Endogenous protease activation of ENaC: effect of serine protease inhibition on ENaC single channel properties

Abstract

Endogenous serine proteases have been reported to control the reabsorption of Na(+) by kidney- and lung-derived epithelial cells via stimulation of electrogenic Na(+) transport mediated by the epithelial Na(+) channel (ENaC). In this study we investigated the effects of aprotinin on ENaC single channel properties using transepithelial fluctuation analysis in the amphibian kidney epithelium, A6. Aprotinin caused a time- and concentration-dependent inhibition (84 +/- 10.5%) in the amiloride-sensitive sodium transport (I(Na)) with a time constant of 18 min and half maximal inhibition constant of 1 microM. Analysis of amiloride analogue blocker-induced fluctuations in I(Na) showed linear rate-concentration plots with identical blocker on and off rates in control and aprotinin-inhibited conditions. Verification of open-block kinetics allowed for the use of a pulse protocol method (Helman, S.I., X. Liu, K. Baldwin, B.L. Blazer-Yost, and W.J. Els. 1998. Am. J. Physiol. 274:C947-C957) to study the same cells under different conditions as well as the reversibility of the aprotinin effect on single channel properties. Aprotinin caused reversible changes in all three single channel properties but only the change in the number of open channels was consistent with the inhibition of I(Na). A 50% decrease in I(Na) was accompanied by 50% increases in the single channel current and open probability but an 80% decrease in the number of open channels. Washout of aprotinin led to a time-dependent restoration of I(Na) as well as the single channel properties to the control, pre-aprotinin, values. We conclude that protease regulation of I(Na) is mediated by changes in the number of open channels in the apical membrane. The increase in the single channel current caused by protease inhibition can be explained by a hyperpolarization of the apical membrane potential as active Na(+) channels are retrieved. The paradoxical increase in channel open probability caused by protease inhibition will require further investigation but does suggest a potential compensatory regulatory mechanism to maintain I(Na) at some minimal threshold value.

Figure 1.

Effect of apically administered aprotinin on amiloride-sensitive I_SC in A6 cell monolayers. A6 cells in Ussing chambers with symmetrical bath solutions continuously gassed with air were voltage clamped at 0 mV and I_SC monitored. After a 25–30-min equilibration period (A) PBS or (B) aprotinin 10 μM was added to the apical bath. I_SC was monitored for 50 min then 10 μM amiloride was added to the apical bath to determine I_Na. The current deflections are responses to 4 mV bipolar pulses used to measure transepithelial resistance. (C) The mean values of I_SC before (shaded bar), 50 min after addition of PBS/aprotinin (hatched bars), and following addition of amiloride (filled bars) indicate substantial decrease of I_SC by aprotinin. The error bars are ± SEM. Significant decrease was found in comparing I_SC values before and 50 min following addition of aprotinin but not PBS with n = 12 and 14 filters for PBS and aprotinin experiments, respectively (*, P < 0.01).

Figure 2.

Time and concentration dependence of the effect of aprotinin on I_Na. (A) I_Na after apical administration of PBS (filled squares) or 10 μM aprotinin (open circles) was plotted as a percentage of I_Na before administration (control) using data recorded at 5-min intervals. The data points plotted represent the mean values of 12 filters each for PBS and aprotinin addition with error bars corresponding to ± SEM. A small time-dependent increase in I_Na could be seen with PBS addition but was not consistently observed in all the filters measured. The solid line through the aprotinin data points represents a fit to an exponential decay % control I_Na = a*exp(−t/τ) + b ignoring the time = 0 data point. The constants a and b represent the percentage of I_Na sensitive and insensitive to 10 μM aprotinin, respectively. The mean control I_Na for the set of 24 filters used in this experiment was 8.2 ± 0.36 μA/cm². (B) The % control I_Na was measured at 50 min after adding the indicated concentration 0, 1, 3, 6, 10 μM of aprotinin (filled squares) and demonstrated a concentration dependence to the aprotinin inhibition of I_Na. The solid line represents a fit to the inhibition curve % control = a*K_1/2/K_1/2 + [aprotinin] + b, where K_1/2 is an inhibition constant; a and b represent the aprotinin sensitive and insensitive percentages of I_Na, respectively. The mean control I_Na for the set of 25 filters used in this experiment was 4.1 ± 0.30 μA/cm². Error bars are ± SEM.

Figure 3.

Aprotinin effect on blocker-induced fluctuation of I_Na in A6 cells. A6 cells in Ussing chambers were continuously voltage clamped at 0 mV and the I_SC monitored on a strip chart for 30 min following addition of PBS (filled squares) or aprotinin 4 μM (open circles). Cumulative step increases in the concentration of CDPC in the apical bath were performed, the I_SC and power density spectra were obtained at each blocker concentration 10, 20, 30, 40, and 50 μM. The values are given as the mean (n = 8 for PBS and n = 6 for aprotinin-treated A6 filters) ± SEM. (A) The I_Na following 30 min of PBS addition was significantly higher than I_Na following aprotinin addition (*, P < 0.01). In both conditions CDPC decreased the I_Na and the decrease could be explained by simple Michaelis-Menten inhibition kinetics (solid lines). (B) The S_o at increasing concentration of CDPC was biphasic and was fitted to a two-state channel blocking model (solid lines) in both cases. The difference in magnitude of S_o between PBS and aprotinin-treated A6 cells was significant (*, P < 0.01). (C) 2πf_c plots with linear regressions (solid lines) from which k_on and k_off were calculated were virtually identical for PBS and aprotinin-treated cells.

Figure 4.

Typical current noise power spectral density in the absence (solid squares; 3.87 μA/cm² I_Na) and presence (open squares; 1.67 μA/cm² I_Na) of aprotinin are shown at 10 μM CDPC (A) and 30 μM CDPC (B). Corner frequencies were 47.6 and 50.2 Hz (A) (at 10 μM CDPC) in and 72.4 and 72.7 Hz (B) (at 30 μM CDPC) in the absence and presence of aprotinin, respectively. The solid lines represents the fit of the data to the sum of Lorentzian plus low frequency “1/f” noise.

Figure 5.

Summary of the changes in the single channel parameters with aprotinin addition and reversal. (A) Summary of reduction in I_Na by aprotinin in the pulse protocol experiments. I_Na of cells continuously perfused with a ringer solution containing 10 μM CDPC was measured at 10-min intervals before addition of aprotinin (filled squares), then the cells were perfused with 10 μM aprotinin and I_Na measured at the indicated times (open squares) after which aprotinin was washed out (open triangles). At 10-min intervals, the CDPC concentration was pulsed to 30 μM and returned to 10 μM for analysis of blocker-induced fluctuations. (B) Summary of changes in i_Na following perfusion of aprotinin and washout. The increase in i_Na caused by aprotinin was significant (*, P < 0.01) and reversible. (C) Summary of changes in P_o. The changes in P_o were significant (*, P < 0.01) at 15 min after perfusing with aprotinin and returned to the pre-aprotinin control values following washout of aprotinin. (D) Summary of changes in N_o following aprotinin perfusion showed a significant decrease in N_o (*, P < 0.01) that was completely reversed upon removal of aprotinin. Values are reported as mean ± SEM for n = 8.

Figure 6.

N_T calculated from the P_o and K_d decreased by ∼80% following 35 min of inhibition by aprotinin (open squares) compared with control conditions (solid squares). Inhibition of N_T was reversed following washout of aprotinin (open triangles) and returned to control values within 30 min of washout.

Figure 7.

Relationship of single channel parameters to I_Na. Individual values of (A) i_Na, (B) P_o, and (C) N_o under control conditions (filled squares) and during aprotinin treatment (open squares) plotted against I_Na. The data points from the wash conditions have been omitted for clarity. The lines are linear regressions to the control period data (solid line) and aprotinin period data (dotted line). The regressions for all three periods are summarized in Table I.

Figure 8.

The effect of aprotinin and aldosterone on the relative abundance of ENaC subunits in the apical membrane of A6 cells. (A) A6 cells grown on permeable supports were treated either with PBS or 10 μM aprotinin for 1 h or 100 nM aldosterone for 6 h. Apical proteins were biotinylated and recovered with streptavidin–agarose beads. The recovered proteins were analyzed with antibodies against α, β, and γ Xenopus ENaC subunits. Representative Western blots are shown with the arrows indicating the migration of molecular mass standards. (B) Mean of relative changes in apical membrane abundance measured by scanning densitometry and normalized to the control density. The bars represent three independent experiments with error bars corresponding to ±SEM.