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Protease modulation of the activity of the epithelial sodium channel expressed in Xenopus oocytes.

Chraïbi A, Vallet V, Firsov D, Hess SK, Horisberger JD - J. Gen. Physiol. (1998)

Bottom Line: A similar effect was observed with chymotrypsin, but not with kallikrein.The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-gammaS, suggesting that this effect was not mediated by G proteins.We conclude that extracellular proteases are able to increase the open probability of the epithelial sodium channel by an effect that does not occur through activation of a G protein-coupled receptor, but rather through proteolysis of a protein that is either a constitutive part of the channel itself or closely associated with it.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland.

ABSTRACT
We have investigated the effect of extracellular proteases on the amiloride-sensitive Na+ current (INa) in Xenopus oocytes expressing the three subunits alpha, beta, and gamma of the rat or Xenopus epithelial Na+ channel (ENaC). Low concentrations of trypsin (2 microg/ml) induced a large increase of INa within a few minutes, an effect that was fully prevented by soybean trypsin inhibitor, but not by amiloride. A similar effect was observed with chymotrypsin, but not with kallikrein. The trypsin-induced increase of INa was observed with Xenopus and rat ENaC, and was very large (approximately 20-fold) with the channel obtained by coexpression of the alpha subunit of Xenopus ENaC with the beta and gamma subunits of rat ENaC. The effect of trypsin was selective for ENaC, as shown by the absence of effect on the current due to expression of the K+ channel ROMK2. The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-gammaS, suggesting that this effect was not mediated by G proteins. Measurement of the channel protein expression at the oocyte surface by antibody binding to a FLAG epitope showed that the effect of trypsin was not accompanied by an increase in the channel protein density, indicating that proteolysis modified the activity of the channel present at the oocyte surface rather than the cell surface expression. At the single channel level, in the cell-attached mode, more active channels were observed in the patch when trypsin was present in the pipette, while no change in channel activity could be detected when trypsin was added to the bath solution around the patch pipette. We conclude that extracellular proteases are able to increase the open probability of the epithelial sodium channel by an effect that does not occur through activation of a G protein-coupled receptor, but rather through proteolysis of a protein that is either a constitutive part of the channel itself or closely associated with it.

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Trypsin-induced increase in amiloride-sensitive Na  current (INa): relationship to the initial INa value. The increase in  INa after a 3–5-min exposure to 2 μg/ml trypsin is reported as a  function of the initial value of INa in 31 oocytes expressing αβγ  Xenopus ENaC. Note that both scales are logarithmic. The straight  line is the regression line of log(increase in INa) versus log(INa). The  corresponding r = 0.5794 (n = 31), indicating a statistically significant inverse relationship (P < 0.001) between these two variables.
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Figure 2: Trypsin-induced increase in amiloride-sensitive Na current (INa): relationship to the initial INa value. The increase in INa after a 3–5-min exposure to 2 μg/ml trypsin is reported as a function of the initial value of INa in 31 oocytes expressing αβγ Xenopus ENaC. Note that both scales are logarithmic. The straight line is the regression line of log(increase in INa) versus log(INa). The corresponding r = 0.5794 (n = 31), indicating a statistically significant inverse relationship (P < 0.001) between these two variables.

Mentions: The amplitude of the increase of the amiloride-sensitive sodium current (INa) was quite variable, ranging from a hardly detectable increase to a sixfold increase in oocytes expressing rat αβγENaC, and a 2- to more than 10-fold increase in oocytes expressing Xenopus αβγENaC. Heteromeric channels composed of the α subunit of the Xenopus ENaC and the β and γ subunits of rat ENaC expressed a small INa, but in these oocytes trypsin induced a very large increase of INa, often >20-fold, following the same time course. The mean values of the baseline INa and of the increase of INa after a 3–5-min exposure to 2 μg/ml trypsin in Xenopus ENaC, (αXβXγX), in rat ENaC (αrβrγr), and in heteromeric rat/Xenopus channels, αXβrγr and αrβXγX, are given in Table I. The response to trypsin was inversely correlated with the initial INa value as shown in Fig. 2, suggesting that trypsin effect was larger in oocytes either expressing a low number of channels or channels with a low open probability.


Protease modulation of the activity of the epithelial sodium channel expressed in Xenopus oocytes.

Chraïbi A, Vallet V, Firsov D, Hess SK, Horisberger JD - J. Gen. Physiol. (1998)

Trypsin-induced increase in amiloride-sensitive Na  current (INa): relationship to the initial INa value. The increase in  INa after a 3–5-min exposure to 2 μg/ml trypsin is reported as a  function of the initial value of INa in 31 oocytes expressing αβγ  Xenopus ENaC. Note that both scales are logarithmic. The straight  line is the regression line of log(increase in INa) versus log(INa). The  corresponding r = 0.5794 (n = 31), indicating a statistically significant inverse relationship (P < 0.001) between these two variables.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1887769&req=5

Figure 2: Trypsin-induced increase in amiloride-sensitive Na current (INa): relationship to the initial INa value. The increase in INa after a 3–5-min exposure to 2 μg/ml trypsin is reported as a function of the initial value of INa in 31 oocytes expressing αβγ Xenopus ENaC. Note that both scales are logarithmic. The straight line is the regression line of log(increase in INa) versus log(INa). The corresponding r = 0.5794 (n = 31), indicating a statistically significant inverse relationship (P < 0.001) between these two variables.
Mentions: The amplitude of the increase of the amiloride-sensitive sodium current (INa) was quite variable, ranging from a hardly detectable increase to a sixfold increase in oocytes expressing rat αβγENaC, and a 2- to more than 10-fold increase in oocytes expressing Xenopus αβγENaC. Heteromeric channels composed of the α subunit of the Xenopus ENaC and the β and γ subunits of rat ENaC expressed a small INa, but in these oocytes trypsin induced a very large increase of INa, often >20-fold, following the same time course. The mean values of the baseline INa and of the increase of INa after a 3–5-min exposure to 2 μg/ml trypsin in Xenopus ENaC, (αXβXγX), in rat ENaC (αrβrγr), and in heteromeric rat/Xenopus channels, αXβrγr and αrβXγX, are given in Table I. The response to trypsin was inversely correlated with the initial INa value as shown in Fig. 2, suggesting that trypsin effect was larger in oocytes either expressing a low number of channels or channels with a low open probability.

Bottom Line: A similar effect was observed with chymotrypsin, but not with kallikrein.The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-gammaS, suggesting that this effect was not mediated by G proteins.We conclude that extracellular proteases are able to increase the open probability of the epithelial sodium channel by an effect that does not occur through activation of a G protein-coupled receptor, but rather through proteolysis of a protein that is either a constitutive part of the channel itself or closely associated with it.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland.

ABSTRACT
We have investigated the effect of extracellular proteases on the amiloride-sensitive Na+ current (INa) in Xenopus oocytes expressing the three subunits alpha, beta, and gamma of the rat or Xenopus epithelial Na+ channel (ENaC). Low concentrations of trypsin (2 microg/ml) induced a large increase of INa within a few minutes, an effect that was fully prevented by soybean trypsin inhibitor, but not by amiloride. A similar effect was observed with chymotrypsin, but not with kallikrein. The trypsin-induced increase of INa was observed with Xenopus and rat ENaC, and was very large (approximately 20-fold) with the channel obtained by coexpression of the alpha subunit of Xenopus ENaC with the beta and gamma subunits of rat ENaC. The effect of trypsin was selective for ENaC, as shown by the absence of effect on the current due to expression of the K+ channel ROMK2. The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-gammaS, suggesting that this effect was not mediated by G proteins. Measurement of the channel protein expression at the oocyte surface by antibody binding to a FLAG epitope showed that the effect of trypsin was not accompanied by an increase in the channel protein density, indicating that proteolysis modified the activity of the channel present at the oocyte surface rather than the cell surface expression. At the single channel level, in the cell-attached mode, more active channels were observed in the patch when trypsin was present in the pipette, while no change in channel activity could be detected when trypsin was added to the bath solution around the patch pipette. We conclude that extracellular proteases are able to increase the open probability of the epithelial sodium channel by an effect that does not occur through activation of a G protein-coupled receptor, but rather through proteolysis of a protein that is either a constitutive part of the channel itself or closely associated with it.

Show MeSH
Related in: MedlinePlus