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.

Vasopressin and protein kinase A activate G protein-sensitive epithelial Na+ channels

1993 ◽  
Vol 265 (1) ◽  
pp. C218-C223 ◽  
Author(s):  
A. G. Prat ◽  
D. A. Ausiello ◽  
H. F. Cantiello
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
G Protein ◽  
Channel Number ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activity ◽  
Intact Cells ◽  
Inside Out ◽  
Kinase A

To determine the molecular steps involved in the vasopressin-induced renal Na+ reabsorption, the patch-clamp technique was utilized to study the role of this hormone in the regulation of apical Na+ channels in renal epithelial A6 cells. Addition of arginine vasopressin (AVP) induced and/or enhanced Na+ channel activity within 5 min of addition under cell-attached conditions. The AVP-induced channel activity was a reflection of both an increase in the average apparent channel number (0.2-1.7) and the percent open time (2-56%). Addition of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, the adenosine 3',5'-cyclic monophosphate (cAMP) analogues, 8-(4-chlorophenylthio)-cAMP and 8-bromo-cAMP, or forskolin elicited a comparable effect to that of AVP. The induced channels had similar properties to Na+ channels previously reported, including a channel conductance of 9 pS, Na(+)-to-K+ selectivity of 3-5:1, and high amiloride sensitivity. The cAMP-dependent protein kinase A (PKA) in the presence of ATP induced and/or enhanced Na+ channel activity in excised inside-out patches with a change in average apparent channel number and percent open probability similar to those observed with either AVP or cAMP analogues in intact cells. Addition of activated pertussis toxin (100 ng/ml) completely blocked the AVP- or PKA-induced Na+ channel activity in excised inside-out patches, whereas incubation of intact cells with the toxin completely prevented the effect of both activators. The data indicate that AVP mediates its effect through a cAMP-dependent pathway involving PKA activation whose target is the G protein pathway that regulates apical epithelial Na+ channel activity.

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.

Protein kinase A and mitogen-activated protein kinase pathways mediate cAMP induction of α-Epithelial Na+channels (α-ENaC)

2008 ◽  
Vol 215 (1) ◽  
pp. 101-110 ◽  
Author(s):  
Shamimunisa B. Mustafa ◽  
Robert Castro ◽  
Alison J. Falck ◽  
Jean A. Petershack ◽  
Barbara M. Henson ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Mitogen Activated Protein Kinase ◽  
Na Channels ◽  
Epithelial Na Channels ◽  
Mitogen Activated Protein ◽  
Camp Induction ◽  
Kinase A

Cytoskeleton modulates gating of voltage-dependent sodium channel in heart

1995 ◽  
Vol 269 (1) ◽  
pp. H203-H214 ◽  
Author(s):  
A. I. Undrovinas ◽  
G. S. Shander ◽  
J. C. Makielski
Keyword(s):  
Membrane Proteins ◽  
Actin Polymerization ◽  
Integral Membrane Proteins ◽  
Na Channel ◽  
Na Channels ◽  
Open Probability ◽  
Whole Cell ◽  
Voltage Dependent ◽  
D 20 ◽  
Inside Out

To investigate the role of the cytoskeleton in cardiac Na+ channel gating, the action of cytochalasin D (Cyto-D), an agent that interferes with actin polymerization, was studied by whole cell voltage clamp and cell-attached and inside-out patches from rat and rabbit ventricular cardiac myocytes. Cyto-D (20-40 microM) reduced whole cell peak Na+ current by 20% within 12 min and slowed current decay without affecting steady-state voltage-dependent availability or recovery from inactivation. Brief treatments (< 10-15 min) of cell-attached patches by Cyto-D (20 microM) in the bath induced short bursts of Na+ channel openings and prolonged decays of ensemble-averaged currents. Bursting of the Na+ channel was more pronounced when the cell suspension was pretreated with Cyto-D (20 microM) for 1 h before seal formation. Application of Cyto-D on the cytoplasmic side of inside-out patches resulted in more dramatic gating changes. Peak open probability was reduced by > 50% within 20 min, and long bursts of openings occurred. Washout of Cyto-D did not restore ensemble-averaged current amplitude, but burst duration decreased toward control values. Cyto-D also induced an additional slower component to open and closed times. These results suggest that Cyto-D, through effects on cytoskeleton, induced cardiac Na+ channels to enter a mode characterized by a lower peak open probability but a greater persistent activity as if the inactivation rate was slowed. The cytoskeleton, in addition to localizing integral membrane proteins, apparently also plays a role in regulating specific detailed functions of integral membrane proteins such as the gating of Na+ channels.

Protein kinase A phosphorylation and G protein regulation of type II pneumocyte Na+ channels in lipid bilayers

1997 ◽  
Vol 272 (4) ◽  
pp. C1262-C1270 ◽  
Author(s):  
B. K. Berdiev ◽  
V. G. Shlyonsky ◽  
O. Senyk ◽  
D. Keeton ◽  
Y. Guo ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Lipid Bilayers ◽  
Na Channel ◽  
Type Ii ◽  
Na Channels ◽  
Channel Activity ◽  
Open Probability ◽  
Atii Cell ◽  
Kinase A

Protein kinase A (PKA)- and G protein-mediated regulation of immunopurified adult rabbit alveolar epithelial type II (ATII) cell proteins that exhibit amiloride-sensitive Na+ channel activity was studied in planar lipid bilayers and freshly isolated ATII cells. Addition of the catalytic subunit of PKA + ATP increased single channel open probability from 0.42 +/- 0.05 to 0.82 +/- 0.07 in a voltage-independent manner, without affecting unitary conductance. This increase in open probability of the channels was mainly due to a decrease in the time spent by the channel in its closed state. The apparent inhibition constant for amiloride increased from 8.0 +/- 1.8 microM under control conditions to 15 +/- 3 microM after PKA-induced phosphorylation; that for ethylisopropylamiloride increased from 1.0 +/- 0.4 to 2.0 +/- 0.5 microM. Neither pertussis toxin (PTX) nor guanosine 5'-O-(3-thiotriphosphate) affected ATII Na+ channel activity in bilayers. Moreover, PTX failed to affect amiloride-inhibitable 22Na+ uptake in freshly isolated ATII cells. In vitro, ADP ribosylation induced by PTX revealed the presence of a specifically ribosylated band at 40-45 kDa in the total solubilized ATII cell protein fraction, but not in the immunopurified fraction. Moreover, the immunopurified channel was downregulated in response to guanosine 5'-O-(3-thiotriphosphate)-mediated activation of the exogenous G alpha(i-2), but not G(oA), G alpha(i-1), or G alpha(i-3), protein added to the channel. This effect occurred only in the presence of actin. These results suggest that amiloride-sensitive Na+ channels in adult alveolar epithelia regulated by PKA-mediated phosphorylation also retain the ability to be regulated by G alpha([i-2), but not G alpha([i-1) or G alpha(i-3), protein.

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.

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