Institute For Zellulare Und Molekulare Physiologie

Erlangen, Germany

Institute For Zellulare Und Molekulare Physiologie

Erlangen, Germany

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L'Hoste S.,University Pierre and Marie Curie | L'Hoste S.,French Institute of Health and Medical Research | L'Hoste S.,French National Center for Scientific Research | Diakov A.,Institute For Zellulare Und Molekulare Physiologie | And 26 more authors.
Biochimica et Biophysica Acta - Biomembranes | Year: 2013

Several Cl- channels have been described in the native renal tubule, but their correspondence with ClC-K1 and ClC-K2 channels (orthologs of human ClC-Ka and ClC-Kb), which play a major role in transcellular Cl - absorption in the kidney, has yet to be established. This is partly because investigation of heterologous expression has involved rat or human ClC-K models, whereas characterization of the native renal tubule has been done in mice. Here, we investigate the electrophysiological properties of mouse ClC-K1 channels heterologously expressed in Xenopus laevis oocytes and in HEK293 cells with or without their accessory Barttin subunit. Current amplitudes and plasma membrane insertion of mouse ClC-K1 were enhanced by Barttin. External basic pH or elevated calcium stimulated currents followed the anion permeability sequence Cl- > Br- > NO3 - > I-. Single-channel recordings revealed a unit conductance of ~ 40 pS. Channel activity in cell-attached patches increased with membrane depolarization (voltage for half-maximal activation: ~ - 65 mV). Insertion of the V166E mutation, which introduces a glutamate in mouse ClC-K1, which is crucial for channel gating, reduced the unit conductance to ~ 20 pS. This mutation shifted the depolarizing voltage for half-maximal channel activation to ~ + 25 mV. The unit conductance and voltage dependence of wild-type and V166E ClC-K1 were not affected by Barttin. Owing to their strikingly similar properties, we propose that the ClC-K1/Barttin complex is the molecular substrate of a chloride channel previously detected in the mouse thick ascending limb (Paulais et al., J Membr. Biol, 1990, 113:253-260). © 2013 Elsevier B.V.


Mansley M.K.,Institute For Zellulare Und Molekulare Physiologie | Neuhuber W.,Friedrich - Alexander - University, Erlangen - Nuremberg | Korbmacher C.,Institute For Zellulare Und Molekulare Physiologie | Bertog M.,Institute For Zellulare Und Molekulare Physiologie
American Journal of Physiology - Renal Physiology | Year: 2015

here is good evidence for a causal link between excessive sympathetic drive to the kidney and hypertension. We hypothesized that sympathetic regulation of tubular Na+ absorption may occur in the aldosterone-sensitive distal nephron, where the fine tuning of renal Na+ excretion takes place. Here, the appropriate regulation of transepithelial Na+ transport, mediated by the amiloride-sensitive epithelial Na+ channel (ENaC), is critical for blood pressure control. To explore a possible effect of the sympathetic transmitter norepinephrine on ENaC-mediated Na+ transport, we performed short-circuit current (Isc) measurements on confluent mCCDcl1 murine cortical collecting duct cells. Norepinephrine caused a complex Isc response with a sustained increase of amiloride-sensitive Isc by ∼44%. This effect was concentration dependent and mediated via basolateral α2-adrenoceptors. In cells pretreated with aldosterone, the stimulatory effect of norepinephrine was reduced. Finally, we demonstrated that noradrenergic nerve fibers are present in close proximity to ENaC-expressing cells in murine kidney slices. We conclude that the sustained stimulatory effect of locally elevated norepinephrine on ENaC-mediated Na+ absorption may contribute to the hypertensive effect of increased renal sympathetic activity. © 2015 the American Physiological Society.


Huber R.,Institute For Zellulare Und Molekulare Physiologie | Krueger B.,Institute For Zellulare Und Molekulare Physiologie | Diakov A.,Institute For Zellulare Und Molekulare Physiologie | Korbmacher J.,Institute For Zellulare Und Molekulare Physiologie | And 8 more authors.
Cellular Physiology and Biochemistry | Year: 2010

Loss-of-function mutations of the epithelial sodium channel (ENaC) may contribute to pulmonary symptoms resembling those of patients with atypical cystic fibrosis (CF). Recently, we identified a loss-of-function mutation in the α-subunit of ENaC (αF61L) in an atypical CF patient without mutations in CFTR. To investigate the functional effect of this mutation, we expressed human wild-type αβγ-ENaC or mutant α F61Lβγ-ENaC in Xenopus laevis oocytes. The αF61L mutation reduced the ENaC mediated whole-cell currents by ñ90%. In contrast, the mutation decreased channel surface expression only by ñ40% and did not alter the single-channel conductance. These findings indicate that the major effect of the mutation is a reduction of the average channel open probability (P o). This was confirmed by experiments using the βS520C mutant ENaC which can be converted to a channel with a P o of nearly one, and by experiments using chymotrypsin to proteolytically activate the channel. These experiments revealed that the mutation reduced the average P o of ENaC by ñ75%. Na + self inhibition of the mutant channel was significantly enhanced, but the observed effect was too small to account for the large reduction in average channel P o. The ENaC-activator S3969 partially rescued the loss-of-function phenotype of the αF61L mutation. We conclude that the αF61L mutation may contribute to respiratory symptoms in atypical CF patients. Copyright © 2010 S. Karger AG, Basel.


PubMed | Institute For Zellulare Und Molekulare Physiologie
Type: Journal Article | Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology | Year: 2010

Loss-of-function mutations of the epithelial sodium channel (ENaC) may contribute to pulmonary symptoms resembling those of patients with atypical cystic fibrosis (CF). Recently, we identified a loss-of-function mutation in the alpha-subunit of ENaC (alphaF61L) in an atypical CF patient without mutations in CFTR. To investigate the functional effect of this mutation, we expressed human wild-type alpha beta gamma-ENaC or mutant alpha(F61L) beta gamma-ENaC in Xenopus laevis oocytes. The alphaF61L mutation reduced the ENaC mediated whole-cell currents by approximately 90%. In contrast, the mutation decreased channel surface expression only by approximately 40% and did not alter the single-channel conductance. These findings indicate that the major effect of the mutation is a reduction of the average channel open probability (P(o)). This was confirmed by experiments using the betaS520C mutant ENaC which can be converted to a channel with a P(o) of nearly one, and by experiments using chymotrypsin to proteolytically activate the channel. These experiments revealed that the mutation reduced the average P(o) of ENaC by approximately 75%. Na(+) self inhibition of the mutant channel was significantly enhanced, but the observed effect was too small to account for the large reduction in average channel P(o). The ENaC-activator S3969 partially rescued the loss-of-function phenotype of the alphaF61L mutation. We conclude that the alphaF61L mutation may contribute to respiratory symptoms in atypical CF patients.


PubMed | Institute For Zellulare Und Molekulare Physiologie
Type: Journal Article | Journal: American journal of physiology. Lung cellular and molecular physiology | Year: 2013

In some patients with atypical cystic fibrosis (CF), only one allele of the CF transmembrane conductance regulator (CFTR) gene is affected. Mutations of the epithelial sodium channel (ENaC) may contribute to the pathophysiology of the disease in these patients. To functionally characterize a mutation in the -subunit of ENaC (V348M) recently identified in a patient with severe CF-like symptoms (Mutesa et al. 2009), we expressed wild-type (wt) ENaC or mutant V348MENaC in Xenopus laevis oocytes. The V348M mutation stimulated amiloride-sensitive whole-cell current (I(ami)) by 40% but had no effect on surface expression or single-channel conductance of ENaC. Instead the mutation increased channel open probability (P(o)). Proteolytic activation of mutant ENaC by chymotrypsin was reduced compared with that of wt ENaC (3.0-fold vs. 4.2-fold), which is consistent with the increased baseline P(o) of mutant ENaC. Similarly, the ENaC activator S3969 stimulated mutant ENaC currents to a lesser degree (by 2.6-fold) than wt ENaC currents (by 3.5-fold). The gain-of-function effect of the V348M mutation was confirmed by whole-cell current measurements in HEK293 cells transiently transfected with wt or mutant ENaC. Computational channel modeling in combination with functional expression of different V348 mutants in oocytes suggests that the V348M mutation increases channel P(o) by destabilizing the closed channel state. Our findings indicate that the gain-of-function effect of the V348M mutation may contribute to CF pathophysiology by inappropriately increasing sodium and fluid absorption in the respiratory tract.

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