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

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

AJP Renal Physiology ◽

10.1152/ajprenal.1989.256.6.f1094 ◽

1989 ◽

Vol 256 (6) ◽

pp. F1094-F1103 ◽

Cited By ~ 29

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.

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Sensitization of the Cardiac Na Channel to α1-Adrenergic Stimulation by Inhalation Anesthetics

Anesthesiology ◽

10.1097/00000542-199801000-00020 ◽

1998 ◽

Vol 88 (1) ◽

pp. 125-133 ◽

Cited By ~ 7

Author(s):

Henry U. Weight ◽

Wai-Meng Kowk ◽

Georg C. Rehmert ◽

Zeljko J. Bosnjak

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

G Proteins ◽

Volatile Anesthetics ◽

Na Channel ◽

Adrenergic Stimulation ◽

Na Current ◽

Patch Clamp Technique ◽

Positive Interaction ◽

Kinase C

Background Alpha1-adrenergic receptor stimulation has been shown to inhibit cardiac Na+ current (INa). Furthermore, some form of synergistic interaction of alpha1-adrenergic effects on INa in combination with volatile anesthetics has been reported. In this study, the authors investigated the possible role of G proteins and protein kinase C in the effects of halothane and isoflurane in the absence and presence of alpha1-adrenergic stimulation on the cardiac INa. Methods The standard whole-cell configuration of the patch-clamp technique was used. INa was elicited by depolarizing test pulses from a holding potential of -80 mV in reduced Na+ solution (10 mM). The experiments were conducted on ventricular myocytes enzymatically isolated from adult guinea pig hearts. Results The inhibitory effect of halothane (1.2 mM) and isoflurane (1 mM) on peak INa was significantly diminished in the presence of guanosine 5'-O-[2-thiodiphosphate (GDPbetaS). In myocytes pretreated with pertussis toxin (PTX), the potency of halothane was significantly enhanced, but the isoflurane effect was unchanged. In the presence of the protein kinase C (PKC) inhibitor bisindolylmaleimide (BIS), the effect of halothane was unchanged. In contrast, the effect of isoflurane on INa in the presence of BIS was significantly enhanced. The positive interaction between methoxamine and halothane was evident in the presence of G protein and PKC inhibitors. In contrast, the effect of methoxamine with isoflurane was additive in the presence of GDPbetaS or BIS. Conclusions Different second messenger systems are involved in the regulation of cardiac Na+ current by volatile anesthetics. The effect of halothane involves a complex interaction with G proteins but is independent of regulation by PKC. In contrast, PKC is involved in the modulation of cardiac INa by isoflurane. In addition, non-PTX-sensitive G proteins may contribute to the effects of isoflurane. The positive interaction between methoxamine and anesthetics are independent of G proteins and PKC for halothane. In the case of isoflurane, the positive interaction with methoxamine is coupled to PTX-insensitive G proteins and PKC.

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Involvement of Tetrodotoxin-Resistant Na+ Current and Protein Kinase C in the Action of Growth Hormone (GH)-Releasing Hormone on Primary Cultured Somatotropes from GH-Green Fluorescent Protein Transgenic Mice

Endocrinology ◽

10.1210/en.2008-0405 ◽

2008 ◽

Vol 149 (9) ◽

pp. 4726-4735 ◽

Cited By ~ 16

Author(s):

Seung-Kwon Yang ◽

Kun Wang ◽

Helena Parkington ◽

Chen Chen

Keyword(s):

Protein Kinase C ◽

Green Fluorescent Protein ◽

Protein Kinase ◽

Fluorescent Protein ◽

Phosphodiesterase Inhibitor ◽

Na Channels ◽

Na Current ◽

Gh Secretion ◽

Kinase C ◽

Green Fluorescent

GHRH depolarizes the membrane of somatotropes, leading to an increase in intracellular free Ca2+ concentration and GH secretion. Na+ channels mediate the rapid depolarization during the initial phase of the action potential, and this regulates Ca2+ influx and GH secretion. GHRH increases a tetrodotoxin-sensitive somatotrope Na+ current that is mediated by cAMP. TTX-resistant (TTX-R) Na+ channels are abundant in sensory neurons and cardiac myocytes, but their occurrence and/or function in somatotropes has not been investigated. Here we demonstrate expression of TTX-R Na+ channels and a TTX-R Na+ current, using patch-clamp method, in green fluorescent protein-GH transgenic mouse somatotropes. GHRH (100nm) increased the TTX-R Na+ current in a reversible manner. The GHRH-induced increase in TTX-R Na+ current was not affected by the cAMP antagonist Rp-cAMP or protein kinase A inhibitors KT5720 or H89. The TTX-R current was increased by 8-bromoadenosine-cAMP (cAMP analog), forskolin (adenylyl-cyclase activator), and 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor), but the additional, GHRH-induced increase in TTX-R Na+ currents was not affected. U-73122 (phospholipase C inhibitor) and protein kinase C (PKC) inhibitors, Gö-6983 and chelerythrine, blocked the effect of GHRH. PKC activators, phorbol dibutyrate and phorbol myristate acetate, increased the TTX-R Na+ current, but GHRH had no further effect on the current. Na+-free extracellular medium significantly reduced GHRH-stimulated GH secretion. We conclude that GHRH-induced increase in the TTX-R Na+ current in mouse somatotropes is mediated by the PKC system. An increase in the TTX-R Na+ current may contribute to the GHRH-induced exocytosis of GH granules from mouse somatotropes.

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

AJP Renal Physiology ◽

10.1152/ajprenal.1996.270.2.f371 ◽

1996 ◽

Vol 270 (2) ◽

pp. F371-F376 ◽

Cited By ~ 15

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.

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