Effects of luminal Na+ on single Na+ channels in A6 cells, a regulatory role for protein kinase C

1989 ◽  
Vol 256 (6) ◽  
pp. F1094-F1103 ◽  
Author(s):  
B. N. Ling ◽  
D. C. Eaton
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Calcium Ionophore ◽  
Direct Interaction ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activity ◽  
Pipette Solution ◽  
Kinase C ◽  
Na Permeability

Na+ "self-inhibition" in tight epithelia describes the reduction in apical Na+ permeability observed with increasing luminal Na+ concentration. Patch clamp was used to examine regulation of self-inhibition at the level of single Na+ channels. After cell-attached patches (pipette solution, 129 mM NaCl) were obtained on amphibian distal nephron cells (A6), the 129 mM NaCl (high Na+) apical bath outside of the patch was replaced with 3 mM NaCl (low Na+). Within minutes there was an increase in open channel probability (Po) and the appearance of one to five "new" channels in patch membranes. A similar increase occurred when apical Na+ entry was blocked by luminal amiloride (10 microM). A23187 (1 microM), a calcium ionophore, added after low Na+ exchange, abolished the rise in channel activity. Increased Po and new channels, induced by either luminal Na+ or amiloride, were also reversed by either 4B-phorbol 12-myristate 13-acetate (PMA; 0.1 microM) or 1-oleyl-2-acetyl glycerol (OAG; 10 microM) over 15-30 min. 4 alpha-Phorbol (0.1 microM), an inactive phorbol, did not reduce channel activity. D-Sphingosine (100 microM), a protein kinase C (PKC) inhibitor, increased Po and new channels. Conclusions: 1) modulation of apical Na+ permeability by luminal Na+ does not require direct interaction of Na+ with the channel protein but, rather, appears to involve an intracellular regulatory pathway, 2) relieving self-inhibition alters both the number and kinetics of single Na+ channels, 3) the effect of low Na+ must be modulated via decreased apical Na+ entry and intracellular Na+, since amiloride yielded similar results, 4) changes in intracellular Na+ probably affect Na+ channel activity via cytosolic Ca2+, 5) the effects of decreasing luminal Na+ are reversed by PKC activators and mimicked by PKC inhibitors suggesting a possible role for PKC in Na+ self-inhibition.

scholarly journals Phosphorylation of S1505 in the cardiac Na+ channel inactivation gate is required for modulation by protein kinase C.

1996 ◽  
Vol 108 (5) ◽  
pp. 375-379 ◽  
Author(s):  
Y Qu ◽  
J C Rogers ◽  
T N Tanada ◽  
W A Catterall ◽  
T Scheuer
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Na Channel ◽  
Na Channels ◽  
Na Current ◽  
Inactivation Curve ◽  
Electrophysiological Properties ◽  
Negative Shift ◽  
Kinase C ◽  
Steady State Inactivation

Inactivation of both brain and cardiac Na+ channels is modulated by activation of protein kinase C (PKC) but in different ways. Previous experiments had shown that phosphorylation of serine 1506 in the highly conserved loop connecting homologous domains III and IV (LIII/IV) of the brain Na+ channel alpha subunit is necessary for all effects of PKC. Here we examine the importance of the analogous serine for the different modulation of the rH1 cardiac Na+ channel. Serine 1505 of rH1 was mutated to alanine to prevent its phosphorylation, and the resulting mutant channel was expressed in 1610 cells. Electrophysiological properties of these mutant channels were indistinguishable from those of wild-type (WT) rH1 channels. Activation of PKC with 1-oleoyl-2-acetyl-sn-glycerol (OAG) reduced WT Na+ current by 49.3 +/- 4.2% (P < 0.01) but S1505A mutant current was reduced by only 8.5 +/- 5.4% (P = 0.29) when the holding potential was -94 mV. PKC activation also caused a -17-mV shift in the voltage dependence of steady-state inactivation of the WT channel which was abolished in the mutant. Thus, phosphorylation of serine 1505 is required for both the negative shift in the inactivation curve and the reduction in Na+ current by PKC. Phosphorylation of S1505/1506 has common and divergent effects in brain and cardiac Na+ channels. In both brain and cardiac Na+ channels, phosphorylation of this site by PKC is required for reduction of peak Na+ current. However, phosphorylation of S1506 in brain Na+ channels slows and destabilizes inactivation of the open channel. Phosphorylation of S1505 in cardiac, but not S1506 in brain, Na+ channels causes a negative shift in the inactivation curve, indicating that it stabilizes inactivation from closed states. Since LIII/IV containing S1505/S1506 is completely conserved, interaction of the phosphorylated serine with other regions of the channel must differ in the two channel types.

Feedback regulation of Na channels in rat CCT. IV. Mediation by activation of protein kinase C

1996 ◽  
Vol 270 (2) ◽  
pp. F371-F376 ◽  
Author(s):  
G. Frindt ◽  
L. G. Palmer ◽  
E. E. Windhager
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Single Channel ◽  
Feedback Inhibition ◽  
Na Channel ◽  
Specific Cell ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Kinase C ◽  
Calmodulin Kinase

The hypothesis that feedback inhibition of the apical Na+ channels in the cortical collecting tubule (CCT) is mediated by activation of a Ca(2+)-dependent protein kinase was tested using the patch-clamp technique. Na+ channel activity was monitored in cell-attached patches in principal cells of split-open rat tubules. Mean number of open channels (NPo) and single-channel current (i) were measured at 37 degrees C during continuous tubule superfusion. Phorbol 12-myristate 13-acetate (PMA; 50 nM), an activator of protein kinase C (PKC), decreased NPo to 33% of the control value. Staurosporine (200 nM), an inhibitor of PKC and of Ca(2+)-calmodulin kinase II, practically abolished the effect of PMA. Ouabain (1 mM), reduced NPo to 29% of control values and decreased i. Ouabain did not downregulate the channels in tubules exposed to staurosporine, although it still reduced i. Incubation of the tubules with 10 microM KN-62, a specific cell membrane-permeable inhibitor of Ca(2+)-calmodulin kinase II, did not interfere with the ouabain-dependent downregulation of the channels. The results support the view that the downregulation caused by ouabain involves the Ca(2+)-dependent phosphorylation of the channel itself or of proteins regulating the channel.

scholarly journals Differential Effects of Protein Kinase C on the Levels of Epithelial Na+Channel Subunit Proteins

2000 ◽  
Vol 275 (33) ◽  
pp. 25760-25765 ◽  
Author(s):  
James D. Stockand ◽  
Hui-Fang Bao ◽  
Julie Schenck ◽  
Bela Malik ◽  
Pam Middleton ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Na Channel ◽  
Differential Effects ◽  
Kinase C ◽  
Epithelial Na Channel

scholarly journals Down-regulation of Na channel α-subunit mRNA by protein kinase C e in adrenal chromaffin cells

1997 ◽  
Vol 73 ◽  
pp. 157
Author(s):  
Toshihiko Yanagita ◽  
Hideyuki Kobayashi ◽  
Keizou Masumoto ◽  
Ryuichi Yamamoto ◽  
Tomoaki Yuhi ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Chromaffin Cells ◽  
Na Channel ◽  
Adrenal Chromaffin Cells ◽  
Down Regulation ◽  
Α Subunit ◽  
Subunit Mrna ◽  
Kinase C ◽  
Adrenal Chromaffin

scholarly journals Protein kinase C in rod outer segments: effects of phosphorylation of the phosphodiesterase inhibitory subunit

1996 ◽  
Vol 317 (1) ◽  
pp. 291-295 ◽  
Author(s):  
Igor P. UDOVICHENKO ◽  
Jess CUNNICK ◽  
Karen GONZALEZ ◽  
Alexander YAKHNIN ◽  
Dolores J. TAKEMOTO
Keyword(s):  
Protein Kinase C ◽  
Catalytic Activity ◽  
Protein Kinase ◽  
Direct Interaction ◽  
Inhibitory Effect ◽  
Rod Outer Segments ◽  
Visual Transduction ◽  
Gtp Hydrolysis ◽  
Outer Segments ◽  
Kinase C

The inhibitory subunit (PDEγ) of the cGMP phosphodiesterase (PDEαβγ2) in rod outer segments (ROS) realizes its regulatory role in phototransduction by inhibition of PDEαβ catalytic activity. The photoreceptor G-protein, transducin, serves as a transducer from the receptor (rhodopsin) to the effector (PDE) and eliminates the inhibitory effect of PDEγ by direct interaction with PDEγ. Our previous study [Udovichenko, Cunnick, Gonzalez and Takemoto (1994) J. Biol. Chem. 269, 9850–9856] has shown that PDEγ is a substrate for protein kinase C (PKC) from ROS and that phosphorylation by PKC increases the ability of PDEγ to inhibit PDEαβ catalytic activity. Here we report that transducin is less effective in activation of PDEαβ(γp)2 (a complex of PDEαβ with phosphorylated PDEγ, PDEγp) than PDEαβγ2. PDEγp also increases the rate constant of GTP hydrolysis of transducin (from 0.16 s-1 for non-phosphorylated PDEγ to 0.21 s-1 for PDEγp). These data suggest that phosphorylation of the inhibitory subunit of PDE by PKC may regulate the visual transduction cascade by decreasing the photoresponse.

scholarly journals A role for protein kinase C in the membrane fusion necessary for platelet granule secretion

Blood ◽  
1989 ◽  
Vol 74 (7) ◽  
pp. 2405-2413 ◽  
Author(s):  
JM Gerrard ◽  
LL Beattie ◽  
J Park ◽  
SJ Israels ◽  
A McNicol ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Membrane Fusion ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ultrastructural Changes ◽  
Ionophore A23187 ◽  
Kinase C ◽  
Granule Secretion ◽  
Dimethyl Amiloride

Abstract The addition of 1-oleoyl-2-acetylglycerol (OAG), or phorbol-12- myristate-13-acetate (PMA) to platelets induced the phosphorylation of a 47,000 dalton protein (47 Kd), fusion of granule membranes with membranes of the surface connected canalicular system, the formation of large vesicles and the secretion of serotonin. 1-(5- isoquinolinesulfonyl)-2-methyl-piperazine (H7), and sphingosine, inhibitors of protein kinase C, significantly inhibited the ultrastructural changes and the phosphorylation of 47 Kd. N-(2- guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), structurally similar to H7, but a weaker inhibitor of protein kinase C, did not attenuate these responses to OAG or to PMA. H7, but not HA1004, also markedly inhibited secretion induced by the synergistic combination of OAG and the calcium ionophore A23187. Amiloride and 5-(N,N dimethyl)- amiloride, inhibitors of the Na+/H+ transporter, did not inhibit the ultrastructural response and the protein phosphorylation induced by OAG, or the secretion induced by the combination of A23187 and OAG. The results link the activation of protein kinase C by diglycerides to the labilization and fusion of granule membranes important for secretion.

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