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 Action potentials in Xenopus oocytes triggered by blue light

2020 ◽  
Vol 152 (5) ◽  
Author(s):  
Florian Walther ◽  
Dominic Feind ◽  
Christian vom Dahl ◽  
Christoph Emanuel Müller ◽  
Taulant Kukaj ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Action Potential ◽  
Blue Light ◽  
Action Potentials ◽  
Xenopus Oocytes ◽  
Na Channel ◽  
Xenopus Laevis Oocytes ◽  
Na Channels ◽  
Excitable Cells ◽  
Voltage Gated

Voltage-gated sodium (Na+) channels are responsible for the fast upstroke of the action potential of excitable cells. The different α subunits of Na+ channels respond to brief membrane depolarizations above a threshold level by undergoing conformational changes that result in the opening of the pore and a subsequent inward flux of Na+. Physiologically, these initial membrane depolarizations are caused by other ion channels that are activated by a variety of stimuli such as mechanical stretch, temperature changes, and various ligands. In the present study, we developed an optogenetic approach to activate Na+ channels and elicit action potentials in Xenopus laevis oocytes. All recordings were performed by the two-microelectrode technique. We first coupled channelrhodopsin-2 (ChR2), a light-sensitive ion channel of the green alga Chlamydomonas reinhardtii, to the auxiliary β1 subunit of voltage-gated Na+ channels. The resulting fusion construct, β1-ChR2, retained the ability to modulate Na+ channel kinetics and generate photosensitive inward currents. Stimulation of Xenopus oocytes coexpressing the skeletal muscle Na+ channel Nav1.4 and β1-ChR2 with 25-ms lasting blue-light pulses resulted in rapid alterations of the membrane potential strongly resembling typical action potentials of excitable cells. Blocking Nav1.4 with tetrodotoxin prevented the fast upstroke and the reversal of the membrane potential. Coexpression of the voltage-gated K+ channel Kv2.1 facilitated action potential repolarization considerably. Light-induced action potentials were also obtained by coexpressing β1-ChR2 with either the neuronal Na+ channel Nav1.2 or the cardiac-specific isoform Nav1.5. Potential applications of this novel optogenetic tool are discussed.

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.

scholarly journals The Human Heart and Rat Brain IIA Na+ Channels Interact with Different Molecular Regions of the β1 Subunit

2002 ◽  
Vol 120 (6) ◽  
pp. 887-895 ◽  
Author(s):  
Thomas Zimmer ◽  
Klaus Benndorf
Keyword(s):  
Rat Brain ◽  
Human Heart ◽  
Xenopus Oocytes ◽  
Extracellular Domain ◽  
Na Channel ◽  
Na Channels ◽  
Membrane Anchor ◽  
Α Subunit ◽  
Extracellular Region ◽  
Recovery From Inactivation

The α subunit of voltage-gated Na+ channels of brain, skeletal muscle, and cardiomyocytes is functionally modulated by the accessory β1, but not the β2 subunit. In the present study, we used β1/β2 chimeras to identify molecular regions within the β1 subunit that are responsible for both the increase of the current density and the acceleration of recovery from inactivation of the human heart Na+ channel (hH1). The channels were expressed in Xenopus oocytes. As a control, we coexpressed the β1/β2 chimeras with rat brain IIA channels. In agreement with previous studies, the β1 extracellular domain sufficed to modulate IIA channel function. In contrast to this, the extracellular domain of the β1 subunit alone was ineffective to modulate hH1. Instead, the putative membrane anchor plus either the intracellular or the extracellular domain of the β1 subunit was required. An exchange of the β1 membrane anchor by the corresponding β2 subunit region almost completely abolished the effects of the β1 subunit on hH1, suggesting that the β1 membrane anchor plays a crucial role for the modulation of the cardiac Na+ channel isoform. It is concluded that the β1 subunit modulates the cardiac and the neuronal channel isoforms by different molecular interactions: hH1 channels via the membrane anchor plus additional intracellular or extracellular regions, and IIA channels via the extracellular region only.

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 Inactivation of cloned Na channels expressed in Xenopus oocytes.

1990 ◽  
Vol 96 (4) ◽  
pp. 689-706 ◽  
Author(s):  
D S Krafte ◽  
A L Goldin ◽  
V J Auld ◽  
R J Dunn ◽  
N Davidson ◽  
...  
Keyword(s):  
Time Course ◽  
Xenopus Oocytes ◽  
Voltage Dependence ◽  
Single Channel ◽  
Single Amino Acid ◽  
Na Channel ◽  
Low Molecular Weight ◽  
Cdna Clones ◽  
Na Channels ◽  
Single Amino Acid Residue

This study investigates the inactivation properties of Na channels expressed in Xenopus oocytes from two rat IIA Na channel cDNA clones differing by a single amino acid residue. Although the two cDNAs encode Na channels with substantially different activation properties (Auld, V. J., A. L. Goldin, D. S. Krafte, J. Marshall, J. M. Dunn, W. A. Catterall, H. A. Lester, N. Davidson, and R. J. Dunn. 1988. Neuron. 1:449-461), their inactivation properties resemble each other strongly but differ markedly from channels induced by poly(A+) rat brain RNA. Rat IIA currents inactivate more slowly, recover from inactivation more slowly, and display a steady-state voltage dependence that is shifted to more positive potentials. The macroscopic inactivation process for poly(A+) Na channels is defined by a single exponential time course; that for rat IIA channels displays two exponential components. At the single-channel level these differences in inactivation occur because rat IIA channels reopen several times during a depolarizing pulse; poly(A+) channels do not. Repetitive stimulation (greater than 1 Hz) produces a marked decrement in the rat IIA peak current and changes the waveform of the currents. When low molecular weight RNA is coinjected with rat IIA RNA, these inactivation properties are restored to those that characterize poly(A+) channels. Slow inactivation is similar for rat IIA and poly(A+) channels, however. The data suggest that activation and inactivation involve at least partially distinct regions of the channel protein.

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.

Selective expression of an amiloride-inhibitable Na+ conductance from mRNA of respiratory epithelium in Xenopus laevis oocytes

1989 ◽  
Vol 257 (4) ◽  
pp. L284-L288
Author(s):  
B. Kroll ◽  
W. Bautsch ◽  
S. Bremer ◽  
M. Wilke ◽  
B. Tummler ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Nasal Polyp ◽  
Primary Cultures ◽  
Respiratory Epithelium ◽  
Membrane Potentials ◽  
Xenopus Laevis Oocytes ◽  
Na Channels ◽  
Epithelial Na Channels ◽  
Selective Expression ◽  
Ion Conductances

Poly(A)+RNA was prepared from primary cultures of human nasal polyp epithelia and from native bovine tracheal epithelia. Six to fifty nanograms mRNA were injected into prophase-arrested immature Xenopus laevis oocytes. One to four days later the oocytes were probed with electrophysiological techniques for induction of novel ion conductances. Oocytes injected with mRNA had lower membrane potentials (Vm) and resistances (Rm) than controls. By use of step changes in extracellular Na+ concentration and applying amiloride, a Na+ conductance could be identified in mRNA-injected oocytes, which was inhibited by submicromolar concentrations of amiloride with the same kinetics (Ki = 1.3 X 10(-7) mol/l) as in the original tissue. After complete inhibition of this conductance by 10(-5) mol/l amiloride, Vm and Rm approached the respective values of controls. The data indicate that oocytes express functional epithelial Na+ channels but apparently no other epithelial ion conductances from injected mRNA of respiratory epithelium.

scholarly journals Specificity for block by saxitoxin and divalent cations at a residue which determines sensitivity of sodium channel subtypes to guanidinium toxins.

1995 ◽  
Vol 106 (2) ◽  
pp. 203-229 ◽  
Author(s):  
I Favre ◽  
E Moczydlowski ◽  
L Schild
Keyword(s):  
Cell Line ◽  
Divalent Cations ◽  
Point Mutations ◽  
Dissociation Rate ◽  
Na Channel ◽  
Na Channels ◽  
Ionic Selectivity ◽  
High Affinity ◽  
Rat Muscle ◽  
Enhanced Sensitivity

bTyrosine 401 of the skeletal muscle isoform (mu 1) of the rat muscle Na channel is an important determinant of high affinity block by tetrodotoxin (TTX) and saxitoxin (STX) in Na-channel isoforms. In mammalian heart Na channels, this residue is substituted by cysteine, which results in low affinity for TTX/STX and enhanced sensitivity to block by Zn2+ and Cd2+. In this study, we investigated the molecular basis for high affinity block of Na channels by STX and divalent cations by measuring inhibition of macroscopic Na+ current for a series of point mutations at residue Tyr401 of the rat mu 1 Na channel expressed in Xenopus oocytes. Substitution of Tyr401 by Gly, Ala, Ser, Cys, Asp, His, Trp, and Phe produced functional Na+ currents without major perturbation of gating or ionic selectivity. High affinity block by STX and neosaxitoxin (NEO) with Ki values in the range of 2.6-18 nM required Tyr, Phe, or Trp, suggestive of an interaction between an aromatic ring and a guanidinium group of the toxin. The Cys mutation resulted in a 7- and 23-fold enhancement of the dissociation rate of STX and NEO, respectively, corresponding to rapid toxin dissociation rates of cardiac Na channels. High affinity block by Zn2+ (Ki = 8-23 microM) required Cys, His, or Asp, three residues commonly found to coordinate directly with Zn2+ in metalloproteins. For the Cys mutant of mu 1 and also for the cardiac isoform Na channel (rh1) expressed in the L6 rat muscle cell line, inhibition of macroscopic Na+ conductance by Zn2+ reached a plateau at 85-90% inhibition, suggesting the presence of a substate current. The Asp mutant also displayed enhanced affinity for inhibition of conductance by Ca2+ (Ki = 0.3 mM vs approximately 40 mM in wild type), but block by Ca2+ was incomplete, saturating at approximately 69% inhibition. In contrast, Cd2+ completely blocked macroscopic current in the Cys mutant and the L6 cell line. These results imply that the magnitude of substate current depends on the particular residue at position 401 and the species of divalent cation. The His mutant also exhibited enhanced sensitivity to block by H+ with a pKa of approximately 7.5 for the His imidazole group. Our findings provide further evidence that residue 401 of mu 1 is located within the outer vestibule of the Na channel but external to the single-filing region for permeant ions.

Sign in / Sign up

Export Citation Format

Share Document