Intact cytoskeleton is required for small G protein dependent activation of the epithelial Na+ channel

Abstract

Background: The Epithelial Na(+) Channel (ENaC) plays a central role in control of epithelial surface hydration and vascular volume. Similar to other ion channels, ENaC activity is regulated, in part, by cortical cytoskeleton. Besides, the cytoskeleton is an established target for small G proteins signaling. Here we studied whether ENaC activity is modulated by changes in the state of the cytoskeleton and whether cytoskeletal elements are involved in small G protein mediated increase of ENaC activity.

Methods and findings: First, the functional importance of the cytoskeleton was established with whole-cell patch clamp experiments recording ENaC reconstituted in CHO cells. Pretreatment with Cytochalasin D (CytD; 10 microg/ml; 1-2 h) or colchicine (500 microM; 1-3 h) to disassembly F-actin and destroy microtubules, respectively, significantly decreased amiloride sensitive current. However, acute application of CytD induced rapid increase in macroscopic current. Single channel measurements under cell-attached conditions revealed similar observations. CytD rapidly increased ENaC activity in freshly isolated rat collecting duct, polarized epithelial mouse mpkCCD(c14) cells and HEK293 cells transiently transfected with ENaC subunits. In contrast, colchicine did not have an acute effect on ENaC activity. Small G proteins RhoA, Rac1 and Rab11a markedly increase ENaC activity. 1-2 h treatment with colchicine or CytD abolished effects of these GTPases. Interestingly, when cells were coexpressed with ENaC and RhoA, short-term treatment with CytD decreased ENaC activity.

Conclusions: We conclude that cytoskeleton is involved in regulation of ENaC and is necessary for small G protein mediated increase of ENaC activity.

Figure 1. Biphasic effects of Cytochalasin D on ENaC activity.

A, Overlays of typical macroscopic current traces before (arrow) and after 10 µM amiloride from voltage-clamped CHO cells transfected with α, β and γ subunits of mENaC. Currents evoked with a voltage ramp (60 to −100 mV from a holding potential of 40 mV). Inward Na⁺ currents are downward. Whole-cell current traces without treatment with Cytochalasin D (CytD, 10 µg/ml) (top) and after 20 min (middle) and 2 hrs (bottom) of treatment with CytD are shown. B, Summary graph of amiloride-sensitive current density at −80 mV for CHO cells expressing α, β and γ subunits of mENaC without treatment with CytD, after 20 min and 2 hrs of treatment with CytD, respectively. The number of observations for each group is shown. *, versus no treatment. C, Typical Western blot probed with anti-Myc antibody containing the plasma membrane fraction and total cell lysate from CHO cells expressing Myc-tagged α, β, and γENaC in the absence and presence of treatment with CytD (10–20 min). Plasma membrane proteins were labeled with sulfo-NHS-LC-biotin and isolated with streptavidin precipitation. Summary graph showing percent ENaC in the plasma membrane in cells expressing ENaC in the absence and presence of treatment with CytD is also shown. Percent ENaC in the membrane was established with densitometry of Western blots with the number of independent experiments indicated. For each experiment, the density of the membrane fraction of ENaC was divided by the total cellular pool of ENaC.

Figure 2. Acute application of Cytochalasin D causes an increase in ENaC activity.

A, Representative current traces from a cell-attached patch in a HEK293 cell transfected with α-, β- and γ-mENaC subunits before and after addition of Cytochalasin D (CytD, 10 µg/ml) to the bath solution. This patch was held at a −60 mV test potential during the course of the experiment. Dashed lines indicate the respective current levels shown to the left. “c” and “o” denote corresponding closed and open current levels. B, Summary graph of ENaC channel activity (NP_o) changes in response to CytD from paired cell-attached experiments performed on HEK293 cells transiently transfected with all three ENaC subunits. *, versus before application of CytD. C and D, Single-channel current-voltage relation for ENaC in cell-attached patches made in HEK293 cells transfected with α-, β- and γ-mENaC subunits before (C) and after (D) 10 min treatment with CytD. Points in the plots are mean ± SEM for at least nine experiments at each potential.

Figure 3. Cytochalasin D rapidly increases ENaC activity in the apical membrane of mpkCCD_c14 principal cells.

A, Continuous current trace from a representative cell-attached patch that was made on the apical membrane of mpkCCD_c14 principal cells before and after treatment with CytD. Areas before (I) and after (II) treatment are shown below with an expanded time scale. This patch was held at a −60 mV test potential during the course of the experiment. “c” and “o” denote closed and open current level, respectively. B, Summary graph of NP_o in cell-attached patches in mpkCCD_c14 cells before (control) and after (+CytD) treatment with CytD. *, versus before application of CytD.

Figure 4. Cytochalasin D rapidly increases ENaC activity in principal cells in isolated split-open rat collecting ducts.

A, Continuous current trace from a representative cell-attached patch that was made on the apical membrane of principal cells in isolated split-open rat collecting ducts before and after treatment with cytochalasin D (CytD). Areas before (I) and after (II) treatment are shown below with an expanded time scale. This patch was held at a −60 mV test potential during the course of the experiment. “c” and “o_i” denote closed and open current levels, respectively. B, Summary graph of NP_o in cell-attached patches from freshly isolated CCD cells before (control) and after acute addition of CytD. *, versus before application of CytD.

Figure 5. Destroying of microtubule network with colchicine decreases ENaC activity.

A, Overlays of typical macroscopic current traces before (arrow) and after 10 µM amiloride from voltage-clamped CHO cells transfected with α-, β- and γ-mENaC subunits without (top) and with treatment (bottom) with colchicine (Colch, 500 µM). B, Summary graph of amiloride-sensitive current density at −80 mV for CHO cells expressing mENaC without treatment with colchicine, after 2 and 24 hrs of treatment with colchicine, respectively. The number of observations for each group is shown. *, versus untreated cells.

Figure 6. Long term treatment with Cytochalasin D and colchicine abolished small G protein-dependent increases in ENaC activity.

A–C, Summary graphs of amiloride-sensitive current density at −80 mV for CHO cells expressing either mENaC alone or coexpressed with small GTPases RhoA (A), Rac1 (B) or Rab11 (C) in the absence and presence of treatment with CytD (10 µg/ml; 2 hrs) or colchicine (500 µM; 2 hrs). The number of observations for each group is shown. *, versus ENaC alone. ^#, versus corresponding small G protein.

Figure 7. Cytochalasin D decreases ENaC activity when the channel coexpressed with RhoA.

A, Summary graph of macroscopic amiloride-sensitive current density for CHO cells expressing either mENaC alone (ENaC) or coexpressing constitutively active RhoA^G14V (+RhoA) before and after treatment with CytD (10 µg/ml; 10 min). The number of observations for each group is shown. B, Continuous current trace from a representative cell-attached patch in HEK293 cells coexpressing α-, β- and γ- mENaC subunits and RhoA^G14V before and after treatment with CytD. Areas before (I) and after (II, III) treatment are shown below with an expanded time scale. This patch was held at a −60 mV test potential during the course of the experiment. “c” and “o_i” denote closed and open current levels, respectively. C, Summary graph of NP_o in cell-attached patches from HEK293 cells coexpressing mENaC and RhoA^G14V before and after treatment with CytD for 10 min. *, versus untreated cells.