Effects of luminal Na+ on single Na+ channels in A6 cells, a regulatory role for 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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Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322.
Eaton
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D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D004075:Diglycerides / Q000494:pharmacology; D004553:Electric Conductivity; D006706:Homeostasis; D007668:Kidney / Q000502:physiology; D007700:Kinetics; D011493:Protein Kinase C / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology*; D015222:Sodium Channels / Q000187:drug effects / Q000502:physiology*; D013110:Sphingosine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004075:Diglycerides; D015222:Sodium Channels; D000001:Calcimycin; D000584:Amiloride; C038237:1-oleoyl-2-acetylglycerol; D012964:Sodium; D011493:Protein Kinase C; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate