanism for ion channel modulation is recognized to be physiologically important for its disruption, in some instances, leads to disease (e.g. Bartter and Andersen syndromes) (2-5). Diverse types of ion channels, including those composed of subunits having only two trans-membrane domains, such as ENaC 2 and inwardly rectifying K ϩ channels (Kir), as well as those with subunits having multiple (four or six) membranespanning domains, such as KCNQ, HERG, and two pore domain K ϩ channels, and transient receptor potential and cyclic nucleotide-gated channels, are modulated by phospholipids (6 -13). Phospholipids, such as PI(4,5)P 2 and PI(3,4,5)P 3 , directly interact with these channels to modulate gating (6,9,14). ENaC is a heteromeric channel composed of three distinct but similar subunits: ␣, ␤, and ␥ (15, 16). Each subunit has an amino-and carboxylterminal cytosolic domain separated by two trans-membrane domains and a large extracellular region. ENaC serves an essential physiological function, since its activity is limiting for Na ϩ absorption across many epithelia, including that in the distal renal nephron (reviewed in Refs. 17-19). Thus, ENaC is well positioned to influence system Na ϩ balance and blood pressure. Indeed, gain and loss of function mutations in ENaC lead to inheritable forms of hypertension and hypotension, respectively, associated with inappropriate salt conservation and wasting at the kidney (20, 21). In addition, ENaC plays a critical role in hydration and fluid reabsorption across many mucosal membranes. Deletion and overexpression of ENaC correspondingly lead to excessively wet and dry air spaces with associated disease (21, 22). The mineralocorticoid aldosterone increases ENaC activity. Several lines of evidence suggest a role for PI(3,4,5)P 3 signaling in regulation of ENaC by aldosterone and other hormones, such as insulin (23-30). Aldosterone increases PI3-K activity and production of its phospholipid products (29). In some preparations, aldosterone activates PI3-K to augment ENaC activity via transcriptional control of the upstream PI3-Kregulator, K-Ras (31-33). Both K-Ras and PI3-K increase ENaC open probability (31)(32)(33). Moreover, we recently demonstrated a direct effect of PI(3,4,5)P 3 on ENaC to increase channel open probability (33).4The phospholipid binding site in several types of ion channels, particularly that for PI(4,5)P 2 binding to Kir and transient receptor potential channels, is becoming cle...

Acute Regulation of the Epithelial Na+ Channel by Phosphatidylinositide 3-OH Kinase Signaling in Native Collecting Duct Principal Cells

“…ENaC, similar to some other types of ion channels, senses membrane phosphoinositide levels and responds with changes in channel P o through direct interactions with these molecules (see reference [27]). Our laboratory showed that ENaC physically interacts with the phosphoinositide products of PI3-K and that this interaction stabilizes ENaC gating to increase P o (24,26). These findings are consistent with such a mechanism contributing to regulation of ENaC activity in both the immortalized mouse principal cell line and principal cells from freshly isolated rat collecting ducts studied here.…”

“…Again, segments before and after LY294002 are shown below at expanded time scales. The results in Figures 6 and 7 demonstrating that the P o of active ENaC within the apical membrane of native and cultured principal cells is tightly coupled to PI3-K activity are consistent with our previous findings in expression studies (24,26).…”

Interplay between PIP 3 and calmodulin regulation of olfactory cyclic nucleotide-gated channels

“…Binding of PIP 2 to both K ir 1 and K ir 6 channels promotes channel activation by stabilizing the open state (32)(33)(34), and PIP 3 has been proposed to activate both epithelial sodium channels and TRPC6 channels by direct binding (35,36). Like PIP 2 -binding regions identified in other ion channels (37)(38)(39), the stretch of amino acids between residues 61-90 of CNGA2 contains multiple basic residues that may be important for the interaction with negatively charged phospholipids.…”

The regulation of selective and nonselective Na+ conductances in H441 human airway epithelial cells

“…Although it is clear that glucocorticoid hormones do induce SGK1 expression (31), SGK1 is an important downstream target of PI3K (29), and data from several absorptive cell types have suggested that insulin can increase the apical abundance of ENaC via a mechanism dependent on a PI3K-mediated increase in SGK1 activity (2,8,46,51). Moreover, PIP 2 and PIP 3 also activate ENaC by binding to the channel complex, and this provides another mechanism by which PI3K-coupled agonists, such as insulin, can stimulate Na ϩ transport (20,42,43,52). However, although the present data show that insulin stimulation/PI3K-P110␣ expression can control G Cl , these maneuvers had no effect on G Na in hormonedeprived cells but augmented the dexamethasone/SGK1-induced G Na .…”