Role of CFTR’s PDZ1-binding domain, NBF1 and Cl− conductance in inhibition of epithelial Na+ channels in Xenopus oocytes

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.m312012200 ◽

2003 ◽

Vol 279 (9) ◽

pp. 8428-8440 ◽

Cited By ~ 27

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

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

The Journal of General Physiology ◽

10.1085/jgp.201210798 ◽

2012 ◽

Vol 140 (4) ◽

pp. 391-402 ◽

Cited By ~ 14

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.

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Functional role of a putative carbonic anhydrase II-binding domain in the electrogenic Na+-HCO3−cotransporter NBCe1 expressed in Xenopus oocytes

Channels ◽

10.4161/chan.5.2.14341 ◽

2011 ◽

Vol 5 (2) ◽

pp. 106-109 ◽

Cited By ~ 15

Author(s):

Hideomi Yamada ◽

Shoko Horita ◽

Masashi Suzuki ◽

Toshiro Fujita ◽

George Seki

Keyword(s):

Carbonic Anhydrase ◽

Functional Role ◽

Xenopus Oocytes ◽

Binding Domain ◽

Carbonic Anhydrase Ii

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Evidence for functional role of εPKC isozyme in the regulation of cardiac Na+ channels

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.5.c1477 ◽

2001 ◽

Vol 281 (5) ◽

pp. C1477-C1486 ◽

Cited By ~ 40

Author(s):

Guang-Qian Xiao ◽

Yongxia Qu ◽

Zhou-Qian Sun ◽

Daria Mochly-Rosen ◽

Mohamed Boutjdir

Keyword(s):

Functional Role ◽

Xenopus Oocytes ◽

Ventricular Myocytes ◽

Na Channels ◽

Kinase C ◽

Electrode Voltage ◽

Specific Antagonist ◽

Pkc Isozymes ◽

Specific Inhibitors

Investigation of the role of individual protein kinase C (PKC) isozymes in the regulation of Na+ channels has been largely limited by the lack of isozyme-selective modulators. Here we used a novel peptide-specific activator (εV1–7) of εPKC and other peptide isozyme-specific inhibitors in addition to the general PKC activator phorbol 12-myristate 13-acetate (PMA) to dissect the role of individual PKCs in the regulation of the human cardiac Na+ channel hH1, heterologously expressed in Xenopus oocytes. Peptides were injected individually or in combination into the oocyte. Whole cell Na+ current ( I Na) was recorded using two-electrode voltage clamp. εV1–7 (100 nM) and PMA (100 nM) inhibited I Na by 31 ± 5% and 44 ± 8% (at −20 mV), respectively. These effects were not seen with the scrambled peptide for εV1–7 (100 nM) or the PMA analog 4α-phorbol 12,13-didecanoate (100 nM). However, εV1–7- and PMA-induced I Na inhibition was abolished by εV1–2, a peptide-specific antagonist of εPKC. Furthermore, PMA-induced I Na inhibition was not altered by 100 nM peptide-specific inhibitors for α-, β-, δ-, or ηPKC. PMA and εV1–7 induced translocation of εPKC from soluble to particulate fraction in Xenopus oocytes. This translocation was antagonized by εV1–2. In native rat ventricular myocytes, PMA and εV1–7 also inhibited I Na; this inhibition was antagonized by εV1–2. In conclusion, the results provide evidence for selective regulation of cardiac Na+channels by εPKC isozyme.

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

AJP Cell Physiology ◽

10.1152/ajpcell.1993.265.1.c224 ◽

1993 ◽

Vol 265 (1) ◽

pp. C224-C233 ◽

Cited By ~ 117

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.

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