Effects of the _d63-subunit [delta-subunit] and of proteolytic channel cleavage on the function of the epithelial sodium channel (ENaC) [Elektronische Ressource] / vorgelegt von Silke Härteis

Pd Dr. Stephan Schwarzinger

Sujets

• Biologie

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2010
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Effects of the δ-subunit and of proteolytic channel
cleavage on the function of the epithelial sodium
channel (ENaC)




Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.







vorgelegt von
Silke Härteis
aus Neumarkt i. d. OPf.






Als Dissertation genehmigt von der Naturwissen-
schaftlichen Fakultät der Friedrich-Alexander-Universität
Erlangen-Nürnberg











Tag der mündlichen Prüfung: 09.12.2009
Vorsitzender der
Promotionskommission: Prof. Dr. Eberhard Bänsch
Erstberichterstatter: Prof. Dr. Christoph Korbmacher
Zweitberichterstatter: Prof. Dr. Johann Helmut BrandstätterContents
Contents
Publications arising from this thesis 3
List of figures 4
Abbreviations 5
Summary 7
Zusammenfassung 9
1. Introduction 12
1.1 Molecular structure and tissue distribution of the epithelial sodium channel
(ENaC) 12
1.2 Body sodium homeostasis critically depends on renal ENaC activity 14
1.3 Pathophysiological relevance of ENaC in cystic fibrosis 16
1.4 Regulation of ENaC 18
1.4.1 Hormonal regulation of ENaC by aldosterone 18
1.4.2 Activation of ENaC by proteolytic processing 19
1.4.3 Regulation of ENaC surface expression by Nedd4-2 20
+1.4.4 Feedback regulation by intra- and extracellular Na 20
1.4.5 Modification of ENaC function by its lipid environment 21
1.5 Aims of thesis 23
2. Materials and methods 25
2.1 Plasmids 25
2.2 Isolation of oocytes and injection of cRNA 25
2.3 Two-electrode voltage-clamp 26
2.4 Surface labeling of oocytes 26
2.5 Detection of ENaC cleavage products at the cell surface 27
2.6 Cholesterol depletion of the plasma membrane of oocytes by MβCD 27
2.7 Preparation of membrane-enriched fractions from oocyte whole-cell lysates 28
2.8 Preparation of non-detergent membrane-enriched fractions and separation
of raft & non-raft membranes by discontinuous sucrose gradient
centrifugation 28
2.9 Western blot analysis 29
2.10 Antibodies 29
2.11 Solutions and chemicals 30
2.12 Peptides 30
2.13 Statistical methods 30
3. Results 31
3.1 The δ-subunit of ENaC enhances channel activity and alters proteolytic
ENaC activation 31
3.1.1 ENaC whole-cell currents are larger in X. laevis oocytes expressing δβγ-
hENaC than in those expressing αβγ-hENaC 31
1
Contents
+ +3.1.2 Replacing extracellular Na by Li increases ΔI in αβγ-hENaC ami
expressing oocytes but decreases ΔI in δβγ-hENaC expressing oocytes 33 ami
3.1.3 Channel surface expression is similar in αβγ- and in δβγ-hENaC
expressing oocytes 35
3.1.4 δβγ-hENaC has a higher average open probability (P ) than αβγ-hENaC 37 O
3.1.5 Co-expression of βγ-hENaC enhances proteolytic cleavage of the α-subunit
but not of the δ-subunit 39
3.1.6 Chymotrypsin can activate δβγ-hENaC but the stimulatory effect is
reduced compared to that on αβγ-hENaC 42
+3.1.7 Reduced Na feedback inhibition may contribute to the increased average
open probability of δβγ-hENaC 42
3.1.8 δβγ-hENaC stimulation by chymotrypsin is associated with the appearance
of a cleavage fragment of δ-hENaC 45
3.1.9 Channels with δ-hENaC require the presence of γ-hENaC to be activated
by chymotrypsin 47
3.1.10 Effect of the synthetic peptide α-13 on αβγ- or δβγ-hENaC 49
3.1.11 Chymotrypsin does not increase surface expression of δβγ-hENaC 51
3.2 Plasmin in nephrotic urine activates ENaC 53
3.2.1 uPA-mediated conversion of plasminogen to plasmin is required for ENaC
activation 53
3.2.2 Combination of uPA and plasminogen resulted in the appearance of a
γ-ENaC cleavage product 55
3.2.3 Plasmin in nephrotic urine stimulates ENaC activity 57
3.3 Cholesterol depletion of the plasma membrane disrupts the association of
ENaC with lipid raft microdomains 59
3.3.1 ENaC expressed in X. laevis oocytes is present in putative lipid raft
fractions 59
3.3.2 MβCD effectively removes cholesterol from the plasma membrane of
living oocytes 61
3.3.3 Cholesterol depletion has no consistent effect on whole-cell currents in
oocytes heterologously expressing ENaC 63
3.4 Stimulatory effect of a small molecule ENaC-activator (S3969) on wild-type
ENaC and on a channel with a loss-of-function mutation 65
4. Discussion 69
4.1 The δ-subunit modifies ENaC function and may contribute to channel
regulation 69
4.2 Activation of ENaC by plasmin as a possible mechanism for renal sodium
retention in nephrotic syndrome 76
4.3 Potential role of the lipid environment for ENaC function and regulation 80
4.4 The ENaC-activator S3969 as pharmacological tool to partially restore the
function of αF61L mutant ENaC 83
Conclusion and perspectives 85
References 88
Acknowledgements Fehler! Textmarke nicht definiert.
Appendix Fehler! Textmarke nicht definiert.
2
Publications arising form this thesis
Publications arising from this thesis
Haerteis S, Krueger B, Korbmacher C & Rauh R (2009).
The δ-subunit of the epithelial sodium channel (ENaC) enhances channel activity and
alters proteolytic ENaC activation.
J Biol Chem 284, 29024-29040.


Svenningsen P, Bistrup C, Friis UG, Bertog M, Haerteis S, Krueger B, Stubbe J, Jensen
OL, Thiesson HC, Uhrenholt TR, Jespersen B, Jensen BL, Korbmacher C & Skøtt O
(2009).
Plasmin in nephrotic urine activates the epithelial sodium channel.
J Am Soc Nephrol 20, 299-310.


Krueger B, Haerteis S, Yang L, Hartner A, Rauh R, Korbmacher C & Diakov A (2009).
Cholesterol depletion of the plasma membrane prevents activation of the epithelial sodium
channel (ENaC) by SGK1.
Cell Physiol Biochem 24, 605-618.


Huber R, Krueger B, Diakov A, Korbmacher J, Haerteis S, Einsiedel J, Gmeiner P, Azad
AK, Cuppens H, Cassiman JJ, Korbmacher C & Rauh R.
Functional characterization of a partial loss-of-function mutation of the epithelial sodium
channel (ENaC) associated with atypical cystic fibrosis.
Cell Physiol Biochem in press.


3
List of figures
List of figures
Fig. 1: ENaC is probably a heterotrimeric channel. 12
Fig. 2: Model of transepithelial ion transport in the aldosterone-sensitive distal nephron.15
Fig. 3: ENaC whole-cell currents are larger in X. laevis oocytes expressing
δβγ-hENaC than in those expressing αβγ-hENaC. 32
+ +Fig. 4: Replacing extracellular Na by Li increases ΔI in αβγ-hENaC expressing ami
oocytes but decreases ΔI in δβγ-hENaC expressing oocytes. 34 ami
Fig. 5: Channel surface expression is similar in αβγ- and in δβγ-hENaC expressing
oocytes. 36
Fig. 6: αβγ-hENaC has a higher average open probability (P ) than αβγ-hENaC. 38 O
Fig. 7: Co-expression of βγ-hENaC enhances proteolytic cleavage of the α-subunit
but not of the δ-subunit. 41
Fig. 8: Chymotrypsin can activate δβγ-hENaC but the stimulatory effect is reduced
compared to that on αβγ-hENaC. 44
Fig. 9: αβγ-hENaC stimulation by chymotrypsin is associated with the appearance of
a cleavage fragment of δ-hENaC. 46
Fig. 10: Channels with δ-hENaC require the presence of γ-hENaC to be activated by
chymotrypsin. 48
Fig. 11: Effect of a 13-mer synthetic peptide on αβγ- or δβγ-hENaC. 50
Fig. 12: Chymotrypsin does not increase surface expression of δβγ-hENaC. 52
Fig. 13: uPA-mediated conversion of plasminogen to plasmin is required for ENaC
activation. 54
Fig. 14: Combination of uPA and plasminogen resulted in the appearance of a γ-ENaC
cleavage product. 56
Fig. 15: Plasmin in nephrotic urine stimulates ENaC activity. 58
Fig. 16: ENaC is present in lipid raft fractions. 60
Fig. 17: MβCD effectively removes cholesterol from the plasma membrane of living
oocytes. 62
Fig. 18: Cholesterol depletion has no consistent effect on ENaC-mediated whole-cell
currents. 64
Fig. 19: ENaC-activator S3969 stimulates δβγ-hENaC. 66
Fig. 20: The novel ENaC-activator S3969 stimulates αF61L mutant ENaC more than
wild-type ENaC. 68

4
Abbreviations
Abbreviations
Ami Amiloride
ASDN Aldosterone-sensitive distal nephron
ASIC1 Acid-sensing ion channel 1
ASL Airway surface liquid
CF Cystic fibrosis
CFTR Cystic fibrosis transmembrane conductance regulator
-Cl Chloride anion
cRNA Complementary ribonucleic acid
ENaC Epithelial sodium channel
Fig. Figure
FLAG An eight amino acid epitope (DYKDDDDK)
HA A nine amino acid hemagglutinin epitope (YPYDVPDYA)
hENaC Human ENaC
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
hNE Human neutrophil elastase
ΔI Amiloride-sensitive current ami
+K Potassium cation
M1 First transmembrane domain of the ENaC subunits
M2 Second transmembrane domain of the ENaC subunits
MβCD Methyl-β-cyclodextrin
MBS Modified Barth’s solution
mENaC Mouse ENaC
MTSET [2-(trimethy