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.

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.

scholarly journals Regulation by Na+ and Ca2+ of renal epithelial Na+ channels reconstituted into planar lipid bilayers.

1995 ◽  
Vol 106 (3) ◽  
pp. 445-466 ◽  
Author(s):  
I I Ismailov ◽  
B K Berdiev ◽  
D J Benos
Keyword(s):  
Protein Kinase ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Single Channels ◽  
Planar Lipid Bilayers ◽  
Na Channels ◽  
Channel Activity ◽  
Open Probability ◽  
Epithelial Na Channels ◽  
Cis And Trans

Purified bovine renal epithelial Na+ channels when reconstituted into planar lipid bilayers displayed a specific orientation when the membrane was clamped to -40 mV (cis-side) during incorporation. The trans-facing portion of the channel was extracellular (i.e., amiloride-sensitive), whereas the cis-facing side was intracellular (i.e., protein kinase A-sensitive). Single channels had a main state unitary conductance of 40 pS and displayed two subconductive states each of 12-13 pS, or one of 12-13 pS and the second of 24-26 pS. Elevation of the [Na+] gradient from the trans-side increased single-channel open probability (Po) only when the cis-side was bathed with a solution containing low [Na+] (< 30 mM) and 10-100 microM [Ca2+]. Under these conditions, Po saturated with increasing [Na+]trans. Buffering of the cis compartment [Ca2+] to nearly zero (< 1 nM) with 10 mM EGTA increased the initial level of channel activity (Po = 0.12 +/- 0.02 vs 0.02 +/- 0.01 in control), but markedly reduced the influence of both cis- and trans-[Na+] on Po. Elevating [Ca2+]cis at constant [Na+] resulted in inhibition of channel activity with an apparent [KiCa2+] of 10-100 microM. Protein kinase C-induced phosphorylation shifted the dependence of channel Po on [Ca2+]cis to 1-3 microM at stationary [Na+]. The direct modulation of single-channel Po by Na+ and Ca2+ demonstrates that the gating of amiloride-sensitive Na2+ channels is indeed dependent upon the specific ionic environment surrounding the channels.

scholarly journals Spatial targeting of type II protein kinase A to filopodia mediates the regulation of growth cone guidance by cAMP

2007 ◽  
Vol 176 (1) ◽  
pp. 101-111 ◽  
Author(s):  
Jianzhong Han ◽  
Liang Han ◽  
Priyanka Tiwari ◽  
Zhexing Wen ◽  
James Q. Zheng
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Growth Cone ◽  
Regulatory Protein ◽  
Cyclic Adenosine Monophosphate ◽  
Spatial Localization ◽  
Adenosine Monophosphate ◽  
Type Ii ◽  
Regulation Of Growth ◽  
Kinase A

The second messenger cyclic adenosine monophosphate (cAMP) plays a pivotal role in axonal growth and guidance, but its downstream mechanisms remain elusive. In this study, we report that type II protein kinase A (PKA) is highly enriched in growth cone filopodia, and this spatial localization enables the coupling of cAMP signaling to its specific effectors to regulate guidance responses. Disrupting the localization of PKA to filopodia impairs cAMP-mediated growth cone attraction and prevents the switching of repulsive responses to attraction by elevated cAMP. Our data further show that PKA targets protein phosphatase-1 (PP1) through the phosphorylation of a regulatory protein inhibitor-1 (I-1) to promote growth cone attraction. Finally, we find that I-1 and PP1 mediate growth cone repulsion induced by myelin-associated glycoprotein. These findings demonstrate that the spatial localization of type II PKA to growth cone filopodia plays an important role in the regulation of growth cone motility and guidance by cAMP.

Effects of prostaglandin E2 on amiloride-blockable Na+ channels in a distal nephron cell line (A6)

1994 ◽  
Vol 267 (5) ◽  
pp. C1414-C1425 ◽  
Author(s):  
K. E. Kokko ◽  
P. S. Matsumoto ◽  
B. N. Ling ◽  
D. C. Eaton
Keyword(s):  
Prostaglandin E2 ◽  
Apical Cell ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activity ◽  
Distal Nephron ◽  
Open Probability ◽  
A6 Cells ◽  
Channel Inhibition ◽  
Camp Levels

We studied the mechanisms by which prostaglandin E2 (PGE2) regulates amiloride-blockable 4-pS Na+ channels in A6 distal nephron cells. With each apical cell-attached patch acting as its own control, acute (3-6 min) basolateral, but not apical, exposure to 1 microM PGE2 inhibited Na+ channel activity by decreasing the open probability (Po). This PGE2-induced inhibition was attenuated by 30 min pretreatment with the protein kinase C (PKC) antagonists 1 microM staurosporine or 100 microM D-sphingosine but was insensitive to pertussis toxin (PTX). Furthermore, the time course for channel inhibition by acute PGE2 correlated with a transient increase in intracellular inositol 1,4,5-trisphosphate (IP3) levels. In contrast, after chronic (10-50 min) exposure of A6 cells to 1 microM basolateral PGE2, channel activity was stimulated compared with controls. This stimulation was due to an increase in the number of apical Na+ channels, similar to the effect of maneuvers that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels in A6 cells (22). Indeed, chronic exposure to basolateral PGE2 correlated with a sustained increase in cAMP levels. In conclusion, 1) the regulation of apical 4-pS highly selective Na+ channel activity by basolateral PGE2 is a complicated biphasic process, which includes inhibition by acute PGE2 and stimulation by chronic PGE2 exposure; 2) acute PGE2 promotes a transient generation of IP3 which activates Ca(2+)-dependent PKC and promotes a decrease in Po; 3) chronic PGE2 promotes a sustained generation of cAMP that leads to an increase in channel density; and 4) both the acute and chronic effects of PGE2 on Na+ channels are PTX-insensitive processes.

Differential Localization of Protein Kinase A Type II Isozymes in the Golgi–Centrosomal Area

1999 ◽  
Vol 249 (1) ◽  
pp. 131-146 ◽  
Author(s):  
Guy Keryer ◽  
Bjørn S. Skålhegg ◽  
Brynjar F. Landmark ◽  
Vidar Hansson ◽  
Tore Jahnsen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Type Ii ◽  
Differential Localization ◽  
Kinase A

Regulation of expression of type II sodium-phosphate cotransporters by protein kinases A and C

1998 ◽  
Vol 275 (2) ◽  
pp. F270-F277 ◽  
Author(s):  
Eleanor D. Lederer ◽  
Sameet S. Sohi ◽  
Jeanine M. Mathiesen ◽  
Jon B. Klein
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Sodium Phosphate ◽  
Broad Band ◽  
Confocal Imaging ◽  
Type Ii ◽  
Ok Cells ◽  
Kinase C ◽  
Kinase A

The purpose of the present study was to determine the effect of protein kinase A and protein kinase C activation on the membrane expression of NaPi-4, the type II sodium-phosphate cotransporter in OK cells. NaPi-4 expression was measured using polyclonal antisera produced in rabbits against a peptide identical to the carboxy-terminal 12-amino acid sequence of NaPi-4. The antisera identified an apically localized protein by confocal imaging of intact OK cells and a broad band of 110–140 kDa by immunoblot analysis of OK cell membranes. Treatment of OK cells with parathyroid hormone (PTH) decreased the intensity of the 110- to 140-kDa band, which was detectable by 2 h, maximal by 4 h at 62%, and sustained for 24 h. 8-Bromo-cAMP (8-BrcAMP) inhibited NaPi-4 expression for up to 24 h by over 90%. However, phorbol 12-myristate 13-acetate inhibited NaPi-4 expression by less than 10%. PTH-(3–34), a fragment which stimulates only protein kinase C, inhibited phosphate transport but also had no effect on NaPi-4 expression. We conclude that protein kinase A but not protein kinase C inhibits sodium-phosphate uptake in OK cells by downregulation of NaPi-4 expression.

Interleukin-1 alpha stimulates dopamine release by activating type II protein kinase A in PC-12 cells

1995 ◽  
Vol 269 (6) ◽  
pp. E1083-E1088
Author(s):  
A. Joseph ◽  
A. Kumar ◽  
N. A. O'Connell ◽  
R. K. Agarwal ◽  
A. R. Gwosdow
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Dopamine Release ◽  
Interleukin 1 ◽  
Intracellular Camp ◽  
Type I ◽  
Camp Accumulation ◽  
Type Ii ◽  
Pc 12 Cells ◽  
Kinase A

A recent study from this laboratory [A. R. Gwosdow, N. A. O'Connell, and A. B. Abou-Samra. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E461-E466, 1992] showed that the inflammatory mediator interleukin-1 alpha (IL-1 alpha) stimulates catecholamine release from primary cultures of rat adrenal cells. The present studies were conducted to determine whether 1) IL-1 alpha stimulates catecholamine/dopamine release from the adrenal medullary cell line PC-12 and 2) the adenosine 3',5'-cyclic monophosphate (cAMP)-protein kinase A (PKA) pathway is involved in IL-1 alpha-induced dopamine release from PC-12 cells. The results indicate that IL-1 alpha significantly (P < 0.05) elevated dopamine release after a 24-h incubation period. IL-1 alpha did not stimulate cAMP accumulation at any time period between 5 min and 2 h. In contrast, forskolin-treated cells elevated (P < 0.05) intracellular cAMP levels and increased dopamine release. Because IL-1 alpha did not affect cAMP accumulation, the effect of IL-1 alpha on PKA activity was investigated. IL-1 alpha increased (P < 0.05) PKA activity at 15 and 30 min and returned to control levels by 1 h. Forskolin also increased (P < 0.05) PKA activity. The type of PKA activated (P < 0.05) by IL-1 alpha was type II PKA. In contrast, forskolin activated (P < 0.05) type I and type II PKA. Inhibition of PKA with the PKA inhibitor H-8 blocked PKA activity and dopamine secretion by both IL-1 alpha and forskolin in PC-12 cells. These observations demonstrate that 1) IL-1 alpha stimulated dopamine release from PC-12 cells by activating PKA, 2) the mechanism of IL-1 alpha activation of PKA does not involve detectable increases in intracellular cAMP accumulation, and 3) IL-1 alpha activates type II PKA, which is used by IL-1 alpha to stimulate dopamine secretion from PC-12 cells.

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