Shab K+ channel slow inactivation

Herein, we report the first characterization of Shab slow inactivation. Open Shab channels inactivate within seconds, with two voltage-independent time constants. Additionally, Shab presents significant closed-state inactivation. We found that with short depolarizing pulses, shorter than the slowest inactivation time constant, the resulting inactivation curve has a marked U-shape, but as pulse duration increases, approaching steady-state conditions, the U-shape vanishes, and the resulting inactivation curves converge to the classical Boltzmann h_∞ curve. Regarding the mechanism of inactivation, we found that external K⁺ and TEA facilitate both open- and closed-state inactivation, while the cavity blocker quinidine hinders inactivation. These results together with our previous observations regarding the K⁺-dependent stability of the K⁺ conductance, suggest the novel hypothesis that inactivation of Shab channels, and possibly that of other Kv channels whose inactivation is facilitated by K⁺, does not involve a significant narrowing of the extracellular entry of the pore. Instead, we hypothesize that there is only a rearrangement of a more internal segment of the pore that affects the central cavity and halts K⁺ conduction.     

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.     

Activation of protein kinase C alters voltage dependence of a Na+ channel.

Phorbol esters and purified protein kinase C (PKC) have been shown to down-modulate the voltage-dependent Na+ channels expressed in Xenopus oocytes injected with chick brain RNA. We used the two-electrode voltage-clamp technique to demonstrate that a Na+ channel expressed in oocytes injected with RNA coding for the alpha subunit of the channel alone (VA200, a variant of rat brain type IIA) is also inhibited by PKC activation. The inhibition of Na+ currents, expressed in oocytes injected with either alpha subunit RNA (rat) or total brain RNA (chick), is voltage-dependent, being stronger at negative potentials. It appears to result mainly from a shift in the activation curve to the right and possibly a decrease in the steepness of the voltage dependence of activation. There is little effect on the inactivation process and maximal Na+ conductance. Thus, PKC modulates the Na+ channel by a mechanism involving changes in voltage-dependent properties of its main, channel-forming alpha subunit.     

A single residue differentiates between human cardiac and skeletal muscle Na+ channel slow inactivation

Slow inactivation determines the availability of voltage-gated sodium channels during prolonged depolarization. Slow inactivation in hNa(V)1.4 channels occurs with a higher probability than hNa(V)1.5 sodium channels; however, the precise molecular mechanism for this difference remains unclear. Using the macropatch technique we show that the DII S5-S6 p-region uniquely confers the probability of slow inactivation from parental hNa(V)1.5 and hNa(V)1.4 channels into chimerical constructs expressed in Xenopus oocytes. Site-directed mutagenesis was used to test whether a specific region within DII S5-S6 controls the probability of slow inactivation. We found that substituting V754 in hNa(V)1.4 with isoleucine from the corresponding position (891) in hNa(V)1.5 produced steady-state slow inactivation statistically indistinguishable from that in wild-type hNa(V)1.5 channels, whereas other mutations have little or no effect on slow inactivation. This result indicates that residues V754 in hNa(V)1.4 and I891in hNa(V)1.5 are unique in determining the probability of slow inactivation characteristic of these isoforms. Exchanging S5-S6 linkers between hNa(V)1.4 and hNa(V)1.5 channels had no consistent effect on the voltage-dependent slow time inactivation constants [tau(V)]. This suggests that the molecular structures regulating rates of entry into and exit from the slow inactivated state are different from those controlling the steady-state probability and reside outside the p-regions.     

Subunit dependence of Na channel slow inactivation and open channel block in cerebellar neurons

Purkinje and cerebellar nuclear neurons both have Na currents with resurgent kinetics. Previous observations, however, suggest that their Na channels differ in their susceptibility to entering long-lived inactivated states. To compare fast inactivation, slow inactivation, and open-channel block, we recorded voltage-clamped, tetrodotoxin-sensitive Na currents in Purkinje and nuclear neurons acutely isolated from mouse cerebellum. In nuclear neurons, recovery from all inactivated states was slower, and open-channel unblock was less voltage-dependent than in Purkinje cells. To test whether specific subunits contributed to this differential stability of inactivation, experiments were repeated in Na(V)1.6-null (med) mice. In med Purkinje cells, recovery times were prolonged and the voltage dependence of open-channel block was reduced relative to control cells, suggesting that availability of Na(V)1.6 is quickly restored at negative potentials. In med nuclear cells, however, currents were unchanged, suggesting that Na(V)1.6 contributes little to wild-type nuclear cells. Extracellular Na(+) prevented slow inactivation more effectively in Purkinje than in nuclear neurons, consistent with a resilience of Na(V)1.6 to slow inactivation. The tendency of nuclear Na channels to inactivate produced a low availability during trains of spike-like depolarization. Hyperpolarizations that approximated synaptic inhibition effectively recovered channels, suggesting that increases in Na channel availability promote rebound firing after inhibition.     

Tyrosine kinase catalyzed phosphorylation and inactivation of the inhibitor protein of the cAMP-dependent protein kinase

The inhibitor protein of the cAMP-dependent protein kinase is a potential high affinity regulator of cAMP function. We now show that it is phosphorylated in Tyr7 by the intrinsic tyrosine kinase activity of epidermal growth factor receptor. The phosphorylated form can be readily separated from the unphosphorylated protein by high pressure liquid chromatography which has permitted the isolation of stoichiometrically phosphorylated protein. Using this method, it has been demonstrated that this phosphorylation, which occurs within the inhibitor protein's active domain, results in a 6 to 9-fold decrease in inhibitory potency. Possibly, a component of growth control could be the coupling of tyrosine kinase activity to cAMP-mediated cellular proliferation via the regulation of the efficacy of the inhibitor protein.     

Redox regulation of cAMP-dependent protein kinase signaling: kinase versus phosphatase inactivation

Many components of cellular signaling pathways are sensitive to regulation by oxidation and reduction. Previously, we described the inactivation of cAMP-dependent protein kinase (PKA) by direct oxidation of a reactive cysteine in the activation loop of the kinase. In the present study, we demonstrate that in HeLa cells PKA activity follows a biphasic response to thiol oxidation. Under mild oxidizing conditions, or short exposure to oxidants, forskolin-stimulated PKA activity is enhanced. This enhancement was blocked by sulfhydryl reducing agents, demonstrating a reversible mode of activation. In contrast, forskolin-stimulated PKA activity is inhibited by more severe oxidizing conditions. Mild oxidation enhanced PKA activity stimulated by forskolin, isoproterenol, or the cell-permeable analog, 8-bromo-cAMP. When cells were lysed in the presence of serine/threonine phosphatase inhibitor, NaF, the PKA-enhancing effect of oxidation was blunted. These results suggest oxidation of a PKA-counteracting phosphatase may be inhibited, thus enhancing the apparent kinase activity. Using an in vivo PKA activity reporter, we demonstrated that mild oxidation does indeed prolong the PKA signal induced by isoproterenol by inhibiting counteracting phosphatase activity. The results of this study demonstrate in live cells a unique synergistic mechanism whereby the PKA signaling pathway is enhanced in an apparent biphasic manner.     

Differential effects of protein kinase C on the levels of epithelial Na+ channel subunit proteins

Regulation of epithelial Na(+) channel (ENaC) subunit levels by protein kinase C (PKC) was investigated in A6 cells. PKC activation altered ENaC subunit levels, differentially decreasing the levels of both beta and gamma, but not alphaENaC. Temporal regulation of beta and gammaENaC by PKC differed; gammaENaC decreased with a time constant of 3.7 +/- 1.0 h, whereas betaENaC decreased in 13.9 +/- 3. 0 h. Activation of PKC also resulted in a decrease in trans-epithelial Na(+) reabsorption for up to 48 h. PMA activation of PKC resulted in negative feedback inhibition of PKC protein levels beginning within 4 h. Both beta and gammaENaC levels, as well as transport tended toward pretreatment values after 48 h of PMA treatment. PKC inhibitors attenuated the effects of PMA on ENaC subunit levels and Na(+) transport. These results directly show for the first time that PKC differentially regulates ENaC subunit levels by decreasing the levels of beta and gamma but not alphaENaC protein. These results imply a PKC-dependent, long term decrease in Na(+) reabsorption.     

Interaction of protein kinase C and cAMP-dependent pathways in the phosphorylation of the Na,K-ATPase

To test the hypothesis that there is cross-talk between the protein kinase C (PKC) and protein kinase A (PKA) pathways in the regulation of the Na,K-ATPase, we measured its phosphorylation in mammalian cell cultures. Phosphorylation of the PKC site, Ser-18, appeared to be due to the activation of the alpha isoform of the kinase. In NRK-52E and L6 cells, this phosphorylation was reduced by prior activation of a cAMP-dependent signaling pathway with forskolin. In principle this would be consistent with direct interaction between the two phosphorylation sites, but further investigation suggested a more indirect mechanism. First, phosphorylation of Ser-938, the PKA site, could not be detected despite the presence of active PKA. Second, there was a major reduction in the phosphorylation of unrelated phosphoproteins as a consequence of elevation of cAMP, suggesting generalized reduction of kinase activity or activation of phosphatase activity. In NRK-52E and L6, phosphorylation of the Na, K-ATPase at Ser-18 paralleled this global change. In C6 cells, in contrast, there was no cAMP effect on Na,K-ATPase phosphorylation at Ser-18 and no global cAMP effect on other phosphoproteins. The cross-talk is evidently mediated by events occurring at the cellular level.     

Activation of intestinal CFTR Cl- channel by heat-stable enterotoxin and guanylin via cAMP-dependent protein kinase.

Heat-stable enterotoxins (STa) produced by pathogenic bacteria induce profound salt and water secretion in the gut, leading to diarrhea. Recently, guanylin, an endogenous peptide with properties similar to STa, was identified. While STa and guanylin bind to the same receptor guanylyl cyclase and raise cell cGMP, the signaling mechanism distal to cGMP remains controversial. Here we show that STa, guanylin and cGMP each activate intestinal Cl- secretion, and that this is abolished by inhibitors of cAMP-dependent protein kinase (PKA), suggesting that PKA is a major mediator of this effect. These agents induce Cl- secretion only in cells expressing the wild-type CFTR, indicating that this molecule is the final common effector of the signaling pathway. The involvement of CFTR suggests a possible cystic fibrosis heterozygote advantage against STa-induced diarrhea.     

Regulation of Na(+)-K(+)-ATPase by cAMP-dependent protein kinase anchored on membrane via its anchoring protein

Na⁺-K⁺- ATPase -subunitsin basolateral membrane vesicles (BLMVs) purified from rat parotidglands were ³²P-labeled within 5 s by incubation with[-³²P]ATP at 37°C in the presence of cAMP, but nolabeling occurred without cAMP. Phosphorylation ofNa⁺-K⁺-ATPase was associated with a decrease inits activity. This -subunit phosphorylation disappeared when BLMVswere briefly incubated with cAMP and subsequent washing before theincubation with [-³²P]ATP, indicating that catalyticsubunit of protein kinase A (PKA) associated to BLMVs via binding withits RII regulatory subunit anchored on the membrane. In theabsence of cAMP, a PKA catalytic subunit readily reassociated with themembrane-bound RII subunit. HT-31 peptide inhibited theNa⁺-K⁺-ATPase phosphorylation by membrane-boundendogenous PKA, indicating an involvement of A-kinase anchoring protein(AKAP). AKAP-150 protein in BLMVs was shown by immunoblotting and anRII overlay assay and was coimmunoprecipitated by anti-RII antibody.These results show that Na⁺-K⁺-ATPase of ratparotid gland acinar cells is regulated in vivo by membrane-anchoredPKA via AKAP rather than by free cytosolic PKA.

     

Conventional protein kinase C isoforms and cross-activation of protein kinase A regulate cardiac Na+ current

We tested the hypothesis that specific isoforms of protein kinase C (PKC) are responsible for modulation of Na+ current (I(Na)) derived from the human cardiac Na+ channel using activators and inhibitors selective for specific PKCs. Experimental results demonstrated that I(Na) suppression was mediated by activation of conventional PKCs (cPKCs) and possibly resulted from channel internalization. In the presence of cPKC inhibition, phorbol ester application unexpectedly increased Na+ current, an effect eliminated by inhibition of protein kinase A. These findings demonstrate complex modulation of cardiac I(Na) by protein kinases and provide further evidence that PKC isoforms have distinct protein targets.     

Phosphorylation of purified rat brain Na+ channel reconstituted into phospholipid vesicles by protein kinase C.

Phosphorylation of voltage-sensitive Na+ channels in neurons by protein kinase C slows Na+ channel inactivation and reduces peak Na+ currents. Na+ channels purified from rat brain and reconstituted into phospholipid vesicles under conditions that restore Na+ channel function were rapidly phosphorylated by protein kinase C on their 260-kDa alpha subunit. The phosphorylation reaction required Ca2+, diolein, and phosphatidylserine for activation of protein kinase C, and the rate of phosphorylation of reconstituted Na+ channels was 3- to 4-fold faster than for Na+ channels in detergent solution. Phosphorylation was on serine residues in three distinct tryptic phosphopeptides designated A, B, and C. Up to 2.5 mol of phosphate were incorporated per mol of Na+ channel. Following maximum phosphorylation by protein kinase C, cAMP-dependent protein kinase was able to incorporate more than 2.25 mol of phosphate per mol of Na+ channel indicating that these two kinases phosphorylate distinct sites. However, prior phosphorylation by cAMP-dependent protein kinase prevented phosphorylation of phosphopeptide B indicating that both kinases phosphorylate the site in this peptide. Phosphopeptide B shown here to be phosphorylated by protein kinase C and phosphopeptide 7 previously shown to be phosphorylated by cAMP-dependent protein kinase co-migrate on two-dimensional phosphopeptide maps and evidently are identical. The reduction in peak Na+ currents caused by both protein kinase C and cAMP-dependent protein kinase may result from phosphorylation of this single common site.     

Predominance of poorly reopening single Na+ channels and lack of slow Na+ inactivation in neonatal cardiocytes

Summary Elementary Na⁺ currents through single cardiac Na⁺ channels were recorded at –50 mV in cell-attached patches from neonatal rat cardiocytes kept at holding potentials between –100 and –120 mV.Na⁺ channel activity may occur as burst-like, closely-timed repetitive openings with shut times close to 0.5–0.6 msec, indicating that an individual Na⁺ channel may reopen several times during step depolarization. A systematic quantiative analysis in 19 cell-attached patches showed that reopening may be quite differently pronounced. The majority, namely 16 patches, contained Na⁺ channels with a low tendency to reopen. This was evidenced from the average value for the mean number of openings per sequence, 2.5. Strikingly different results were obtained in a second group of three patches. Here, a mean number of openings per sequence of 3.42, 3.72, and 5.68 was found. Ensemble averages from the latter group of patches revealed macroscopic Na⁺ currents with a biexponential decay phase. Reconstructed Na⁺ currents from patches with poorly reopening Na⁺ channels were devoid of a slow decay component. This strongly suggests that reopening may be causally related to slow Na⁺ inactivation. Poorly pronounced reopening and, consequently, the lack of slow Na⁺ inactivation could be characteristic features of neonatal cardiac Na⁺ channels.     

Comparison of phosphorylation sites in protamines between protein kinase C and cAMP-dependent protein kinase

Phosphorylation sites of protamines by protein kinase C and cAMP-dependent protein kinase (protein kinase A) were studied. Using clupeine Y1 as a substrate, protein kinase C phosphorylates both Ser and Thr residues, whereas protein kinase A phosphorylates only Ser residue(s). Protein kinase C phosphorylates all Ser and Thr residues of clupeine Y2 and Z, however protein kinase A phosphorylates mainly Ser9 and slightly Thr5 in clupeine Y2 and Ser6 and Ser10 in clupeine Z. These results suggest that protein kinase C recognizes more sites than those of protein kinase A and may participate in protamine phosphorylation in vivo.     

Regulation of the epithelial Na+ channel by the protein kinase CK2

CK2 is a ubiquitous, pleiotropic, and constitutively active Ser/Thr protein kinase that controls protein expression, cell signaling, and ion channel activity. Phosphorylation sites for CK2 are located in the C terminus of both beta- and gamma-subunits of the epithelial Na(+) channel (ENaC). We examined the role of CK2 on the regulation of both endogenous ENaC in native murine epithelia and in Xenopus oocytes expressing rENaC. In Ussing chamber experiments with mouse airways, colon, and cultured M1-collecting duct cells, amiloride-sensitive Na(+) transport was inhibited dose-dependently by the selective CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). In oocytes, ENaC currents were also inhibited by TBB and by the structurally unrelated inhibitors heparin and poly(E:Y). Expression of a trimeric channel lacking both CK2 sites (alphabeta(S631A)gamma(T599A)) produced a largely attenuated amiloride-sensitive whole cell conductance and rendered the mutant channel insensitive to CK2. In Xenopus oocytes, CK2 was translocated to the cell membrane upon expression of wt-ENaC but not of alphabeta(S631A)gamma(T599A)-ENaC. Phosphorylation by CK2 is essential for ENaC activation, and to a lesser degree, it also controls membrane expression of alphabetagamma-ENaC. Channels lacking the Nedd4-2 binding motif in beta-ENaC (R561X, Y618A) no longer required the CK2 site for channel activity and siRNA-knockdown of Nedd4-2 eliminated the effects of TBB. This implies a role for CK2 in inhibiting the Nedd4-2 pathway. We propose that the C terminus of beta-ENaC is targeted by this essential, conserved pleiotropic kinase that directs its constitutive activity toward many cellular protein complexes.     

Modulation of Na+ transport and epithelial sodium channel expression by protein kinase C in rat alveolar epithelial cells

Although the amiloride-sensitive epithelial sodium channel (ENaC) plays an important role in the modulation of alveolar liquid clearance, the precise mechanism of its regulation in alveolar epithelial cells is still under investigation. Protein kinase C (PKC) has been shown to alter ENaC expression and activity in renal epithelial cells, but much less is known about its role in alveolar epithelial cells. The objective of this study was to determine whether PKC activation modulates ENaC expression and transepithelial Na+ transport in cultured rat alveolar epithelial cells. Alveolar type II cells were isolated and cultured for 3 to 4 d before they were stimulated with phorbol 12-myristate 13-acetate (PMA 100 nmol/L) for 4 to 24 h. PMA treatment significantly decreased alpha, beta, and gammaENaC expression in a time-dependent manner, whereas an inactive form of phorbol ester had no apparent effect. This inhibitory action was seen with only 5-min exposure to PMA, which suggested that PKC activation was very important for the reduction of alphaENaC expression. The PKC inhibitors bisindolylmaleimide at 2 micromol/L and G?6976 at 2 micromol/L diminished the PMA-induced suppression of alphaENaC expression, while rottlerin at 1 micromol/L had no effect. PMA elicited a decrease in total and amiloride-sensitive current across alveolar epithelial cell monolayers. This decline in amiloride-sensitive current was not blocked by PKC inhibitors except for a partial inhibition with bisindolylmaleimide. PMA induced a decrease in rubidium uptake, indicating potential Na+-K+-ATPase inhibition. However, since ouabain-sensitive current in apically permeabilized epithelial cells was similar in PMA-treated and control cells, the inhibition was most probably related to reduced Na+ entry at the apical surface of the cells. We conclude that PKC activation modulates ENaC expression and probably ENaC activity in alveolar epithelial cells. Ca2+-dependent PKC is potentially involved in this response.     

Caldesmon phosphorylation in intact human platelets by cAMP-dependent protein kinase and protein kinase C

Caldesmon is a calmodulin- and actin-binding protein present in both smooth and non-muscle tissue. The present study demonstrates that platelet caldesmon is a substrate for cAMP-dependent protein kinase (protein kinase A). Purified platelet caldesmon has an apparent molecular mass of 82 kDa on sodium dodecyl sulfate-polyacrylamide gels and can be phosphorylated in vitro by the catalytic subunit of protein kinase A to a level of 2 mol of phosphate/mol of caldesmon. Phosphorylation of caldesmon by protein kinase A results in a shift in the apparent molecular mass of the protein to 86 kDa. When caldesmon was immunoprecipitated from intact platelets treated with prostacyclin (PGI2) the same shift in apparent molecular mass of caldesmon was observed. Comparison of two-dimensional tryptic phosphopeptide maps of caldesmon phosphorylated in vitro by protein kinase A with caldesmon immunoprecipitated from intact platelets verified that protein kinase A was responsible for the observed increase in caldesmon phosphorylation in PGI2-treated platelets. The present study demonstrates that although caldesmon is basally phosphorylated in the intact platelet, activation of protein kinase A by PGI2 results in the significant incorporation of phosphate into two new sites. In addition, the effects of phorbol ester, collagen, and thrombin on caldesmon phosphorylation were also examined. Although phorbol ester treatment results in a significant increase in caldesmon phosphorylation apparently by protein kinase C, treatment of intact platelets with thrombin or collagen does not result in an increase in caldesmon phosphorylation.     

Specific and nonspecific effects of protein kinase C on the epithelial Na (+) channel

The Xenopus oocyte expression system was used to explore the mechanisms of inhibition of the cloned rat epithelial Na(+) channel (rENaC) by PKC (Awayda, M.S., I.I. Ismailov, B.K. Berdiev, C.M. Fuller, and D.J. Benos. 1996. J. Gen. Physiol. 108:49-65) and to determine whether human ENaC exhibits similar regulation. Effects of PKC activation on membrane and/or channel trafficking were determined using impedance analysis as an indirect measure of membrane area. hENaC-expressing oocytes exhibited an appreciable activation by hyperpolarizing voltages. This activation could be fit with a single exponential, described by a time constant (tau) and a magnitude (DeltaI (V)). A similar but smaller magnitude of activation was also observed in oocytes expressing rENaC. This activation likely corresponds to the previously described effect of hyperpolarizing voltage on gating of the native Na(+) channel (Palmer, L.G., and G. Frindt. 1996. J. Gen. Physiol. 107:35-45). Stimulation of PKC with 100 nM PMA decreased DeltaI(V) in hENaC-expressing oocytes to a plateau at 57.1 +/- 4.9% (n = 6) of baseline values at 20 min. Similar effects were observed in rENaC-expressing oocytes. PMA decreased the amiloride-sensitive hENaC slope conductance (g(Na)) to 21.7 +/- 7.2% (n = 6) of baseline values at 30 min. This decrease was similar to that previously reported for rENaC. This decrease of g (Na) was attributed to a decrease of membrane capacitance (C (m)), as well as the specific conductance (g(m)/C(m )). The effects on g(m)/C(m) reached a plateau within 15 min, at approximately 60% of baseline values. This decrease is likely due to the specific ability of PKC to inhibit ENaC. On the other hand, the decrease of C(m) was unrelated to ENaC and is likely an effect of PKC on membrane trafficking, as it was observed in ENaC-expressing as well as control oocytes. At lower PMA concentrations (0.5 nM), smaller changes of C(m) were observed in rENaC- and hENaC-expressing oocytes, and were preceded by larger changes of g(m ) and by changes of g(m)/C(m), indicating specific effects on ENaC. These findings indicate that PKC exhibits multiple and specific effects on ENaC, as well as nonspecific effects on membrane trafficking. Moreover, these findings provide the electrophysiological basis for assessing channel-specific effects of PKC in the Xenopus oocyte expression system.     

Regulation of D-myo-inositol 1,4,5-trisphosphate 3-kinase by cAMP-dependent protein kinase and protein kinase C

The Ca2(+)-mobilizing second messenger D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is converted to the putative messenger D-myo-inositol 1,3,4,5-tetrakisphosphate by Ins(1,4,5)P3 3-kinase. We found that cAMP-dependent protein kinase and protein kinase C phosphorylate, and thereby modulate, the activity of Ins(1,4,5)P3 3-kinase. cAMP-dependent kinase introduced a stoichiometric amount of phosphate at serine 109 of the 53-kDa polypeptide and caused a 1.8-fold increase in Vmax, whereas the protein kinase C-dependent phosphorylation reduced the Vmax to one-fourth of that of the unphosphorylated enzyme. Upon prolonged incubation, protein kinase C introduced phosphate at multiple sites in Ins(1,4,5)P3 3-kinase, and the resulting inactivation of the enzyme appeared to be well-correlated with the simultaneous phosphorylation of two major sites, serine 109 and serine 175. The Km for Ins(1,4,5)P3 was not affected significantly after phosphorylation by either protein kinase. We propose, therefore, that the phosphorylation of Ins(1,4,5)P3 3-kinase by cAMP-dependent kinase and protein kinase C constitutes mechanisms of cross-talk between cellular signaling pathways that use various second messengers such as inositol phosphates, diacylglycerol, Ca2+, and cAMP.