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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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Effect of G protein stimulation with  GTP-γS. (a) Original current recording showing  the effect of 1 μM epinephrine on the conductance of an oocyte expressing the β-adrenergic receptor and CFTR. The holding potential was alternating between −40 and −60 mV with a 1 Hz  frequency. (b) The same protocol was applied to  an oocyte injected 13 min before with 50 nl of a  1.8-mM GTP-γS solution. (c) Effect of trypsin (2  μg/ml for 3.5 min) on the amiloride-sensitive  Na+ current (INa) at −100 mV after intracellular  injection with GTP-γS (50 nl, 1.8 mM). (d) The effect of a 3-min trypsin treatment (left) on INa (before trypsin, white bars; after trypsin, black bars) in  oocytes expressing αβγXENaC. Nine oocytes  were injected with GTP-γS, and seven control oocytes were noninjected. The effect of trypsin was  not affected by previous GTP-γS intracellular injection. (right) The whole oocyte conductance in  oocytes expressing CFTR and the β2-adrenergic  receptor before (open bars) and after (filled bars)  stimulation with 1 μM epinephrine. In control oocytes (n = 11) (i.e., without previous GTP-γS injection), epinephrine induced an increase of the  whole oocyte conductance (P < 0.005, paired t  test). Intracellular GTP-γS injection (n = 13) increased the oocyte conductance (P < 0.05, unpaired t test) and completely prevented the effect  of epinephrine.
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Figure 5: Effect of G protein stimulation with GTP-γS. (a) Original current recording showing the effect of 1 μM epinephrine on the conductance of an oocyte expressing the β-adrenergic receptor and CFTR. The holding potential was alternating between −40 and −60 mV with a 1 Hz frequency. (b) The same protocol was applied to an oocyte injected 13 min before with 50 nl of a 1.8-mM GTP-γS solution. (c) Effect of trypsin (2 μg/ml for 3.5 min) on the amiloride-sensitive Na+ current (INa) at −100 mV after intracellular injection with GTP-γS (50 nl, 1.8 mM). (d) The effect of a 3-min trypsin treatment (left) on INa (before trypsin, white bars; after trypsin, black bars) in oocytes expressing αβγXENaC. Nine oocytes were injected with GTP-γS, and seven control oocytes were noninjected. The effect of trypsin was not affected by previous GTP-γS intracellular injection. (right) The whole oocyte conductance in oocytes expressing CFTR and the β2-adrenergic receptor before (open bars) and after (filled bars) stimulation with 1 μM epinephrine. In control oocytes (n = 11) (i.e., without previous GTP-γS injection), epinephrine induced an increase of the whole oocyte conductance (P < 0.005, paired t test). Intracellular GTP-γS injection (n = 13) increased the oocyte conductance (P < 0.05, unpaired t test) and completely prevented the effect of epinephrine.

Mentions: To evaluate the potential role of G protein in the response to trypsin, we followed the amiloride-sensitive current before and after trypsin treatment in oocytes injected with GTP-γS, a nonspecific activator of all G proteins. As a positive control, we coexpressed human CFTR and the human β2 adrenergic receptor and tested for activation of a chloride current by 1 μM epinephrine. Epinephrine induced a fast increase of the conductance in oocytes expressing CFTR and the β2 adrenergic receptor (Fig. 5 a). When oocytes were injected with GTP-γS (0.09 nmol = 50 nl of a 1.8-mM GTP-γS solution) 10–30 min before the electrophysiological measurement, the initial conductance of the oocytes expressing CFTR was larger and the response to epinephrine was abolished (Fig. 5 b), indicating that GTP-γS injection was efficient to activate G proteins. In contrast, when we studied oocytes expressing Xenopus ENaC, the baseline amiloride-sensitive current was similar in the GTP-γS–injected and water-injected groups, and trypsin induced a large increase of the amiloride-sensitive current (Fig. 5 c) similar to that observed in the control group. The mean values of INa before and after trypsin and of the oocyte conductance (G) before and after epinephrine stimulation are shown in Fig. 5 d.


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)

Effect of G protein stimulation with  GTP-γS. (a) Original current recording showing  the effect of 1 μM epinephrine on the conductance of an oocyte expressing the β-adrenergic receptor and CFTR. The holding potential was alternating between −40 and −60 mV with a 1 Hz  frequency. (b) The same protocol was applied to  an oocyte injected 13 min before with 50 nl of a  1.8-mM GTP-γS solution. (c) Effect of trypsin (2  μg/ml for 3.5 min) on the amiloride-sensitive  Na+ current (INa) at −100 mV after intracellular  injection with GTP-γS (50 nl, 1.8 mM). (d) The effect of a 3-min trypsin treatment (left) on INa (before trypsin, white bars; after trypsin, black bars) in  oocytes expressing αβγXENaC. Nine oocytes  were injected with GTP-γS, and seven control oocytes were noninjected. The effect of trypsin was  not affected by previous GTP-γS intracellular injection. (right) The whole oocyte conductance in  oocytes expressing CFTR and the β2-adrenergic  receptor before (open bars) and after (filled bars)  stimulation with 1 μM epinephrine. In control oocytes (n = 11) (i.e., without previous GTP-γS injection), epinephrine induced an increase of the  whole oocyte conductance (P < 0.005, paired t  test). Intracellular GTP-γS injection (n = 13) increased the oocyte conductance (P < 0.05, unpaired t test) and completely prevented the effect  of epinephrine.
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Figure 5: Effect of G protein stimulation with GTP-γS. (a) Original current recording showing the effect of 1 μM epinephrine on the conductance of an oocyte expressing the β-adrenergic receptor and CFTR. The holding potential was alternating between −40 and −60 mV with a 1 Hz frequency. (b) The same protocol was applied to an oocyte injected 13 min before with 50 nl of a 1.8-mM GTP-γS solution. (c) Effect of trypsin (2 μg/ml for 3.5 min) on the amiloride-sensitive Na+ current (INa) at −100 mV after intracellular injection with GTP-γS (50 nl, 1.8 mM). (d) The effect of a 3-min trypsin treatment (left) on INa (before trypsin, white bars; after trypsin, black bars) in oocytes expressing αβγXENaC. Nine oocytes were injected with GTP-γS, and seven control oocytes were noninjected. The effect of trypsin was not affected by previous GTP-γS intracellular injection. (right) The whole oocyte conductance in oocytes expressing CFTR and the β2-adrenergic receptor before (open bars) and after (filled bars) stimulation with 1 μM epinephrine. In control oocytes (n = 11) (i.e., without previous GTP-γS injection), epinephrine induced an increase of the whole oocyte conductance (P < 0.005, paired t test). Intracellular GTP-γS injection (n = 13) increased the oocyte conductance (P < 0.05, unpaired t test) and completely prevented the effect of epinephrine.
Mentions: To evaluate the potential role of G protein in the response to trypsin, we followed the amiloride-sensitive current before and after trypsin treatment in oocytes injected with GTP-γS, a nonspecific activator of all G proteins. As a positive control, we coexpressed human CFTR and the human β2 adrenergic receptor and tested for activation of a chloride current by 1 μM epinephrine. Epinephrine induced a fast increase of the conductance in oocytes expressing CFTR and the β2 adrenergic receptor (Fig. 5 a). When oocytes were injected with GTP-γS (0.09 nmol = 50 nl of a 1.8-mM GTP-γS solution) 10–30 min before the electrophysiological measurement, the initial conductance of the oocytes expressing CFTR was larger and the response to epinephrine was abolished (Fig. 5 b), indicating that GTP-γS injection was efficient to activate G proteins. In contrast, when we studied oocytes expressing Xenopus ENaC, the baseline amiloride-sensitive current was similar in the GTP-γS–injected and water-injected groups, and trypsin induced a large increase of the amiloride-sensitive current (Fig. 5 c) similar to that observed in the control group. The mean values of INa before and after trypsin and of the oocyte conductance (G) before and after epinephrine stimulation are shown in Fig. 5 d.

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