Entity

Time filter

Source Type


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. Source


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. Source


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. Source

Discover hidden collaborations