Molecular determinants of PI(4,5)P2 and PI(3,4,5)P3 regulation of the epithelial Na+ channel

Abstract

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) are physiologically important second messengers. These molecules bind effector proteins to modulate activity. Several types of ion channels, including the epithelial Na(+) channel (ENaC), are phosphoinositide effectors capable of directly interacting with these signaling molecules. Little, however, is known of the regions within ENaC and other ion channels important to phosphoinositide binding and modulation. Moreover, the molecular mechanism of this regulation, in many instances, remains obscure. Here, we investigate modulation of ENaC by PI(3,4,5)P(3) and PI(4,5)P(2) to begin identifying the molecular determinants of this regulation. We identify intracellular regions near the inner membrane interface just following the second transmembrane domains in beta- and gamma- but not alpha-ENaC as necessary for PI(3,4,5)P(2) but not PI(4,5)P(2) modulation. Charge neutralization of conserved basic amino acids within these regions demonstrated that these polar residues are critical to phosphoinositide regulation. Single channel analysis, moreover, reveals that the regions just following the second transmembrane domains in beta- and gamma-ENaC are critical to PI(3,4,5)P(3) augmentation of ENaC open probability, thus, defining mechanism. Unexpectedly, intracellular domains within the extreme N terminus of beta- and gamma-ENaC were identified as being critical to down-regulation of ENaC activity and P(o) in response to depletion of membrane PI(4,5)P(2). These regions of the channel played no identifiable role in a PI(3,4,5)P(3) response. Again, conserved positive-charged residues within these domains were particularly important, being necessary for exogenous PI(4,5)P(2) to increase open probability. We conclude that beta and gamma subunits bestow phosphoinositide sensitivity to ENaC with distinct regions of the channel being critical to regulation by PI(3,4,5)P(3) and PI(4,5)P(2). This argues that these phosphoinositides occupy distinct ligand-binding sites within ENaC to modulate open probability.

Figure 1.

ENaC mutants used to probe phosphoinositide regulation. (A) Schematic representation of wild-type and mutant mENaC subunits. The N and C termini are intracellular and separated by two transmembrane domains (TM1 and TM2; shown as gray boxes) and a large extracellular region. The relative positions of deletion (black boxes) and substitution (black lines) mutations are indicated. (B) Regions of note (between molecular weight markers 75 and 105 kD) from typical Western blots (right) containing whole-cell lysates (T) and membrane fractions (M; isolated from 4x T) from CHO cells expressing wild-type and mutant ENaC. Shown in the inset is a full blot from cells expressing wild-type ENaC and the negative eGFP control. All blots probed with anti-Myc antibody to identify the ENaC subunit of interest (α-ENaC in top blot of inset). Blots were subsequently stripped and counterprobed with anti–Fra-2 (bottom blot of inset) to ensure good separation of M from T. The summary graph to the left reports relative membrane levels. Summary data from three to six experiments for each group. (C) This graph summarizes the activity of recombinant ENaC expressed in CHO cells containing all wild-type (wt) and the respective mutant subunits. Activity quantified in whole-cell voltage clamp experiments as the amiloride-sensitive macroscopic current density at −80 mV. The numbers of observations for each group shown. *, significantly different using a one-way ANOVA plus the Dunnett's subtest comparing treatment groups to the control wild-type group.

Figure 2.

The regions just following TM2 in β- and γ-ENaC subunits are necessary for PI3-K regulation. Summary graph of the relative increase in ENaC activity in the presence of constitutively active PI3-K for wild-type channels and channels containing a single type of mutant subunit coexpressed with complementary wild-type subunits. Activity of recombinant wild-type and mutant ENaC quantified in whole-cell voltage clamp experiments on CHO cells. All conditions the same as in Fig. 1 C. *, significant increase in activity compared with that in the absence of PI3-K. **, significantly different compared with wild type using an ANOVA plus the Dunnett's subtest.

Figure 3.

ENaC containing wild-type and mutant subunits used to probe regulation by phosphoinositides are not different at the single channel level in resting CHO cells. (A) Representative current traces (left) and associated all-point histograms (right) from excised, outside-out patches (V_p = 0 mV) made from CHO cells expressing ENaC containing all wild-type subunits (top) and a single mutant subunit coexpressed with complementary wild-type subunits. Inward current is downwards. As shown in the latter portions of the traces from patches containing wild-type and β2D mutant channels, 10 μM amiloride was routinely added to the bathing solution. This was a standard procedure used to confirm that these Na⁺-selective, nonvoltage-activated, ∼5 pS channels with hallmark slow gating kinetics were indeed recombinant ENaC. Such channels were absent in nontransfected cells. (B) Summary single channel current–voltage relations for wild-type and mutant ENaC. Data is from, at least, four independent excised, outside-out patches for each group. (C) Summary graph of resting P_o for wild-type and mutant ENaC in CHO cells. Summary data is from excised, outside-out patches similar to those in A.

Figure 4.

A typical response of ENaC to exogenous PI(3,4,5)P₃. Shown is a representative current trace from an excised, outside-out patch (V_p = 0 mV) formed from a CHO cell expressing wild-type ENaC before and after addition of 20 μM exogenous diC8 PI(3,4,5)P₃. PI(3,4,5)P₃ added to the bathing solution in the presence of histone H1 carrier. Amiloride subsequently added to the bath solution toward the end of the experiment. This representative patch contains, at least, five ENaC. Shown at top is a continuous trace. Shown below (left) at an expanded timescale are regions of the trace before (1. control; middle) and after (2. PIP₃; bottom) addition of exogenous phosphoinositide. Respective all-point histograms for the regions shown at an expanded time-scale are to the right. All other conditions the same as Fig. 3 A.

Figure 5.

ENaC containing mutant α2D but not β2D subunits have a typical response to exogenous PI(3,4,5)P₃: an increase in P_o. (A) Representative current traces containing a single ENaC from excised, outside-out patches (V_p = 0 mV) from CHO cells expressing ENaC containing the α2D (A) and β2D (B) mutant subunits before and after addition of 20 μM PI(3,4,5)P₃. Shown at top are continuous traces. Shown below (left) at expanded timescales are regions of these traces before (1. control) and after (2. PIP₃) addition of phosphoinositide. Respective all-point histograms for the regions shown at an expanded timescale are to the right. All other conditions the same as Fig. 4.

Figure 6.

Positive-charged residues just following TM2 in β and γ subunits are necessary for PI3-K and PI(3,4,5)P₃-dependent increases in ENaC open probability. Summary graphs of the effects of PI(3,4,5)P₃ on P_o for recombinant ENaC expressed in CHO cells containing all three wild-type subunits (A) and single mutant subunits in the presence of the two other complementary wild-type subunits (B–D). Results are from paired excised, outside-out patch clamp experiments similar to those in Fig. 5. *, significantly greater compared with the absence of PI(3,4,5)P₃. Summary graphs of the effects on P_o of expressing recombinant ENaC in CHO cells in the absence and presence of constitutively active PI3-K. ENaC containing all three wild-type subunits (E) and single mutant subunits in the presence of the two other complementary wild-type subunits (F–H) were expressed. Results are from unpaired excised, outside-out patch clamp experiments similar to those in Fig. 3. *, significantly greater compared with the absence of PI3-K.

Figure 7.

Mutant and wild-type ENaC have similar responses to coexpression with PI(4)P5-K. Summary graph of the relative increase in ENaC activity in the presence of coexpressed PI(4)P5-K for wild-type channels and channels containing a single type of mutant subunit expressed with complementary wild-type subunits. Activity of recombinant wild-type and mutant ENaC quantified in whole-cell voltage clamp experiments on CHO cells. All conditions the same as in Fig. 1 B. All channels had significantly greater activity in the presence of PI(4)P5-K and the relative increases in activity were not different compared with wild-type.

Figure 8.

Vanadate decreases apical membrane PI(4,5)P₂ levels in mpkCCD_c14 principal cells within a confluent monolayer. (A) Fluorescence micrographs showing typical changes in apical membrane PI(4,5)P₂ levels in mpkCCD_c14 principal cells treated with vehicle (top) and 100 μM VO₄ (bottom). Cell are within confluent monolayers having high transepithelia resistances and avid Na⁺ reabsorption. PI(4,5)P₂ was followed with the GFP-PLC-δ-PH reporter. Fluorescence emissions from the reporter in the apical membrane were optically isolated with TIRF microscopy. Cells are shown before (1) and 5 (2) and 15 (3) min after treatment. (B) Summary graph showing the time course of decrease in the relative levels of PI(4,5)P₂ within the apical membrane of mpkCCD_c14 cells in response to VO₄ (n = 5) and LY294002 (n = 9). Apical membrane PI(4,5)P₂ levels were quantified from experiments similar to the typical experiments shown in Fig. 8 A.

Figure 9.

Decreases in ENaC open probability in mpkCCD_c14 principal cells treated with vanadate parallel decreases in apical membrane PI(4,5)P₂ levels. (A) Representative cur rent trace of ENaC in a cell-attached patch (−V_p = -60 mV) made on the apical membrane of a mpkCCD_c14 principal cell within a confluent monolayer before and after addition of vanadate. The major cation in the pipette solution was Li⁺. Inward current is downwards. Shown at top is a continuous current trace. Shown below are portions of this trace before (1) and after vanadate (2) at an expanded timescale. Corresponding all-point histograms for these regions are shown in B. Results for decreases in ENaC P_o following treatment with vanadate from six such paired experiments are summarized in C. *, significantly less P_o compared with before addition of vanadate.

Figure 10.

Positive-charged residues within the N termini of β and γ subunits are necessary for PI(4,5)P₂ regulation of ENaC activity. Summary graph of relative activity of recombinant wild-type and mutant ENaC following treatment with vanadate. Mutant channels contained, as indicated, one or two types of mutant subunits. Activity quantified in whole-cell voltage clamp experiments on CHO cells. All conditions the same as in Fig. 1 C. *, significantly smaller decrease in relative activity in response to vanadate compared with wild type using an ANOVA plus the Dunnett's subtest.

Figure 11.

Positive-charged residues within the N terminus of γ-ENaC are critical for a response to decreases in membrane PI(4,5)P₂ levels. Representative current traces containing recombinant ENaC in excised, outside-out patches (V_p = −60 mV) from CHO cells expressing wild-type (A) and γNS (B) ENaC before and after addition of 100 μM VO₄. Shown at top are continuous traces. Shown below at expanded timescales are regions of these traces before (1) and after (2) vanadate. Summary graphs of the effects of vanadate on P_o for wild type (C; n = 8 with an average value of N = 1.5 ± 0.22), α2D (D; n = 4 with an average value of N = 2.25 ± 0.45), and γNS (E; n = 8 with an average value of N = 2.3 ± 0.33) ENaC. *, significant decrease in P_o in response to VO₄.

Figure 12.

Positive-charged residues within the N termini of β- and γ-ENaC are necessary for PI(4,5)P₂ regulation of ENaC P_o. (A) Representative ENaC current traces from excised, inside-out patches (−V_p = −60 mV) made on CHO cells expressing wild-type (A) and γNS + βND (B) channels before and after addition of 20 μM PI(4,5)P₂. Shown at top are continuous traces. Shown below at expanded timescales are regions of these traces before rundown (1) and just before (2) and after (3) addition of PI(4,5)P₂. Respective summary graphs in C and D. (For both wt and mutant channels, n = 11 with average values of N = 3.4 ± 0.60 and 2.0 ± 0.38, respectively. Two data points in the summary graph for wt channels were from traces containing six and eight channels.) *, vs. before PI(4,5)P₂.