Wang XP et al. (2022), Bile acids regulate the epithelial Na+ channel ...

XB-ART-59319

J Physiol 2022 Nov 23;60021:4695-4711. doi: 10.1113/JP283318.

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Bile acids regulate the epithelial Na+ channel in native tissues through direct binding at multiple sites.

Wang XP , Tomilin V , Nickerson AJ , Tian R , Ertem M , McKernan A , Lei X , Pochynyuk O , Kashlan OB .

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Bile acids, originally known to emulsify dietary lipids, are now established signalling molecules that regulate physiological processes. Signalling targets several proteins that include the ion channels involved in regulating intestinal motility and bile viscosity. Studies show that bile acids regulate the epithelial sodium channel (ENaC) in cultured cell models and heterologous expression systems. ENaC plays both local and systemic roles in regulating extracellular fluids. Here we investigated whether bile acids regulate ENaC expressed in native tissues. We found that taurocholic acid and taurohyodeoxycholic acid regulated ENaC in both the distal nephron and distal colon. We also tested the hypothesis that regulation occurs through direct binding. Using photoaffinity labelling, we found evidence for specific binding to both the β and γ subunits of the channel. In functional experiments, we found that the α subunit was sufficient for regulation. We also found that regulation by at least one bile acid was voltage-sensitive, suggesting that one binding site may be closely associated with the pore-forming helices of the channel. Our data provide evidence that bile acids regulate ENaC by binding to multiple sites to influence the open probability of the channel. KEY POINTS: Recent studies have shown that bile acids regulate the epithelial sodium channel (ENaC) in vitro. Here we investigated whether bile acids regulate ENaC in native tissues and whether bile acids directly bind the channel. We found that bile acids regulate ENaC expressed in the mouse cortical collecting duct and mouse colon by modulating open probability. Photoaffinity labelling experiments showed specific binding to the β and γ subunits of the channel, while channels comprising only α subunits were sensitive to taurocholic acid in functional experiments using Xenopus oocytes. Taurocholic acid regulation of ENaC was voltage-dependent, providing evidence for binding to pore-forming helices. Our data indicate that bile acids are ENaC regulatory effectors that may have a role in the physiology and pathophysiology of several systems.

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Species referenced: Xenopus laevis
GO keywords: sodium channel activity [+]

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	Figure 4. Effect of subunit composition on ENaC response to t-CA. A, currents from Xenopus oocytes expressing ENaC subunits shown were assessed by two-electrode voltage clamp at -100 mV. Recordings were performed in a buffer containing 110 mM Li+, and supplemented with 1 mM t-CA or 100 Î¼M amiloride, as indicated. Currents at baseline and in the presence of t-CA or amiloride were measured at the end of each treatment period. B, amiloride-sensitive currents (blue) in the absence (â) or presence (+) of t-CA were transformed as log(-current) and plotted. Absolute currents (red) were similarly plotted for uninjected oocytes. Summary statistics are means (SD). Transformed amiloride-sensitive currents were analysed by repeated measures two-way ANOVA, with Sidak's multiple comparisons test to examine the effect of t-CA. Transformed absolute currents from uninjected oocytes were analysed by paired Student's t test. The number of oocytes measured for each group is shown in parentheses. [Colour figure can be viewed at wileyonlinelibrary.com]
	Figure 5. ENaC regulation by t-CA is voltage-dependent. A, oocytes expressing Î±, Î², and Î³ ENaC subunits were bathed in a 110 mM Na+ solution supplemented with t-CA or amiloride as indicated. After 60 s for each t-CA dose, or 20 s for amiloride, currents were recorded while varying voltage in 20 mV steps for 4 s, as shown in the representative experiment. Steady-state currents at each voltage (recorded at 3.8 s) were subsequently analysed. B, the amiloride-sensitive IâV curve from the representative experiment is plotted for each t-CA dose, with an inset highlighting greater relative stimulation at less negative potentials. C, the relative t-CA doseâresponse for each voltage is plotted and was determined as âI = IX/I0 â 1, where I0 and IX are the amiloride-sensitive currents at baseline and a given t-CA dose, respectively. Data shown are means (SD), n = 13 oocytes. Individual points are shown in panel D. DâE, the t-CA doseâresponse data were replotted as a function of voltage, with individual data points in D and mean values in E. Linear regression was performed for each t-CA dose (D), and gave R2 values of 0.19 for 30 Î¼M t-CA (P = 0.38), 0.98 for 100 Î¼M t-CA (P = 0.0002) and 0.99 for both 300 and 1000 Î¼M t-CA (P < 0.0001). F, relative âI vs voltage data were fit to various models of regulation. The simplest model considered (Model A, dashed lines in panel E) had three states, a closed state (C), an open state (O), and an open state with the bile acid bound at one site (O-B). Equilibrium constants between these states are Ko = [O]/[C] and K1 = [O][B]/[O-B], and K1 was permitted to have voltage-dependence according to K1V = K1Â·ezFÎ´V/RT. Additional binding sites were defined analogously. Models A, B and C are nested within Model D by setting the indicated binding constant(s) to â. The fit to the best model (Model D, solid lines in panel E) is shown. [Colour figure can be viewed at wileyonlinelibrary.com]
	Figure 6. ENaC regulation by t-HDCA is voltage-independent. A, oocytes expressing Î±, Î² and Î³ ENaC subunits were bathed in a 110 mM Na+ solution supplemented with t-HDCA or amiloride as indicated. After 60 s for each t-HDCA dose, or 20 s for amiloride, currents were recorded while varying voltage in 20 mV steps for 4 s, as shown in the representative experiment. Steady-state currents at each voltage (recorded at 3.8 s) were subsequently analysed. B, the amiloride-sensitive IâV curve from the representative experiment is plotted for each t-HDCA dose. C, the relative t-HDCA doseâresponse at each voltage is plotted and was determined as âI = IX/I0 â 1, where I0 and IX are the amiloride-sensitive currents at baseline and a given t-HDCA dose, respectively. Data shown are means (SD), n = 6 oocytes. D, the t-HDCA doseâresponse data were replotted as a function of voltage, with individual measurements shown as coloured circles. Data at each voltage were fit by linear regression, with values of R2 = 0.001, 0.005 and 0.03 for 100, 300 and 1000 Î¼M t-HDCA, respectively. None of the slopes were significantly non-zero: P = 0.83 for 100 Î¼M t-HDCA, P = 0.68 for 300 Î¼M t-HDCA and P = 0.28 for 1000 Î¼M t-HDCA. [Colour figure can be viewed at wileyonlinelibrary.com]

References [+] :

Ahn, Cloning and functional expression of the mouse epithelial sodium channel. 1999, Pubmed, Xenbase