Epithelial Na channels and short-term renal response to salt deprivation

2002 ◽  
Vol 283 (4) ◽  
pp. F717-F726 ◽  
Author(s):  
Gustavo Frindt ◽  
Tiffany McNair ◽  
Anke Dahlmann ◽  
Emily Jacobs-Palmer ◽  
Lawrence G. Palmer
Keyword(s):  
Distal Tubule ◽  
Treated Group ◽  
Na Channel ◽  
Na Channels ◽  
Nacl Transport ◽  
Drug Concentrations ◽  
Epithelial Na Channels ◽  
Late Evening ◽  
Excretion Rates

To test the role of epithelial Na channels in the day-to-day regulation of renal Na excretion, rats were infused via osmotic minipumps with the Na channel blocker amiloride at rates that achieved drug concentrations of 2–5 μM in the lumen of the distal nephron. Daily Na excretion rates were unchanged, although amiloride-treated animals tended to excrete more Na in the afternoon and less in the late evening than controls. When the rats were given a low-Na diet, Na excretion rates were elevated in the amiloride-treated group within 4 h and remained higher than controls for at least 48 h. Adrenalectomized animals responded similarly to the low-Na diet. In contrast, rats infused with polythiazide at rates designed to inhibit NaCl transport in the distal tubule were able to conserve Na as well as did the controls. Injection of aldosterone (2 μg/100 g body wt) decreased Na excretion in control animals after a 1-h delay. This effect was largely abolished in amiloride-treated rats. On the basis of quantitative analysis of the results, we conclude that activation of amiloride-sensitive channels by mineralocorticoids accounts for 50–80% of the immediate natriuretic response of the kidney to a reduction in Na intake. Furthermore, the channels are necessary to achieve minimal rates of Na excretion during more chronic Na deprivation.

Activation of epithelial Na+ channels by protein kinase A requires actin filaments

1993 ◽  
Vol 265 (1) ◽  
pp. C224-C233 ◽  
Author(s):  
A. G. Prat ◽  
A. M. Bertorello ◽  
D. A. Ausiello ◽  
H. F. Cantiello
Keyword(s):  
Protein Kinase ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activation ◽  
Epithelial Na Channels ◽  
Inside Out ◽  
Kinase A

We have recently demonstrated a novel role for "short" actin filaments, a distinct species of polymerized actin different from either monomeric (G-actin) or long actin filaments (F-actin), in the activation of epithelial Na+ channels. In the present study, the role of actin in the activation of apical Na+ channels by the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A (PKA) was investigated by patch-clamp techniques in A6 epithelial cells. In excised inside-out patches, addition of deoxyribonuclease I, which prevents actin polymerization, inhibited Na+ channel activation mediated by PKA. Disruption of endogenous actin filament organization with cytochalasin D for at least 1 h prevented the PKA-mediated activation of Na+ channels but not activation following the addition of actin to the cytosolic side of the patch. To assess the role of PKA on actin filament organization, actin was used as a substrate for the specific phosphorylation by the PKA. Actin was phosphorylated by PKA with an equilibrium stoichiometry of 2:1 mol PO4-actin monomer. Actin was phosphorylated in its monomeric form, but only poorly once polymerized. Furthermore, phosphorylated actin reduced the rate of actin polymerization. Thus actin allowed to polymerize for at least 1 h in the presence of PKA and ATP to obtain phosphorylated actin filaments induced Na+ channel activity in excised inside-out patches, in contrast to actin polymerized either in the absence of PKA or in the presence of PKA plus a PKA inhibitor (nonphosphorylated actin filaments). This was also confirmed by using purified phosphorylated G-actin incubated in a polymerizing buffer for at least 1 h at 37 degrees C. These data suggest that the form of actin required for Na+ channel activation (i.e., "short" actin filaments) may be favored by the phosphorylation of G-actin and may thus mediate or facilitate the activation of Na+ channels by PKA.

Block of Human Heart hH1 Sodium Channels by the Enantiomers of Bupivacaine

2000 ◽  
Vol 93 (4) ◽  
pp. 1022-1033 ◽  
Author(s):  
Carla Nau ◽  
Sho-Ya Wang ◽  
Gary R. Strichartz ◽  
Ging Kuo Wang
Keyword(s):  
Human Heart ◽  
Point Mutations ◽  
Cell Voltage ◽  
Site Directed Mutagenesis ◽  
Na Channel ◽  
Amino Acid Residues ◽  
Na Channels ◽  
State Dependent ◽  
Positively Charged

Background S(-)-bupivacaine reportedly exhibits lower cardiotoxicity but similar local anesthetic potency compared with R(+)-bupivacaine. The bupivacaine binding site in human heart (hH1) Na+ channels has not been studied to date. The authors investigated the interaction of bupivacaine enantiomers with hH1 Na+ channels, assessed the contribution of putatively relevant residues to binding, and compared the intrinsic affinities to another isoform, the rat skeletal muscle (mu1) Na+ channel. Methods Human heart and mu1 Na+ channel alpha subunits were transiently expressed in HEK293t cells and investigated during whole cell voltage-clamp conditions. Using site-directed mutagenesis, the authors created point mutations at positions hH1-F1760, hH1-N1765, hH1-Y1767, and hH1-N406 by introducing the positively charged lysine (K) or the negatively charged aspartic acid (D) and studied their influence on state-dependent block by bupivacaine enantiomers. Results Inactivated hH1 Na+ channels displayed a weak stereoselectivity with a stereopotency ratio (+/-) of 1.5. In mutations hH1-F1760K and hH1-N1765K, bupivacaine affinity of inactivated channels was reduced by approximately 20- to 40-fold, in mutation hH1-N406K by approximately sevenfold, and in mutations hH1-Y1767K and hH1-Y1767D by approximately twofold to threefold. Changes in recovery of inactivated mutant channels from block paralleled those of inactivated channel affinity. Inactivated hH1 Na+ channels exhibited a slightly higher intrinsic affinity than mu1 Na+ channels. Conclusions Differences in bupivacaine stereoselectivity and intrinsic affinity between hH1 and mu1 Na+ channels are small and most likely of minor clinical relevance. Amino acid residues in positions hH1-F1760, hH1-N1765, and hH1-N406 may contribute to binding of bupivacaine enantiomers in hH1 Na+ channels, whereas the role of hH1-Y1767 remains unclear.

Localization of amiloride-sensitive sodium channels in A6 cells by atomic force microscopy

1997 ◽  
Vol 272 (4) ◽  
pp. C1295-C1298 ◽  
Author(s):  
P. R. Smith ◽  
A. L. Bradford ◽  
S. Schneider ◽  
D. J. Benos ◽  
J. P. Geibel
Keyword(s):  
Na Channel ◽  
High Resolution Imaging ◽  
Na Channels ◽  
Renal Epithelial Cells ◽  
Gold Particles ◽  
Force Microscopy ◽  
Atomic Force ◽  
Epithelial Na Channels ◽  
Resolution Imaging ◽  
Epithelial Na Channel

Atomic force microscopy (AFM) was used for high-resolution imaging of the apical distribution of epithelial Na+ channels in A6 renal epithelial cells. A6 cells grown on coverslips were labeled with antibodies generated against an amiloride-sensitive epithelial Na+ channel complex purified from bovine renal medulla that had been conjugated to 8-nm colloidal gold particles before preparation for AFM. AFM revealed that there was a marked increase in the height of the microvilli in cells labeled with the anti-epithelial Na+ channel antibodies compared with unlabeled cells or cells labeled with rabbit nonimmune immunoglobulin G conjugated to colloidal gold particles. We interpret this apparent increase in microvillar height to be due to anti-epithelial Na+ channel antibody binding to the apical microvilli. These data demonstrate that epithelial Na+ channels are restricted to the apical microvilli in Na+-transporting renal epithelial cells. Furthermore, they demonstrate the applicability of using AFM for high-resolution imaging of the cell surface distribution of epithelial ion channels.

scholarly journals The Role of Pre-H2 Domains of α- and δ-Epithelial Na+Channels in Ion Permeation, Conductance, and Amiloride Sensitivity

2003 ◽  
Vol 279 (9) ◽  
pp. 8428-8440 ◽  
Author(s):  
Hong-Long Ji ◽  
LaToya R. Bishop ◽  
Susan J. Anderson ◽  
Catherine M. Fuller ◽  
Dale J. Benos
Keyword(s):  
Ion Permeation ◽  
Na Channels ◽  
Epithelial Na Channels

scholarly journals High Ca2+ permeability of a peptide-gated DEG/ENaC from Hydra

2012 ◽  
Vol 140 (4) ◽  
pp. 391-402 ◽  
Author(s):  
Stefan Dürrnagel ◽  
Björn H. Falkenburger ◽  
Stefan Gründer
Keyword(s):  
Xenopus Oocytes ◽  
Divalent Cations ◽  
Na Channel ◽  
Low Permeability ◽  
Xenopus Laevis Oocytes ◽  
Na Channels ◽  
Transient Component ◽  
Epithelial Na Channels ◽  
Hydra Magnipapillata ◽  
Secondary Activation

Degenerin/epithelial Na+ channels (DEG/ENaCs) are Na+ channels that are blocked by the diuretic amiloride. In general, they are impermeable for Ca2+ or have a very low permeability for Ca2+. We describe here, however, that a DEG/ENaC from the cnidarian Hydra magnipapillata, the Hydra Na+ channel (HyNaC), is highly permeable for Ca2+ (PCa/PNa = 3.8). HyNaC is directly gated by Hydra neuropeptides, and in Xenopus laevis oocytes expressing HyNaCs, RFamides elicit currents with biphasic kinetics, with a fast transient component and a slower sustained component. Although it was previously reported that the sustained component is unselective for monovalent cations, the selectivity of the transient component had remained unknown. Here, we show that the transient current component arises from secondary activation of the Ca2+-activated Cl− channel (CaCC) of Xenopus oocytes. Inhibiting the activation of the CaCC leads to a simple on–off response of peptide-activated currents with no apparent desensitization. In addition, we identify a conserved ring of negative charges at the outer entrance of the HyNaC pore that is crucial for the high Ca2+ permeability, presumably by attracting divalent cations to the pore. At more positive membrane potentials, the binding of Ca2+ to the ring of negative charges increasingly blocks HyNaC currents. Thus, HyNaC is the first member of the DEG/ENaC gene family with a high Ca2+ permeability.

scholarly journals β spectrin-dependent and domain specific mechanisms for Na+ channel clustering

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Cheng-Hsin Liu ◽  
Ryan Seo ◽  
Tammy Szu-Yu Ho ◽  
Michael Stankewich ◽  
Peter J Mohler ◽  
...  
Keyword(s):  
Nervous System ◽  
Motor Impairment ◽  
Cytoskeletal Proteins ◽  
System Structure ◽  
Na Channel ◽  
Na Channels ◽  
Domain Specific ◽  
Nervous System Function ◽  
Axon Initial Segments

Previously, we showed that a hierarchy of spectrin cytoskeletal proteins maintains nodal Na+ channels (Liu et al., 2020). Here, using mice lacking β1, β4, or β1/β4 spectrins, we show this hierarchy does not function at axon initial segments (AIS). Although β1 spectrin, together with AnkyrinR (AnkR), compensates for loss of nodal β4 spectrin, it cannot compensate at AIS. We show AnkR lacks the domain necessary for AIS localization. Whereas loss of β4 spectrin causes motor impairment and disrupts AIS, loss of β1 spectrin has no discernable effect on central nervous system structure or function. However, mice lacking both neuronal β1 and β4 spectrin show exacerbated nervous system dysfunction compared to mice lacking β1 or β4 spectrin alone, including profound disruption of AIS Na+ channel clustering, progressive loss of nodal Na+ channels, and seizures. These results further define the important role of AIS and nodal spectrins for nervous system function.

Characterization and localization of epithelial Na+ channels in toad urinary bladder

1994 ◽  
Vol 266 (4) ◽  
pp. C1105-C1111 ◽  
Author(s):  
T. R. Kleyman ◽  
P. R. Smith ◽  
D. J. Benos
Keyword(s):  
Urinary Bladder ◽  
Cellular Localization ◽  
Model Systems ◽  
Apical Plasma Membrane ◽  
Na Channel ◽  
Toad Urinary Bladder ◽  
Na Channels ◽  
Single Class ◽  
Biochemical Characteristics ◽  
Epithelial Na Channels

The toad urinary bladder and epithelial cell lines derived from the urinary bladder, including TBM, serve as model systems for the study of transepithelial Na+ transport. We examined biochemical characteristics of epithelial Na+ channels in toad urinary bladder and TBM cells and their cellular localization in the urinary bladder. The radiolabeled amiloride analogue [3H]benzamil bound to a single class of high-affinity binding sites in membrane vesicles from toad urinary bladder with a dissociation constant (Kd) of 10 nM. Photoactive benzamil analogues specifically labeled a 135,000-Da polypeptide in toad urinary bladder and TBM cells. A monoclonal anti-Na+ channel antibody directed against the amiloride-binding component of the channel specifically recognized a 135,000-Da polypeptide in TBM cells. Polyclonal anti-Na+ channel antibodies generated against purified bovine epithelial Na+ channel specifically recognized a 235,000-Da polypeptide in toad urinary bladder and localized Na+ channels to the apical plasma membrane of urinary bladder epithelial cells. The biochemical characteristics and the cellular localization of epithelial Na+ channels in toad urinary bladder are similar to those previously described in mammalian kidney and in the A6 cell line.

Activation of epithelial Na channels during short-term Na deprivation

2001 ◽  
Vol 280 (1) ◽  
pp. F112-F118 ◽  
Author(s):  
Gustavo Frindt ◽  
Shyama Masilamani ◽  
Mark A. Knepper ◽  
Lawrence G. Palmer
Keyword(s):  
Control Group ◽  
Na Channel ◽  
Na Channels ◽  
Charge Balance ◽  
Short Term ◽  
Patch Clamp Technique ◽  
Epithelial Na Channels ◽  
Group 5 ◽  
Collecting Tubules

The role of epithelial Na channels in the response of the kidney to short-term Na deprivation was studied in rats. Animals were fed either a control-Na (3.9 g/kg) or a low-Na ( 3.8 mg/kg) diet for 15 h. Urinary excretion of Na (μmol/min), measured in conscious animals in metabolic cages, was 0.45 ± 0.07 in controls and 0.04 ± 0.01 in Na-deprived animals. Glomerular filtration rate, measured as the clearance of creatinine, was unaffected by the change in diet, suggesting that the reduced Na excretion was the result of increased Na reabsorption. K excretion (μmol/min), increased after the 15-h period of Na deprivation from 0.70 ± 0.10 to 1.86 ± 0.19. Thus the decrease in urine Na was compensated for, in terms of electrical charge balance, by an increase in urine K. Plasma aldosterone increased from 0.50 ± 0.08 to 1.22 ± 0.22 nM. Principal cells from cortical collecting tubules isolated from the animals were studied by using the patch-clamp technique. Whole cell amiloride-sensitive currents were negligible in the control group (5 ± 4 pA/cell) but substantial in the Na-deprived group (140 ± 28 pA/cell). The abundance of the epithelial Na channel subunits, α, β, and γ in the kidney was estimated by using immunoblots. There was no change in the overall abundance of any of the subunits after the 15-h Na deprivation. However, the apparent molecular mass of a fraction of the γ-subunits decreased as was previously reported for long-term Na deprivation. Calculations of the rate of Na transport mediated by the Na channels indicated that activation of the channels during short-term Na deprivation could account in large part for the increased Na reabsorption under these conditions.

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