PAR1 (Protease-Activated Receptor 1) Pepducin Therapy Targeting Myocardial Necrosis in Coronary Artery Disease and Acute Coronary Syndrome Patients Undergoing Cardiac Catheterization: A Randomized, Placebo-Controlled, Phase 2 Study.

OBJECTIVE: Arterial thrombosis leading to ischemic injury worsens the prognosis of many patients with cardiovascular disease. PZ-128 is a first-in-class pepducin that reversibly inhibits PAR1 (protease-activated receptor 1) on platelets and other vascular cells by targeting the intracellular surface of the receptor. The TRIP-PCI (Thrombin Receptor Inhibitory Pepducin in Percutaneous Coronary Intervention) trial was conducted to assess the safety and efficacy of PZ-128 in patients undergoing cardiac catheterization with intent to perform percutaneous coronary intervention. Approach and Results: In this randomized, double-blind, placebo-controlled, phase 2 trial, 100 patients were randomly assigned (2:1) to receive PZ-128 (0.3 or 0.5 mg/kg), or placebo in a 2-hour infusion initiated just before the start of cardiac catheterization, on top of standard oral antiplatelet therapy. Rates of the primary end point of bleeding were not different between the combined PZ-128 doses (1.6%, 1/62) and placebo group (0%, 0/35). The secondary end points of major adverse coronary events at 30 and 90 days did not significantly differ but were numerically lower in the PZ-128 groups (0% and 2% in the PZ-128 groups, 6% and 6% with placebo, p=0.13, p=0.29, respectively). In the subgroup of patients with elevated baseline cardiac troponin I, the exploratory end point of 30-day major adverse coronary events + myocardial injury showed 83% events in the placebo group versus 31% events in the combined PZ-128 drug groups, an adjusted relative risk of 0.14 (95% CI, 0.02-0.75); P=0.02. CONCLUSIONS: In this first-in-patient experience, PZ-128 added to standard antiplatelet therapy appeared to be safe, well tolerated, and potentially reduced periprocedural myonecrosis, thus providing the basis for further clinical trials. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02561000.

PAR2 controls cholesterol homeostasis and lipid metabolism in nonalcoholic fatty liver disease.

OBJECTIVE: Increases in hepatic and plasma cholesterol occur in patients with nonalcoholic fatty liver disease (NAFLD), although the reason for this is not well understood. We investigated whether Protease-Activated Receptor 2 (PAR2) plays a role in cholesterol and lipid homeostasis in NAFLD. METHODS: Human liver biopsies (n = 108) were quantified for PAR2 expression from NAFLD cases randomly selected and stratified by liver fibrosis stage, the primary predictor for clinical outcomes, while controlling for age, gender, and BMI between fibrosis groups. Demographic data and laboratory studies on plasma samples were obtained within 6 months of liver biopsy. Wild-type and PAR2-KO (C57BL/6 F2rl1-/-) mice were fed either normal or high fat diet for 16 weeks and plasma and liver assayed for lipids and soluble metabolites. RESULTS: Severity of NAFLD and plasma cholesterol levels significantly correlated with hepatocyte PAR2 expression in NAFLD patients. Conversely, PAR2 deficiency in mice resulted in reduced expression of key hepatic genes involved in cholesterol synthesis, a 50% drop in plasma and total liver cholesterol, and induced a reverse cholesterol transport system that culminated in 25% higher fecal bile acid output. PAR2-deficient mice exhibited enhanced fatty acid ß-oxidation with a ketogenic shift and an unexpected increase in liver glycogenesis. Mechanistic studies identified Gi-Jnk1/2 as key downstream effectors of protease-activated PAR2 in the regulation of lipid and cholesterol homeostasis in liver. CONCLUSIONS: These data indicate that PAR2 may be a new target for the suppression of plasma cholesterol and hepatic fat accumulation in NAFLD and related metabolic conditions.

Modeling a Ca(2+) channel/BKCa channel complex at the single-complex level.

BKCa-channel activity often affects the firing properties of neurons, the shapes of neuronal action potentials (APs), and in some cases the extent of neurotransmitter release. It has become clear that BKCa channels often form complexes with voltage-gated Ca(2+) channels (CaV channels) such that when a CaV channel is activated, the ensuing influx of Ca(2+) activates its closely associated BKCa channel. Thus, in modeling the electrical properties of neurons, it would be useful to have quantitative models of CaV/BKCa complexes. Furthermore, in a population of CaV/BKCa complexes, all BKCa channels are not exposed to the same Ca(2+) concentration at the same time. Thus, stochastic rather than deterministic models are required. To date, however, no such models have been described. Here, however, I present a stochastic model of a CaV2.1/BKCa(α-only) complex, as might be found in a central nerve terminal. The CaV2.1/BKCa model is based on kinetic modeling of its two component channels at physiological temperature. Surprisingly, The CaV2.1/BKCa model predicts that although the CaV channel will open nearly every time during a typical cortical AP, its associated BKCa channel is expected to open in only 30% of trials, and this percentage is very sensitive to the duration of the AP, the distance between the two channels in the complex, and the presence of fast internal Ca(2+) buffers. Also, the model predicts that the kinetics of the BKCa currents of a population of CaV2.1/BKCa complexes will not be limited by the kinetics of the CaV2.1 channel, and during a train of APs, the current response of the complex is expected to faithfully follow even very rapid trains. Aside from providing insight into how these complexes are likely to behave in vivo, the models presented here could also be of use more generally as components of higher-level models of neural function.

Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels.

Small conductance Ca(2+)-sensitive potassium (SK2) channels are voltage-independent, Ca(2+)-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca(2+) permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3' terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca(2+) and Ca(2+)-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca(2+) influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.

Suppression of arterial thrombosis without affecting hemostatic parameters with a cell-penetrating PAR1 pepducin.

BACKGROUND: Thrombin-dependent platelet activation is heightened in the setting of percutaneous coronary intervention and may cause arterial thrombosis with consequent myocardial necrosis. Given the high incidence of adverse effects in patients with acute coronary syndromes, there remains an unmet need for the development of new therapeutics that target platelet activation without unduly affecting hemostasis. The thrombin receptor, PAR1, has recently emerged as a promising new target for therapeutic intervention in patients with acute coronary syndromes. METHODS AND RESULTS: We report the development of a first-in-class intracellular PAR1 inhibitor with optimized pharmacokinetic properties for use during percutaneous coronary intervention in patients with acute coronary syndromes. PZ-128 is a cell-penetrating pepducin inhibitor of PAR1 that targets the receptor-G-protein interface on the inside surface of platelets. The structure of PZ-128 closely resembles the predicted off-state of the corresponding juxtamembrane region of the third intracellular loop of PAR1. The onset of action of PZ-128 was rapid and suppressed PAR1 aggregation and arterial thrombosis in guinea pigs and baboons and strongly synergized with oral clopidogrel. There was full recovery of platelet function by 24 hours. Importantly, PZ-128 had no effect on bleeding or coagulation parameters in primates or in blood from patients undergoing percutaneous coronary intervention. CONCLUSIONS: Based on the efficacy data in nonhuman primates with no noted adverse effects on hemostasis, we anticipate that the rapid onset of platelet inhibition and reversible properties of PZ-128 are well suited to the acute interventional setting of percutaneous coronary intervention and may provide an alternative to long-acting small-molecule inhibitors of PAR1.

Ca(2+)-regulated ion channels.

Due to its high external and low internal concentration the Ca(2+) ion is used ubiquitously as an intracellular signaling molecule, and a great many Ca(2+)-sensing proteins have evolved to receive and propagate Ca(2+) signals. Among them are ion channel proteins, whose Ca(2+) sensitivity allows internal Ca(2+) to influence the electrical activity of cell membranes and to feedback-inhibit further Ca(2+) entry into the cytoplasm. In this review I will describe what is understood about the Ca(2+) sensing mechanisms of the three best studied classes of Ca(2+)-sensitive ion channels: Large-conductance Ca(2+)-activated K(+) channels, small-conductance Ca(2+)-activated K(+) channels, and voltage- gated Ca(2+) channels. Great strides in mechanistic understanding have be made for each of these channel types in just the past few years.

Measuring the influence of the BKCa {beta}1 subunit on Ca2+ binding to the BKCa channel.

The large-conductance Ca(2+)-activated potassium (BK(Ca)) channel of smooth muscle is unusually sensitive to Ca(2+) as compared with the BK(Ca) channels of brain and skeletal muscle. This is due to the tissue-specific expression of the BK(Ca) auxiliary subunit beta1, whose presence dramatically increases both the potency and efficacy of Ca(2+) in promoting channel opening. beta1 contains no Ca(2+) binding sites of its own, and thus the mechanism by which it increases the BK(Ca) channel's Ca(2+) sensitivity has been of some interest. Previously, we demonstrated that beta1 stabilizes voltage sensor activation, such that activation occurs at more negative voltages with beta1 present. This decreases the work that Ca(2+) must do to open the channel and thereby increases the channel's apparent Ca(2+) affinity without altering the real affinities of the channel's Ca(2+) binding sites. To explain the full effect of beta1 on the channel's Ca(2+) sensitivity, however, we also proposed that there must be effects of beta1 on Ca(2+) binding. Here, to test this hypothesis, we have used high-resolution Ca(2+) dose-response curves together with binding site-specific mutations to measure the effects of beta1 on Ca(2+) binding. We find that coexpression of beta1 alters Ca(2+) binding at both of the BK(Ca) channel's two types of high-affinity Ca(2+) binding sites, primarily increasing the affinity of the RCK1 sites when the channel is open and decreasing the affinity of the Ca(2+) bowl sites when the channel is closed. Both of these modifications increase the difference in affinity between open and closed, such that Ca(2+) binding at either site has a larger effect on channel opening when beta1 is present.

Measurements of the BKCa channel's high-affinity Ca2+ binding constants: effects of membrane voltage.

It has been established that the large conductance Ca(2+)-activated K(+) channel contains two types of high-affinity Ca(2+) binding sites, termed the Ca(2+) bowl and the RCK1 site. The affinities of these sites, and how they change as the channel opens, is still a subject of some debate. Previous estimates of these affinities have relied on fitting a series of conductance-voltage relations determined over a series of Ca(2+) concentrations with models of channel gating that include both voltage sensing and Ca(2+) binding. This approach requires that some model of voltage sensing be chosen, and differences in the choice of voltage-sensing model may underlie the different estimates that have been produced. Here, to better determine these affinities we have measured Ca(2+) dose-response curves of channel activity at constant voltage for the wild-type mSlo channel (minus its low-affinity Ca(2+) binding site) and for channels that have had one or the other Ca(2+) binding site disabled via mutation. To accurately determine these dose-response curves we have used a series of 22 Ca(2+) concentrations, and we have used unitary current recordings, coupled with changes in channel expression level, to measure open probability over five orders of magnitude. Our results indicate that at -80 mV the Ca(2+) bowl has higher affinity for Ca(2+) than does the RCK1 site in both the opened and closed conformations of the channel, and that the binding of Ca(2+) to the RCK1 site is voltage dependent, whereas at the Ca(2+) bowl it is not.

Modulation of the conductance-voltage relationship of the BK Ca channel by mutations at the putative flexible interface between two RCK domains.

Calcium-dependent gating of the large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel is conferred by the large cytosolic carboxyl terminus containing two domains of the regulator of K(+) conductance (RCK) and the high-affinity Ca(2+)-binding site (the Ca(2+)-bowl). In our previous study, we located the putative second RCK domain (RCK2) and demonstrated that it interacts directly with RCK1 via a hydrophobic "assembly interface". In this study, we tested the structural model of the other interface, the "flexible interface", by strategically positioning charge pairs across the putative interface. Several charge mutations on RCK2 affected the voltage-dependent activation of the channel. In particular, the Gly-to-Asp substitution at position 803 profoundly affected channel activation by stabilizing the open conformation of the channel with minimal effects on its Ca(2+) affinity and voltage sensitivity. Various mutations at Gly-803 shifted the channel's conductance-voltage curve either left or right over a 145-mV range. Since this residue is predicted to be in the first loop of RCK2 these results strongly suggest that this loop plays a critical role in determining the intrinsic equilibrium constant for channel opening, and they support the hypothesis that this loop is part of an interface that mediates conformational coupling between RCK1 and RCK2.

RACK1 is a BKCa channel binding protein.

The large conductance calcium-activated potassium channel, or BK(Ca) channel, plays an important feedback role in a variety of physiological processes, including neurotransmitter release and smooth muscle contraction. Some reports have suggested that this channel forms a stable complex with regulators of its function, including several kinases and phosphatases. To further define such signaling complexes, we used the yeast two-hybrid system to screen a human aorta cDNA library for proteins that bind to the BK(Ca) channel's intracellular, COOH-terminal "tail". One of the interactors we identified is the protein receptor for activated C kinase 1 (RACK1). RACK1 is a member of the WD40 protein family, which also includes the G protein beta-subunits. Consistent with an important role in BK(Ca)-channel regulation, RACK1 has been shown to be a scaffolding protein that interacts with a wide variety of signaling molecules, including cSRC and PKC. We have confirmed the interaction between RACK1 and the BK(Ca) channel biochemically with GST pull-down and coimmunoprecipitation experiments. We have observed some co-localization of RACK1 with the BK(Ca) channel in vascular smooth muscle cells with immunocytochemical experiments, and we have found that RACK1 has effects on the BK(Ca) channel's biophysical properties. Thus RACK1 binds to the BK(Ca) channel and it may form part of a BK(Ca)-channel regulatory complex in vascular smooth muscle.

Gating and ionic currents reveal how the BKCa channel's Ca2+ sensitivity is enhanced by its beta1 subunit.

Large-conductance Ca(2+)-activated K(+) channels (BK(Ca) channels) are regulated by the tissue-specific expression of auxiliary beta subunits. Beta1 is predominantly expressed in smooth muscle, where it greatly enhances the BK(Ca) channel's Ca(2+) sensitivity, an effect that is required for proper regulation of smooth muscle tone. Here, using gating current recordings, macroscopic ionic current recordings, and unitary ionic current recordings at very low open probabilities, we have investigated the mechanism that underlies this effect. Our results may be summarized as follows. The beta1 subunit has little or no effect on the equilibrium constant of the conformational change by which the BK(Ca) channel opens, and it does not affect the gating charge on the channel's voltage sensors, but it does stabilize voltage sensor activation, both when the channel is open and when it is closed, such that voltage sensor activation occurs at more negative voltages with beta1 present. Furthermore, beta1 stabilizes the active voltage sensor more when the channel is closed than when it is open, and this reduces the factor D by which voltage sensor activation promotes opening by approximately 24% (16.8-->12.8). The effects of beta1 on voltage sensing enhance the BK(Ca) channel's Ca(2+) sensitivity by decreasing at most voltages the work that Ca(2+) binding must do to open the channel. In addition, however, in order to fully account for the increase in efficacy and apparent Ca(2+) affinity brought about by beta1 at negative voltages, our studies suggest that beta1 also decreases the true Ca(2+) affinity of the closed channel, increasing its Ca(2+) dissociation constant from approximately 3.7 microM to between 4.7 and 7.1 microM, depending on how many binding sites are affected.

Mapping the BKCa channel's "Ca2+ bowl": side-chains essential for Ca2+ sensing.

There is controversy over whether Ca(2+) binds to the BK(Ca) channel's intracellular domain or its integral-membrane domain and over whether or not mutations that reduce the channel's Ca(2+) sensitivity act at the point of Ca(2+) coordination. One region in the intracellular domain that has been implicated in Ca(2+) sensing is the "Ca(2+) bowl". This region contains many acidic residues, and large Ca(2+)-bowl mutations eliminate Ca(2+) sensing through what appears to be one type of high-affinity Ca(2+)-binding site. Here, through site-directed mutagenesis we have mapped the residues in the Ca(2+) bowl that are most important for Ca(2+) sensing. We find acidic residues, D898 and D900, to be essential, and we find them essential as well for Ca(2+) binding to a fusion protein that contains a portion of the BK(Ca) channel's intracellular domain. Thus, much of our data supports the conclusion that Ca(2+) binds to the BK(Ca) channel's intracellular domain, and they define the Ca(2+) bowl's essential Ca(2+)-sensing motif. Overall, however, we have found that the relationship between mutations that disrupt Ca(2+) sensing and those that disrupt Ca(2+) binding is not as strong as we had expected, a result that raises the possibility that, when examined by gel-overlay, the Ca(2+) bowl may be in a nonnative conformation.

Elimination of the BK(Ca) channel's high-affinity Ca(2+) sensitivity.

We report here a combination of site-directed mutations that eliminate the high-affinity Ca(2+) response of the large-conductance Ca(2+)-activated K(+) channel (BK(Ca)), leaving only a low-affinity response blocked by high concentrations of Mg(2+). Mutations at two sites are required, the "Ca(2+) bowl," which has been implicated previously in Ca(2+) binding, and M513, at the end of the channel's seventh hydrophobic segment. Energetic analyses of mutations at these positions, alone and in combination, argue that the BK(Ca) channel contains three types of Ca(2+) binding sites, one of low affinity that is Mg(2+) sensitive (as has been suggested previously) and two of higher affinity that have similar binding characteristics and contribute approximately equally to the power of Ca(2+) to influence channel opening. Estimates of the binding characteristics of the BK(Ca) channel's high-affinity Ca(2+)-binding sites are provided.