Modulation of cardiac Na+ channels expressed in a mammalian cell line and in ventricular myocytes by protein kinase C.

Abstract

Cardiac rH1 Na+ channel a subunits were expressed in cells of the Chinese hamster lung 1610 cell line by transfection, and a stable cell line exprsing cardiac Na+ channels (SNa-rHl) was isolated. Mean Na+ currents of 2.2 ± 1.0 nA were recorded, which corresponds to a cell surface density of approximately 1-2 channels active at the peak of the Na+ current per pam2. The expressed cardiac Na+ current was tetrodotoxin resistant (Kd = 1.8 FM) and had voltagedependent properties similar to those of the Na+ current in neonatal ventricular myocytes. Activation of protein kinase C by 1-oleoyl-2-acetyl-sn-glycerol (OAG) (10 saM) decreased this current -33% at a holding potential of -114 mV and 56% at -94 mV. This reduction in peak current was caused in part by an 8-to 14-mV shift of steady-state inactivation in the hyperpolarized direction. Na+ channel activation was unchanged. Effects of OAG in SNa-rH1 cells and in neonatal rat cardiac myocytes were similar, except that the time course of inactivation was slowed either transiently or persistently when protein kinase C was activated in myocytes bathed in low-Ca2+ (1 M) or Ca2+-free solution but was unaffected in SNa-rH1 cells. The effects of OAG on cardiac Na+ current were blocked in cells that had been previously microlijected with a peptide inhibitor of protein kinase C but not with a peptide inhibitor of cAMP-dependent protein kinase, indicating that protein kinase C is responsible for the effects of OAG. Single-channel recordings from SNa-rHi cells showed that the probability of channel opening was reduced by OAG, but the conductance was unaffected. OAG did not induce the late Na+ channel openings observed with PKC modulation ofneuronal and skeletal muscle Na+ channels. Thus, the substantial reduction in Na+ current at normal diastolic depolarizations with 10 pM OAG is due to failure of channel opening in response to depolarization. Such Na+ current reductions may have profound effects on cardiac cell excitability.The rapid upstroke of the cardiac action potential due to the Na+ current through voltage-dependent Na+ channels is responsible for the rapid spread of activation through the atria and ventricles that initiates and coordinates contraction of the heart. Cardiac function depends critically on the amplitude, timing, and voltage dependence of the Na+ current, and interventions that modulate Na+ current have potent physiological effects. The functional properties of mammalian cardiac Na+ channels are distinctive. They are less sensitive to tetrodotoxin (TTX) than most Na+ channels, with a Kd of approximately 1-10 ,uM (1), and their kinetics of activation and inactivation are slower and more complex (2). These properties are mediated by a distinct Na+ channel a subunit (rH1) that was originally characterized by cloning from newborn rat heart (3) and denervated rat skeletal muscle (4). A closely related Na+ channel has been described in human cardiac muscle (5). Expression of these a subunit cDNAs in Xenopus oocytes (5-7) yields Na+ currents with functiona...

Effects of PKC and PKA Inhibitors on the cAMP-Stimulant-Induced Enhancement of Tetrodotoxin-Resistant Na+ (Nav1.8) Currents

“…Similarly, we found that application of 0.5 μM Ro-31-8425 application blocked an increase in Nav1.8 currents induced by either 8-Br-cAMP or the cAMP analogue forskolin [23]. This was supported by the fact that application of a PKC inhibitor (staurosporine or PKC [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] ) caused a marked inhibition of the forskolin-induced increase in the G V1/2 base (i.e. the percentage change in G at baseline V 1/2 ) [24,36,46].…”

“…The application of the PKC inhibitor PKC [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] significantly suppresses the forskolin-induced increase in the Nav1.8 current but the PKA inhibitor WIPTIDE has no significant effect on the PKC activator phorbol12, 13-dibutyrate (PDBu)-induced increase in the current [36]. When considering these results together, it is possible that PKCinduced phosphorylation of the channel protein at serine 1506 is required to enable PKA-induced phosphorylation of other sites on the channel protein suggested by Li et al [40] in a relationship between PKC and PKA on the convergent regulation of Na + channels.…”

“…The slow onset of Nav1.9 channel opening probably implies that this type of Na + channels makes a minor contribution to the action potential amplitude [25]. The majority of voltage-gated sodium channels (VGSCs) are inhibited by either protein kinase C (PKC) or protein kinase A (PKA) [26][27][28][29][30][31][32]. The VGSC into the skeletal and cardiac muscles is not significantly affected by the activation of PKA [33,34].…”

Modulation of the Cardiac Sodium Channel Nav1.5 by Fibroblast Growth Factor Homologous Factor 1B

“…The present study clearly shows that co-expression of FHF1B produces a significant hyperpolarizing shift of the voltage dependence of inactivation of Na v 1.5. This shift is similar to that attributed to the phosphorylation of Ser 1505 in the cytoplasmic linker joining domains 3 and 4 by protein kinase C (48,49). Other residues, for example Ser 526 and Ser 529 in the cytoplasmic loop linking domains 1 and 2 (L1) of Na v 1.5, have been reported to be phosphorylated by cAMP-dependent protein kinase (50).…”

Effects of aldosterone and mineralocorticoid receptor antagonism on cardiac ion channels in the view of upstream therapy of atrial fibrillation

“…In turn, this lowers the amount of the oxidized form of glutathione and increases the reduced form. Thus, this shift of cellular redox state might finally alter important cardiac ion currents like transients I to (Rozanski and Xu 2002), I Ca,L (Hool 2008) or I Na (Qu et al 1994). However, these theoretical considerations have to be confirmed in vivo, not only as Wagner et al (2008) provided evidence that the activation of the MR per se depends on the intracellular redox state of cardiac myocytes.…”