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and Its Adaptor Protein p62 in Voltage-Gated Potassium Channel Modulation in Pulmonary ArteriesDepartment of Pharmacology, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain
Received April 12, 2007; accepted August 15, 2007
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Abstract |
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and its adaptor protein p62 in the modulation of KV channels. We report that the thromboxane A2 analog 9,11-dideoxy-11
,9-epoxymethano-prostaglandin F2 methyl acetate (U46619
[GenBank]
) inhibited KV currents in isolated mice pulmonary artery myocytes and the KV current carried by human cloned KV1.5 channels expressed in Ltk– cells. Using protein kinase C (PKC)–/– and p62–/– mice, we demonstrate that these two proteins are involved in the KV channel inhibition. PKC coimmunoprecipitated with KV1.5, and this interaction was markedly reduced in p62–/– mice. Pulmonary arteries from PKC–/– mice also showed a diminished [Ca2+]i and contractile response, whereas genetic inactivation of p62–/– resulted in an absent [Ca2+]i response, but it preserved contractile response to U46619.
[GenBank]
These data demonstrate that PKC and its adaptor protein p62 play a key role in the modulation of KV channel function in pulmonary arteries. These observations identify PKC and/or p62 as potential therapeutic targets for the treatment of pulmonary hypertension.
; Archer et al., 1998; Yuan et al., 1998b). These channels are common targets of pulmonary vasoconstrictor stimuli such as hypoxia, thromboxane A2 (TXA2), 5-hydroxytryptamine, endothelin-1 or angiotensin-II (Archer et al., 1998; Shimoda et al., 2001; Cogolludo et al., 2003, 2006). In addition, decreased expression or function of KV channels in PASMCs has been involved in the pathogenesis of pulmonary arterial hypertension (PH) (Weir et al., 1996, Yuan et al., 1998a; Pozeg et al., 2003). From the variety of KV channels expressed in PASMC (Platoshyn et al., 2006), special interest has been paid to KV1.5, because decreased expression or activity and mutations of KV1.5 occur in human (Yuan et al., 1998a; Remillard et al., 2007) and experimental (Archer et al., 1998; Pozeg et al., 2003) idiopathic and hypoxic PH, and in vivo gene transfer of KV1.5 reduces PH (Pozeg et al., 2003).
TXA2 is a prostanoid synthesized by cyclooxygenase with potent vasoconstrictor, mitogenic, and platelet aggregant properties via activation of thromboxane-endoperoxide (TP) receptors (Halushka et al., 1989). The vasoconstrictor effects of TXA2 are particularly pronounced in the pulmonary vascular bed, where it participates in the control of vessel tone under physiological and pathological situations, including PH. We have previously reported that in intact PAs and freshly isolated PASMCs, TXA2, via activation of TP receptors, inhibits KV channels, leading to membrane depolarization, activation of L-type Ca2+ channels, and vasoconstriction. Furthermore, using a protein kinase C (PKC) pseudosubstrate inhibitory peptide (PKC-PI), we provided evidence for the role of this kinase as a link between TP receptor activation and KV channel inhibition (Cogolludo et al., 2003, 2005). PKC (together with PKC
/
) belongs to the atypical PKC (aPKC) subclass. Both aPKCs play key roles in different signaling pathways regulating cell growth, survival, and differentiation (Moscat and Díaz-Meco, 2000). The aPKCs share with other members of their family a conserved catalytic domain, but they display a clearly distinct regulatory region because they have been shown to be independent of Ca2+, diacylglycerol, and phorbol esters, all of which are potent activators of other PKC isoforms. PKC is activated by phosphatidylinositols, arachidonic acid, and other lipids (Hirai and Chida, 2003) as well as by a variety of mediators, including insulin (Liu et al., 2006), thromboxane A2 (Shizukuda and Buttrick, 2002; Cogolludo et al., 2003, 2005), angiotensin II (Gayral et al., 2006; Godeny and Sayeski, 2006), or proinflammatory cytokines (Frey et al., 2006).
The mechanism underlying the activation of aPKCs responsible for its diverse physiological functions remains unclear, but several groups have identified a number of aPKC-interacting proteins, including p62 (also called ZIP1 or sequestosome 1), Par-4, Par-6, and MEK5 (Moscat and Diaz-Meco, 2000). It is noteworthy that nerve growth factor and catecholamines have been reported to increase the expression of p62, enabling the formation of the PKC-p62-KV
complex, which results in a hyperpolarizing shift in the KV current activation curve (Gong et al., 1999; Kim et al., 2004, 2005).
The role of PKC on pulmonary vasoconstriction has been widely reported (Ward et al., 2004); however, many of these studies have been conducted with PKC modulators of dubious selectivity, thereby limiting their conclusions. Molecular biology and genetic approaches and the currently available isoform-selective PKC inhibitors have made possible the elucidation of the involvement of specific PKC isoforms in cellular processes (such as vascular contractility) (Salamanca and Khalil, 2005). However, recent evidence suggests that some considered isoform-specific PKC inhibitors, such as myristoylated PKC pseudosubstrate peptide, may exert other effects unrelated to inhibition of PKC; thus, they should be used with caution (Krotova et al., 2006).
Therefore, in the present study, we aimed to further characterize the signaling pathway modulating KV currents in PAs. Using PKC–/– and p62–/– mice, we provide evidence for the interaction of PKC with KV channels, which further support the role of this interaction in TXA2-induced effects. In addition, we hypothesized that the PKC-KV-L-type Ca2+ channels pathway might involve other proteins such as p62. This possibility was tested by analyzing the modulation of KV channels in wild-type and p62 homozygous null mice.
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Materials and Methods |
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Animals. Lungs from PKC–/– (mixed C57BL/6 and SV129J background), p62–/– (C57BL/6), and corresponding wild-type mice (6–8 weeks old; either sex) were generously supplied by Drs. J. Moscat and M. T. Diaz-Meco (both from the Genome Research Institute, University of Cincinnati, Cincinnati, OH). These mice were generated as described previously (Leitges et al., 2001; Duran et al., 2004). PAs from male Wistar rats (250–300 g) were also used in these experiments.
Tissue Preparation and Cell Isolation. Second-order branches of the PA (internal diameter, 
0.5 mm) isolated from mice were dissected into a nominally calcium-free physiological salt solution (PSS) of the following composition: 130 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 10 mM glucose, and 10 mM HEPES, pH 7.3, with NaOH. Endothelium denuded PAs were cut into small segments (2 x 2 mm), and cells were isolated in Ca2+-free PSS containing 1 mg/ml papain, 0.8 mg/ml dithiothreitol, and 0.7 mg/ml albumin. Cells were stored in Ca2+-free PSS (4°C) and used within 8 h of isolation.
Electrophysiological Studies. Membrane currents were measured using the whole-cell configuration of the patch-clamp technique (Cogolludo et al., 2003) normalized for cell capacitance and expressed in picoamperes per picofarad. Membrane potential (Em) was measured under current-clamp configuration. KV currents were recorded under essentially Ca2+-free conditions using an external Ca2+-free PSS and a Ca2+-free pipette (internal) solution (see Solutions and Chemicals). Ltk– cells stably expressing hKV1.5 channels (Valenzuela et al., 1995) were superfused with PSS containing 1 mM CaCl2. Currents were evoked after the application of 200-ms depolarizing pulses from –60 mV to test potentials from –60 to +40 mV in 10-mV increments. All experiments were performed at room temperature (22–24°C).
[Ca2+]i Recording. PA rings were incubated for 80 min at room temperature in Krebs' solution containing the fluorescent dye fura-2 acetoxymethyl ester (5 x 10–6 M) and 0.05% cremophor EL, and then they were mounted in a fluorimeter (model CAF 110; Jasco, Tokyo, Japan). PA rings were alternatively illuminated (128 Hz) with two excitation wavelengths (340 and 380 nm), and the emitted fluorescence was filtered at 505 nm (Pérez-Vizcaíno et al., 1999). The ratio of emitted fluorescence (F340/F380) obtained at the two excitation wavelengths was used as an indicator of [Ca2+]i. Arteries were stimulated with 30 and 300 nM U46619
[GenBank]
, added in a cumulative manner. In preliminary experiments in wild-type mice, these concentrations produced 
60 and 80% of the maximal response, respectively. The [Ca2+]i signal in each vessel was calibrated according to the Grynkiewicz equation by sequential addition of 15 µM ionomycin and 10 mM EGTA at the end of the experiment.
Coimmunoprecipitation and Western Blot Analysis. Mice lungs were rapidly frozen in liquid nitrogen. In some experiments, rat PA were placed in warm Krebs' solution and then in the absence or presence of 1 µM U46619
[GenBank]
for 30 s and then rapidly frozen. Frozen tissues were homogenized in a glass potter in 200 µl of a buffer of the following composition: 10 mM HEPES, pH 8, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 6 µM aprotinin, 9 µM leupeptin, 11 µM N-p-tosyl-L-lysine chloromethyl ketone, 5 mM NaF, 10 mM Na2MoO4, 1 mM NaVO4, 0.5 mM phenylmethanesulfonyl fluoride, and 10 nM okadaic acid. Homogenates were centrifuged at 13,000g for 5 min at 4°C, and the supernatant fraction was collected. For immunoprecipitation, 60 µg of protein was incubated for 2 h with anti-PKC or anti-KV1.5 antibody at 4°C, followed by the addition of protein A/G beads and further incubation overnight. These immune complexes or 20 µg of the homogenates from mice lungs or rat PA were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane for Western blotting as described previously (Cogolludo et al., 2003). Membranes were probed for KV1.5-, PKC-, and p62-like immunoreactivity.
Solutions and Chemicals. For the single cell electrophysiological studies, the composition of the Ca2+-free bath solution (external Ca2+-free PSS) was as follows: 130 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 10 mM glucose, and 10 mM HEPES, buffered to pH 7.3 with NaOH. The Ca2+-free pipette (internal) solution contained 110 mM KCl, 1.2 mM MgCl2, 5 mM Na2ATP, 10 mM HEPES, and 10 mM EGTA, pH adjusted to 7.3 with KOH. The Krebs' solution used for tissue bath experiments included 118 mM NaCl, 4.75 mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4, 2.0 mM CaCl2, 1.2 mM KH2PO4, and 11 mM glucose. This solution was gassed with a 95% O2, 5% CO2 mixture at 37°C. U46619
[GenBank]
was obtained from Sigma Chemical Co. (Tres Cantos, Spain). [1S-1,2,5]-[5-Methyl-2-(1-methylethyl) cyclohexyl] diphenyl phosphine oxide (DPO-1) was from Tocris Cookson Inc. (Bristol, UK), secondary horseradish peroxidase-conjugated antibodies and fura-2 acetoxymethyl ester were from Calbiochem (Barcelona, Spain), rabbit anti-KV1.5 was from Alomone Labs (Jerusalem, Israel), goat anti-PKC was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and guinea pig anti-p62 was from Progen (Heidelberg, Germany).
Statistical Analysis. Data are expressed as means ± S.E.M.; n indicates the number of arteries or cells tested. All experiments were conducted in arteries or cells from at least four different animals. Statistical analysis was performed using Student's t test for paired or unpaired observations. Differences were considered statistically significant when p was less than 0.05.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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in KV Current Inhibition Induced by TXA2. A family of KV currents [IK(V)] were obtained in mice PASMCs when eliciting depolarizing steps from –60 to +40 mV (Fig. 1, A and B) from a holding potential of –60 mV. The magnitude of the currents, the threshold voltage for activation, and the current-voltage relationship (Fig. 1, C and D) was similar in PASMCs from wild-type and PKC–/– mice (e.g., current density at +40 mV was 9.1 ± 1.9 and 8.7 ± 0.8 pA/pF, respectively). Current inactivation was also similar in both strains (i.e., at 200 ms, the current decayed by 11.5 ± 3 and 12.1 ± 3.8%, respectively). Currents were recorded before (control) and after addition of the TXA2 analog U46619.
[GenBank]
U46619 (100 nM) caused a significant inhibition of KV currents in the whole range of channel activation in PASMCs from wild-type mice (Fig. 1A). The degree of current inactivation at +40 mV was increased by U46619
[GenBank]
(i.e., at 200 ms, the current decayed by 25.6 ± 4.8%; p < 0.05). In addition, U46619
[GenBank]
induced membrane depolarization in wild-type PASMCs (Fig. 1E). However, U46619
[GenBank]
had no effect on either KV currents or membrane potentials in PASMC from PKC–/– mice (Fig. 1, B, D, and F).
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Role of PKC in [Ca2+]i Increase and Contraction Induced by TXA2. Changes in [Ca2+]i and contraction induced by U46619
[GenBank]
were simultaneously analyzed in fura-2-loaded PAs from wild-type and from PKC–/– mice. Basal levels of [Ca2+]i in PKC–/– (203 ± 40 nM; n = 6) were not significantly different from those in wild-type mice (160 ± 40 nM; n = 6). Stimulation of endothelium-denuded PA rings with 30 and 300 nM U46619
[GenBank]
induced a sustained elevation in [Ca2+]i and a contractile response in PAs from wild-type and PKC–/– animals (Fig. 2, A and B). However, the increase in [Ca2+]i (Fig. 2C) and the contractile response (Fig. 2D) was significantly reduced in PKC–/– mice compared with wild-type mice.
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-p62-KV1.5 interaction, we analyzed the effects of 100 nM U46619
[GenBank]
on KV currents in p62–/– and the corresponding wild-type mice. The magnitude of the currents, the threshold voltage for activation, the current-voltage relationship, and the current inactivation (Fig. 3, C and D) were similar in PASMCs from wild-type and p62–/– mice (e.g., current density at +40 mV was 10.9 ± 1.3 and 11.6 ± 1.7 pA/pF, respectively; and at 200 ms, current decayed by 12.7 ± 2.8 and 14.3 ± 2.9%, respectively). As expected, U46619
[GenBank]
caused a significant inhibition in the whole range of channel activation and depolarized the membrane in PASMCs from wild-type mice (Fig. 3, A, C, and E). Current inactivation at +40 mV was also increased by U46619
[GenBank]
(i.e., at 200 ms, the current decayed by 21.7 ± 2.9%; p < 0.05). However, the TXA2 analog had no effect on KV currents in PASMCs from p62–/– mice (Fig. 3, B, D, and E).
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Basal levels of [Ca2+]i in p62–/– (184 ± 35 nM; n = 5) were not significantly different from those in wild-type mice (170 ± 45 nM; n = 6). We found that genetic inactivation of p62 abolished the increase in [Ca2+]i induced by U46619 [GenBank] (Fig. 4, B and C). However, the contractile response induced by the two concentrations of U46619 [GenBank] tested was remarkably similar in p62–/– and wild-type mice (Fig. 4D).
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). The KV1.5 channel blocker DPO-1 (Lagrutta et al., 2006) inhibited KV currents in rat PASMCs. In the presence of DPO-1, U46619
[GenBank]
produced no further inhibitory effects (Fig. 5B). Therefore, we analyzed a possible interaction between PKC, KV1.5 channels and p62. Rat pulmonary arteries were incubated for 30 s in the absence (control) or presence of U46619.
[GenBank]
Homogenates were immunoprecipitated with anti-PKC or anti-KV1.5 antibodies, and the content of KV1.5, PKC, or p62 in the immunoprecipitates was analyzed via Western blot. Figure 5C shows that in immunoprecipitates of KV1.5 both PKC and p62 were present. The KV1.5-PKC and the KV1.5-p62 association were 135 ± 13% (n = 8; p = 0.06, not significant) and 163 ± 31% (n = 7; p < 0.05), respectively, in U46619
[GenBank]
-treated versus untreated arteries. The KV1.5-PKC interaction was also observed in immunoprecipitates of PKC immunoblotted with the anti-KV1.5 antibody (data not shown).
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Interaction of PKC with KV Channels: Role of p62. To determine the potential role of the PKC scaffold protein p62, the PKC-KV1.5 interaction was analyzed by coimmunoprecipitation in lungs from wild-type and p62–/– mice. Genetic inactivation of p62 in mice did not modify the expression levels of either PKC or KV1.5 channels in PASMCs (Fig. 6A). In immunoprecipitates of PKC from wild-type mice immunoblotted with the anti-KV1.5 antibody, a band of approx. 80 kDa was observed, which presumably reflects the mature (glycosylated) form of the channel expressed in the membrane (Li et al., 2000). However, p62-deficient mice showed a weak PKC-KV1.5 coimmunoprecipitation (Fig. 6B).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-selective inhibitor PKC-PI, we suggested that PKC was involved in the KV channel inhibition and the contractile response induced by TXA2 in rat pulmonary artery myocytes (Cogolludo et al., 2003, 2005). Herein, we confirmed the role of PKC in native KV currents by using PASMCs from PKC–/– mice. Consistent with the essential role of KV1.5 channels in the pulmonary vasculature, we show that the KV1.5 inhibitor DPO-1 inhibited KV currents in native rat PASMCs by approx. 50% and that the TXA2 analog U46619
[GenBank]
had no further inhibitory effects. In addition, cloned human KV1.5 channels expressed in Ltk– cells were also inhibited by U46619.
[GenBank]
Moreover, our results demonstrate the interaction between PKC and KV1.5 in both rat PAs and mouse lungs, which was minimal in p62–/– mice. Deletion of p62 abolished KV channel inhibition and Ca2+ responses induced by TXA2, further supporting the role of p62 as a key mediator between PKC and KV1.5. However, our study also showed that the contractile response induced by U46619
[GenBank]
in PA was similar in wild-type and p62–/– mice.
In both rat and newborn porcine PASMCs, U46619
[GenBank]
inhibited KV currents, depolarized cell membrane, increased [Ca2+]i through CaL channels, and induced a contractile response (Cogolludo et al., 2003, 2005). U46619
[GenBank]
had no direct effect on CaL channels in voltage-clamped cells, indicating that increased Ca2+ entry through CaL channels is secondary to membrane depolarization. Herein, we demonstrated that, in mice, U46619
[GenBank]
also inhibits KV currents in PASMCs and induces a [Ca2+]i response and vasoconstriction in isolated PA. The degree of KV channel inhibition in mice PASMCs (25% at 100 nM U46619
[GenBank]
) was similar to that observed in porcine and in rat PA, and it was accompanied by a significant membrane depolarization. In rat and porcine PAs, all these effects were inhibited by calphostin C and PKC-PI (Cogolludo et al., 2003, 2005). These experiments suggested a role for PKC as a link between TP receptors and KV channels, which was confirmed in the present study using PKC–/– mice. The magnitude and current-voltage relationship of KV currents were similar in the wild-type and knock-out animals, suggesting no changes in the channel proteins underlying KV currents. Thus, genetic inactivation or pharmacological inhibition of PKC abolished the effects of U46619
[GenBank]
on KV currents or membrane potential in PASMCs. In contrast, both approaches only partially inhibited (50–70%) the Ca2+ signal induced by U46619
[GenBank]
in rat and mice PAs, indicating that, in addition to the PKC-KV-CaL pathway, mechanisms increasing [Ca2+]i (e.g., Ca2+ release from intracellular stores) are also activated in response to U46619
[GenBank]
(Snetkov et al., 2006).
The present experiments also indicate that in mice, PKC contributes to the vasoconstriction induced by TP receptor activation. These results are in agreement with those obtained in rats and newborn piglets using PKC-PI (Cogolludo et al., 2003, 2005). However, in 2-week-old piglets (Cogolludo et al., 2005), PKC-PI and the Ca2+ channel blocker nifedipine almost fully inhibited U46619
[GenBank]
-induced increases in [Ca2+]i, but they had no effect on U46619
[GenBank]
-induced contractile responses; i.e., there was a contractile response in the absence of changes in [Ca2+]i. Therefore, in these animals, the up-regulation of Ca2+-independent mechanisms for contraction (Somlyo and Somlyo, 2000) makes PKC and the [Ca2+]i signal redundant.
KV currents recorded in native PASMCs reflect the contribution of multiple KV channel proteins [e.g., in human PAs, 22 transcripts of KV subunits: KV1.1 to KV1.7, KV1.10, KV2.1, KV3.1, KV3.3, KV3.4, KV4.1, KV4.2, KV5.1, KV6.1 to -6.3, KV9.1, KV9.3, KV10.1, and KV11.1, and three of KV subunits KV1 to -3 have been identified by reverse transcription-polymerase chain reaction]. However, KV1.5 subunits are thought to be major contributors of the native KV currents in PAs from different species, and their activity is regulated by vasoactive factors such as 5-hydroxytryptamine (Cogolludo et al., 2006) and hypoxia (Platoshyn et al., 2006). Therefore, we analyzed the effects of U46619
[GenBank]
on the KV current carried by human cloned KV1.5 channels expressed in mouse fibroblast (Ltk–) cells. This cell line expresses endogenously the KV2.1 subunit, which assembles with the transfected hKv1.5 protein (Uebele et al., 1996). U46619
[GenBank]
induced a weak but significant inhibitory effect on this current, suggesting that KV1.5 channels are involved in the effects of TP receptor activation in native PASMCs. The small inhibition in this cell type probably reflects a lower efficacy of the signaling pathway compared with rat or mouse PASMCs. Furthermore, after pharmacological inhibition of KV1.5 channels with DPO-1, U46619
[GenBank]
had no further inhibitory effects on KV currents in rat PASMCs.
In the present article, we show that PKC coimmunoprecipitates with KV1.5 channels. In a previous study (Cogolludo et al., 2003), we reported that U46619
[GenBank]
induced the translocation of PKC from the cytosolic to the membrane fraction. Therefore, TP receptor-induced KV channel inhibition is associated with the translocation of PKC to the plasma membrane where it interacts with KV1.5 channels. This PKC-KV1.5 interaction is not necessarily a direct protein-protein interaction; it seems more likely that it is mediated by adaptor proteins. In this regard, it has been described that PKC can interact with the subunit KV2 of the KV channel via the p62 adaptor protein (Gong et al., 1999). In immunoprecipitation experiments, we found that p62 was present in the KV1.5-PKC complex. Even when the complex was constitutive, the association of p62 with KV1.5 increased significantly by U46619.
[GenBank]
Furthermore, the PKC-KV1.5 coimmunoprecipitation was strongly reduced in p62–/– mouse lung, indicating that p62 physically associates PKC into the KV channel complex.
KV subunits function as molecular chaperones, and they can directly regulate channel inactivation, voltage dependence, and current amplitude (Martens et al., 1999). p62 overexpression stimulates PKC-dependent phosphorylation of KV2 (Gong et al., 1999), and it induces a hyperpolarizing shift of KV current activation in pheochromocytoma cells (Kim et al., 2004). Thus, we analyzed the effect of genetic inactivation of p62 on KV currents and its modulation by TP receptor activation. KV currents in PASMCs from p62–/– were similar to wild type. As expected, U46619
[GenBank]
had no effect on KV currents in p62–/– PASMCs, indicating that the p62-dependent PKC-KV1.5 interaction is required for the inhibitory effect of TP receptor activation on KV current.
Thus, genetic inactivation of p62 had a similar effect to genetic or pharmacological inactivation of PKC regarding KV current modulation. We were surprised to find that p62 gene deletion fully inhibited the Ca2+ response induced by U46619
[GenBank]
in isolated PAs compared with a 50 to 70% inhibition by PKC inactivation. More intriguingly, the contractile response to U46619
[GenBank]
was not affected in PA from p62–/– mice. This contractile response in the absence of changes in [Ca2+]i must then be attributed to Ca2+-independent mechanisms (i.e., Ca2+ sensitization; Somlyo and Somlyo, 2000). This response to U46619
[GenBank]
in p62–/– mice PA is similar to that observed in 2-week-old piglet PAs after inhibition of PKC (i.e., contraction without [Ca2+]i signal) (Cogolludo et al., 2005). In these animals, there is an up-regulation of Rho kinase (Bailly et al., 2004), a key enzyme in Ca2+-sensitizing mechanisms. In addition, Rho kinase inhibitors were more effective inhibiting U46619
[GenBank]
contractions in these piglets than in newborn piglets or adult rats (Cogolludo et al., 2005). Thus, we speculate that the chronic down-regulation of the PKC-p62-KV-CaL-dependent pathway, either at the level of KV channel activity (as occurs in older piglets) or p62 (p62–/– mice), but not PKC (PKC–/– mice), is compensated by up-regulation of Ca2+ sensitization mechanisms.
In conclusion, PKC modulates KV channel function, and it is involved in pulmonary vasoconstriction induced by TP receptor activation. The interaction between PKC and KV1.5 and the inhibitory effect of U46619
[GenBank]
in cloned human KV1.5 channels suggest that these specific channel subtypes are functional targets for PKC. The adaptor protein p62 is required for the PKC-KV1.5 interaction and hence for the inhibition of KV currents after TP receptor activation.
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Footnotes |
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L.M. and G.F. contributed equally to this work.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: KV, voltage-gated K+; PASMC, pulmonary artery smooth muscle cell; CaL, voltage-dependent L-type Ca2+ channel; TXA2, thromboxane A2; PH, pulmonary hypertension; TP, thromboxane-endoperoxide; PKC, protein kinase C; PA, pulmonary artery; IP, inhibitory peptide; aPKC, atypical PKC; PSS, physiological salt solution; h, human; U46619
[GenBank]
, 9,11-dideoxy-11,9-epoxymethano-prostaglandin F2; DPO-1, [1S-1,2,5]-[5-methyl-2-(1-methylethyl) cyclohexyl] diphenyl phosphine oxide.
Address correspondence to: Dr. Francisco Perez-Vizcaino, Department of Pharmacology, School of Medicine, Universidad Complutense, 28040 Madrid, Spain. E-mail: fperez{at}med.ucm.es
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