Differential modulation of TTX-sensitive and TTX-resistant Na+ channels in spinal cord astrocytes following activation of protein kinase C

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.

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Opposite effects of angiotensin II and the protein kinase C activator OAG on cardiac Na+ channels

The Journal of Membrane Biology ◽

10.1007/bf00231895 ◽

1992 ◽

Vol 130 (2) ◽

Cited By ~ 16

Author(s):

I. Benz ◽

J.W. Herzig ◽

M. Kohlhardt

Keyword(s):

Protein Kinase C ◽

Angiotensin Ii ◽

Protein Kinase ◽

Na Channels ◽

Kinase C

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Molecular Mechanism of Convergent Regulation of Brain Na+ Channels by Protein Kinase C and Protein Kinase A Anchored to AKAP-15

Molecular and Cellular Neuroscience ◽

10.1006/mcne.2002.1162 ◽

2002 ◽

Vol 21 (1) ◽

pp. 63-80 ◽

Cited By ~ 61

Author(s):

Angela R. Cantrell ◽

Victoria C. Tibbs ◽

Frank H. Yu ◽

Brian J. Murphy ◽

Elizabeth M. Sharp ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Protein Kinase A ◽

Molecular Mechanism ◽

Na Channels ◽

Kinase C ◽

Kinase A

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

The Journal of General Physiology ◽

10.1085/jgp.108.5.375 ◽

1996 ◽

Vol 108 (5) ◽

pp. 375-379 ◽

Cited By ~ 63

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.

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