Monoterpenes Differently Regulate Acid-Sensitive and Mechano-Gated K2P Channels.

Potassium K2P ("leak") channels conduct current across the entire physiological voltage range and carry leak or "background" currents that are, in part, time- and voltage-independent. The activity of K2P channels affects numerous physiological processes, such as cardiac function, pain perception, depression, neuroprotection, and cancer development. We have recently established that, when expressed in Xenopus laevis oocytes, K2P2.1 (TREK-1) channels are activated by several monoterpenes (MTs). Here, we show that, within a few minutes of exposure, other mechano-gated K2P channels, K2P4.1 (TRAAK) and K2P10.1 (TREK-2), are opened by monoterpenes as well (up to an eightfold increase in current). Furthermor\e, carvacrol and cinnamaldehyde robustly enhance currents of the alkaline-sensitive K2P5.1 (up to a 17-fold increase in current). Other members of the K2P potassium channels, K2P17.1, K2P18.1, but not K2P16.1, were also activated by various MTs. Conversely, the activity of members of the acid-sensitive (TASK) K2P channels (K2P3.1 and K2P9.1) was rapidly decreased by monoterpenes. We found that MT selectively decreased the voltage-dependent portion of the current and that current inhibition was reduced with the elevation of external K+ concentration. These findings suggest that penetration of MTs into the outer leaflet of the membrane results in immediate changes at the selectivity filter of members of the TASK channel family. Thus, we suggest MTs as promising new tools for the study of K2P channels' activity in vitro as well as in vivo.

A regulatory domain in the K2P2.1 (TREK-1) carboxyl-terminal allows for channel activation by monoterpenes.

Potassium K2P ('leak') channels conduct current across the entire physiological voltage range and carry leak or 'background' currents that are, in part, time- and voltage-independent. K2P2.1 channels (i.e., TREK-1, KCNK2) are highly expressed in excitable tissues, where they play a key role in the cellular mechanisms of neuroprotection, anesthesia, pain perception, and depression. Here, we report for the first time that human K2P2.1 channel activity is regulated by monoterpenes (MTs). We found that cyclic, aromatic monoterpenes containing a phenol moiety, such as carvacrol, thymol and 4-IPP had the most profound effect on current flowing through the channel (up to a 6-fold increase). By performing sequential truncation of the carboxyl-terminal domain of the channel and testing the activity of several channel regulators, we identified two distinct regulatory domains within this portion of the protein. One domain, as previously reported, was needed for regulation by arachidonic acid, anionic phospholipids, and temperature changes. Within a second domain, a triple arginine residue motif (R344-346), an apparent PIP2-binding site, was found to be essential for regulation by holding potential changes and important for regulation by monoterpenes.

Nocturnal Atrial Fibrillation Caused by Mutation in KCND2, Encoding Pore-Forming (α) Subunit of the Cardiac Kv4.2 Potassium Channel.

BACKGROUND: Paroxysmal atrial fibrillation (AF) can be caused by gain-of-function mutations in genes, encoding the cardiac potassium channel subunits KCNJ2, KCNE1, and KCNH2 that mediate the repolarizing potassium currents Ik1, Iks, and Ikr, respectively. METHODS: Linkage analysis, whole-exome sequencing, and Xenopus oocyte electrophysiology studies were used in this study. RESULTS: Through genetic studies, we showed that autosomal dominant early-onset nocturnal paroxysmal AF is caused by p.S447R mutation in KCND2, encoding the pore-forming (α) subunit of the Kv4.2 cardiac potassium channel. Kv4.2, along with Kv4.3, contributes to the cardiac fast transient outward K+ current, Ito. Ito underlies the early phase of repolarization in the cardiac action potential, thereby setting the initial potential of the plateau phase and governing its duration and amplitude. In Xenopus oocytes, the mutation increased the channel's inactivation time constant and affected its regulation: p.S447 resides in a protein kinase C (PKC) phosphorylation site, which normally allows attenuation of Kv4.2 membrane expression. The mutant Kv4.2 exhibited impaired response to PKC; hence, Kv4.2 membrane expression was augmented, enhancing potassium currents. Coexpression of mutant and wild-type channels (recapitulating heterozygosity in affected individuals) showed results similar to the mutant channel alone. Finally, in a hybrid channel composed of Kv4.3 and Kv4.2, simulating the mature endogenous heterotetrameric channel underlying Ito, the p.S447R Kv4.2 mutation exerted a gain-of-function effect on Kv4.3. CONCLUSIONS: The mutation alters Kv4.2's kinetic properties, impairs its inhibitory regulation, and exerts gain-of-function effect on both Kv4.2 homotetramers and Kv4.2-Kv4.3 heterotetramers. These effects presumably increase the repolarizing potassium current Ito, thereby abbreviating action potential duration, creating arrhythmogenic substrate for nocturnal AF. Interestingly, Kv4.2 expression was previously shown to demonstrate circadian variation, with peak expression at daytime in murine hearts (human nighttime), with possible relevance to the nocturnal onset of paroxysmal AF symptoms in our patients. The atrial-specific phenotype suggests that targeting Kv4.2 might be effective in the treatment of nocturnal paroxysmal AF, avoiding adverse ventricular effects.

Molding the business end of neurotoxins by diversifying evolution.

A diverse range of organisms utilize neurotoxins that target specific ion channels and modulate their activity. Typically, toxins are clustered into several multigene families, providing an organism with the upper hand in the never-ending predator-prey arms race. Several gene families, including those encoding certain neurotoxins, have been subject to diversifying selection forces, resulting in rapid gene evolution. Here we sought a spatial pattern in the distribution of both diversifying and purifying selection forces common to neurotoxin gene families. Utilizing the mechanistic empirical combination model, we analyzed various toxin families from different phyla affecting various receptors and relying on diverse modes of action. Through this approach, we were able to detect clear correlations between the pharmacological surface of a toxin and rapidly evolving domains, rich in positively selected residues. On the other hand, patches of negatively selected residues were restricted to the nontoxic face of the molecule and most likely help in stabilizing the tertiary structure of the toxin. We thus propose a mutual evolutionary strategy of venomous animals in which adaptive molecular evolution is directed toward the toxin active surface. Furthermore, we propose that the binding domains of unstudied toxins could be readily predicted using evolutionary considerations.

Symptomatic relief of botulinum neurotoxin/a intoxication with aminopyridines: a new twist on an old molecule.

Botulinum neurotoxins (BoNT) are the etiological agents responsible for botulism, a disease characterized by peripheral neuromuscular blockade and a characteristic flaccid paralysis of humans. BoNT/A is the most toxic protein known to man and has been classified by the Centers of Disease Control (CDC) as one of the six highest-risk threat agents for bioterrorism. Of particular concern is the apparent lack of clinical interventions that can reverse cellular intoxication. Efforts to uncover molecules that can act within an intoxicated cell so as to provide symptomatic relief to BoNT/A are paramount. Aminopyridines have shown clinical efficacy for multiple sclerosis treatment as well as BoNT/A intoxication; yet, aminopyridines for BoNT/A treatment has been abandoned because of blood brain barrier (BBB) penetration producing undesired neurotoxic side effects. Two aminopyridines (5 and 11) exhibited inhibitory activity toward Shaker-IR voltage-gated potassium (K(V)1.x) channels with potencies similar to that of the previous "gold-standard", 3,4-diaminopyridine (3,4-DAP), including reversal of symptoms from BoNT-induced paralysis in phrenic nerve-hemidiaphragm preparations. Importantly, pharmacokinetic experiments revealed a lack of BBB penetration of 5, which is a significant advancement toward resolving the neurotoxicity issues associated with prolonged 3,4-DAP treatments. Finally, 5 was found to be as effective as 3,4-DAP in rescuing BoNT-poisoned mice in the mouse lethality assay, signifying an optimized balance between the undesired permeability across the BBB and the required permeability across lipid cellular membranes. The results demonstrate that 5 is the most promising small molecule K(+) channel inhibitor discovered to date for the treatment of BoNT/A intoxication.

Molecular mechanisms underlying membrane-potential-mediated regulation of neuronal K2P2.1 channels.

The activity of background K(2P) channels adjusts the resting membrane potential to enable plasticity of excitable cells. Here we have studied the regulation of neuronal K(2P)2.1 (KCNK2, TREK-1) channel activity by resting membrane potential. When heterologously expressed, K(2P)2.1 currents gradually increased at hyperpolarizing potentials and declined at depolarizing potentials, with a midpoint potential of -60 mV. As K(2P) channels are not equipped with an integral voltage sensor, we sought extrinsic cellular components that could convert changes in the membrane electrical field to cellular activity that would indirectly modify K(2P)2.1 currents. We propose that membrane depolarization activated the Gq protein-coupled receptor pathway, in the apparent absence of ligand, resulting in phosphatidylinositol-4,5-bisphosphate (PIP(2)) depletion through the action of phospholipase C. Our results suggest a novel mechanism in which an indirect pathway confers membrane potential regulation onto channels that are not intrinsically voltage sensitive to enhance regulation of neuronal excitability levels.

Gating the pore of potassium leak channels.

A key feature of potassium channel function is the ability to switch between conducting and non-conducting states by undergoing conformational changes in response to cellular or extracellular signals. Such switching is facilitated by the mechanical coupling of gating domain movements to pore opening and closing. Two-pore domain potassium channels (K(2P)) conduct leak or background potassium-selective currents that are mostly time- and voltage-independent. These channels play a significant role in setting the cell resting membrane potential and, therefore modulate cell responsiveness and excitability. Thus, K(2P) channels are key players in numerous physiological processes and were recently shown to also be involved in human pathologies. It is well established that K(2P) channel conductance, open probability and cell surface expression are significantly modulated by various physical and chemical stimuli. However, in understanding how such signals are translated into conformational changes that open or close the channels gate, there remain more open questions than answers. A growing line of evidence suggests that the outer pore area assumes a critical role in gating K(2P) channels, in a manner reminiscent of C-type inactivation of voltage-gated potassium channels. In some K(2P) channels, this gating mechanism is facilitated in response to external pH levels. Recently, it was suggested that K(2P) channels also possess a lower activation gate that is positively coupled to the outer pore gate. The purpose of this review is to present an up-to-date summary of research describing the conformational changes and gating events that take place at the K(2P) channel ion-conducting pathway during the channel regulation.

Pain-associated signals, acidosis and lysophosphatidic acid, modulate the neuronal K(2P)2.1 channel.

Pain is a physiological state promoting protective responses to harmful episodes. However, pain can become pathophysiological and become a chronic disruptive condition, damaging quality of life. The mammalian K(2P)2.1 (KCNK2, TREK-1) channel, expressed in sensory neurons of the dorsal root ganglia, was previously identified as a polymodal molecular sensor involved in pain perception. Here, we report that two pain-associated signals, external acidosis and lysophosphatidic acid (LPA), known to rise during injury, inflammation and cancer, profoundly down-modulate human K(2P)2.1 activity. The pH regulatory effect was mediated by activation of proton-sensitive G-protein coupled receptors and phospholipase C. Physiological concentrations of LPA overcame the effects of known K(2P)2.1 activators, such as arachidonic acid, lysophosphatidylcholine and temperature, by activating cell-surface receptors stimulating the G(q) pathway. Furthermore, we identified three K(2P)2.1 carboxy-terminal residues that mediate both pH and LPA regulatory effects. Our results highlight the important role of K(2P)2.1 channels as receptors for mediators known to cause nociception.

Inverse coupling in leak and voltage-activated K+ channel gates underlies distinct roles in electrical signaling.

Voltage-activated (Kv) and leak (K(2P)) K(+) channels have key, yet distinct, roles in electrical signaling in the nervous system. Here we examine how differences in the operation of the activation and slow inactivation pore gates of Kv and K(2P) channels underlie their unique roles in electrical signaling. We report that (i) leak K(+) channels possess a lower activation gate, (ii) the activation gate is an important determinant controlling the conformational stability of the K(+) channel pore, (iii) the lower activation and upper slow inactivation gates of leak channels cross-talk and (iv) unlike Kv channels, where the two gates are negatively coupled, these two gates are positively coupled in K(2P) channels. Our results demonstrate how basic thermodynamic properties of the K(+) channel pore, particularly conformational stability and coupling between gates, underlie the specialized roles of Kv and K(2P) channel families in electrical signaling.

Voltage-dependent anion channel 1-based peptides interact with hexokinase to prevent its anti-apoptotic activity.

In brain and tumor cells, the hexokinase isoforms, HK-I and HK-II, bind to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane. The VDAC domains interacting with these anti-apoptotic proteins were recently defined using site-directed mutagenesis. Now, we demonstrate that synthetic peptides corresponding to the VDAC1 N-terminal region and selected sequences bound specifically, in a concentration- and time-dependent manner, to immobilized HK-I, as revealed by real time surface plasmon resonance technology. The same VDAC1-based peptides also detached HK bound to brain or tumor-derived mitochondria. Moreover, expression of the VDAC1-based peptides in cells overexpressing HK-I or HK-II prevented HK-mediated protection against staurosporine-induced release of cytochrome c and subsequent cell death. One loop-shaped VDAC1-based peptide corresponding to a selected sequence and fused to a cell-penetrating peptide entered the cell and prevented the anti-apoptotic effects of HK-I and HK-II. This peptide detached mitochondrial-bound HK better than did the same peptide in its linear form. Both cell-expressed and exogenously added cell-penetrating peptide detached mitochondrial-bound HK-I-GFP. These results point to HK-I and HK-II as promoting tumor cell survival through binding to VDAC1, thereby inhibiting cytochrome c release and apoptotic cell death. Moreover, VDAC1-based peptides interfering with HK-mediated anti-apoptotic activity may potentiate the efficacy of conventional chemotherapeutic agents.

Assembling an arsenal, the scorpion way.

BACKGROUND: For survival, scorpions depend on a wide array of short neurotoxic polypeptides. The venoms of scorpions from the most studied group, the Buthida, are a rich source of small, 23-78 amino acid-long peptides, well packed by either three or four disulfide bridges that affect ion channel function in excitable and non-excitable cells. RESULTS: In this work, by constructing a toxin transcripts data set from the venom gland of the scorpion Buthus occitanus israelis, we were able to follow the evolutionary path leading to mature toxin diversification and suggest a mechanism for leader peptide hyper-conservation. Toxins from each family were more closely related to one another than to toxins from other species, implying that fixation of duplicated genes followed speciation, suggesting early gene conversion events. Upon fixation, the mature toxin-coding domain was subjected to diversifying selection resulting in a significantly higher substitution rate that can be explained solely by diversifying selection. In contrast to the mature peptide, the leader peptide sequence was hyper-conserved and characterized by an atypical sub-neutral synonymous substitution rate. We interpret this as resulting from purifying selection acting on both the peptide and, as reported here for the first time, the DNA sequence, to create a toxin family-specific codon bias. CONCLUSION: We thus propose that scorpion toxin genes were shaped by selective forces acting at three levels, namely (1) diversifying the mature toxin, (2) conserving the leader peptide amino acid sequence and intriguingly, (3) conserving the leader DNA sequences.

Maternally inherited Birk Barel mental retardation dysmorphism syndrome caused by a mutation in the genomically imprinted potassium channel KCNK9.

We describe a maternally transmitted genomic-imprinting syndrome of mental retardation, hypotonia, and unique dysmorphism with elongated face. We mapped the disease-associated locus to approximately 7.27 Mb on chromosome 8q24 and demonstrated that the disease is caused by a missense mutation in the maternal copy of KCNK9 within this locus. KCNK9 is maternally transmitted (imprinted with paternal silencing) and encodes K(2P)9.1, a member of the two pore-domain potassium channel (K(2P)) subfamily. The mutation fully abolishes the channel's currents--both when functioning as a homodimer or as a heterodimer with K(2P)3.1.

A novel mechanism for human K2P2.1 channel gating. Facilitation of C-type gating by protonation of extracellular histidine residues.

The mammalian K2P2.1 potassium channel (TREK-1, KCNK2) is highly expressed in excitable tissues, where it plays a key role in the cellular mechanisms of neuroprotection, anesthesia, pain perception, and depression. Here, we report that external acidification, within the physiological range, strongly inhibits the human K2P2.1 channel by inducing "C-type" closure. We have identified two histidine residues (i.e. His-87 and His-141), located in the first external loop of the channel, that govern the response of the channel to external pH. We demonstrate that these residues are within physical proximity to glutamate 84, homologous to Shaker Glu-418, KcsA Glu-51, and KCNK0 Glu-28 residues, all previously argued to stabilize the outer pore gate in the open conformation by forming hydrogen bonds with pore-adjacent residues. We thus propose a novel mechanism for pH sensing in which protonation of His-141 and His-87 generates a local positive charge that serves to draw Glu-84 away from its natural interactions, facilitating the collapse of the selectivity filter region. In accordance with this proposed mechanism, low pH modified K2P2.1 selectivity toward potassium. Moreover, the proton-mediated effect was inhibited by external potassium ions and was enhanced by a mutation (S164Y) known to accelerate C-type gating. Furthermore, proton-induced current inhibition was more pronounced at negative potentials. Thus, voltage-dependent C-type gating acceleration by protons represents a novel mechanism for K2P2.1 outward rectification.

Isolation of the first toxin from the scorpion Buthus occitanus israelis showing preference for Shaker potassium channels.

We have purified BoiTx1, the first toxin from the venom of the Israeli scorpion, Buthus occitanus israelis, and studied its activity and genomic organization. BoiTx1 is a 37 amino acid-long peptide contained six conserved cysteines, and is classified as an alpha-KTx3.10 toxin. The pharmacological effects of BoiTx1 were studied on various cloned K(+) channels expressed in Xenopus laevis oocytes. BoiTx1 inhibited currents through Drosophila Shaker channels with an IC(50) value of 3.5+/-0.5nM, yet had much lesser effect on its mammalian orthologs. Thus, BoiTx1 is the first member of the alpha-KTx3 family that preferentially affects insect potassium channels.

Fluctuations in Xenopus oocytes protein phosphorylation levels during two-electrode voltage clamp measurements.

The biophysical and pharmacological properties of ion channels and transporters are often studied in exogenous expression systems using either the two-electrode voltage clamp (TEVC) in Xenopus oocytes or the patch clamp techniques. Cells machinery is trusted to produce active proteins that are correctly phosphorylated and glycosylated. However, native physiological cellular processes that might be altered during the course of the experiment are often ignored. Here, we detected and quantified the effects of various electrophysiological recording conditions on the phosphorylation levels of Xenopus oocytes proteins, including membrane proteins, as phosphorylation/dephosphorylation events modulate ion channels gating and cell surface expression. Two strategies were chosen to determine relative protein phosphorylation levels: a direct detection with a phospho-Ser/Thr PKA substrate antibody, and a functional method employing two different leak potassium channels as indicators, chosen based on their opposite responses to protein kinase phosphorylation. We report that holding potential, and bath solution properties such as pH, osmolarity, temperature and ion composition, dramatically affect protein phosphorylation levels in Xenopus oocytes. Our results might explain some of the fluctuations in the biophysical properties of expressed channels, often observed during electrophysiological measurements. Minimizing possible misinterpretations could be achieved using either mutated, kinase insensitive, channels or kinases/phosphatases modulators.

Azido ruthenium: a new photoreactive probe for calcium-binding proteins.

Ca2+, involved in almost all processes of cell life, mediates its activity through reversible interaction with specific binding sites in proteins. Although several Ca2+-dependent activities are known, many of the proteins responsible remain unidentified. Here we describe the synthesis, purification, characterization and potential uses of a new Ca2+-like reagent, azido ruthenium (AzRu), which can be photoactivated. AzRu strongly inhibits Ca2+-dependent activities. AzRu can be used to probe proteins in solution or embedded in membranes. AzRu has no effect on Ca2+-independent or Mg2+-dependent activity. After exposure to ultraviolet irradiation, AzRu binds covalently and specifically to Ca2+-binding proteins, thus providing a new approach for identifying and purifying Ca2+-binding proteins, for characterizing their Ca2+-binding sites and for exploring previously unknown Ca2+-dependent processes. In this protocol we also include a description of the preparation of [103Ru]AzRu, which can be used for labeling Ca2+-binding sites in proteins and identifying previously unknown Ca2+-binding proteins. The preparation of AzRu takes approximately 2-3 days.

Genetic polymorphism and expression of a highly potent scorpion depressant toxin enable refinement of the effects on insect Na channels and illuminate the key role of Asn-58.

We isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus an extremely active anti-insect selective depressant toxin, Lqh-dprIT(3). Cloning of Lqh-dprIT(3) revealed a gene family encoding eight putative polypeptide variants (a-h) differing at three positions (37A/G, 50D/E, and 58N/D). All eight toxin variants were expressed in a functional form, and their toxicity to blowfly larvae, binding affinity for cockroach neuronal membranes, and CD spectra were compared. This analysis links Asn-58, which appears in variants a-d, to a toxin conformation associated with high binding affinity for insect sodium channels. Variants e-h, bearing Asp-58, exhibit a different conformation and are less potent. The importance of Asn-58, which is conserved in other depressant toxins, was further validated by construction and analysis of an N58D mutant of the well-characterized depressant toxin, LqhIT(2). Current and voltage clamp assays using the cockroach giant axon have shown that despite the vast difference in potency, the two types of Lqh-dprIT(3) variants (represented by Lqh-dprIT(3)-a and Lqh-dprIT(3)-e) are capable of blocking the action potentials (manifested as flaccid paralysis in blowfly larvae) and shift the voltage dependence of activation to more negative values, which typify the action of beta-toxins. Moreover, the stronger and faster shift in voltage dependence of activation and lack of tail currents observed in the presence of Lqh-dprIT(3)-a suggest an extremely efficient trapping of the voltage sensor compared to that of Lqh-dprIT(3)-e. The current clamp assays revealed that repetitive firing of the axon, which is reflected in contraction paralysis of blowfly larvae, can be obtained with either the less potent Lqh-dprIT(3)-e or the highly potent Lqh-dprIT(3)-a at more negative membrane potentials. Thus, the contraction symptoms in flies are likely to be dominated by the resting potential of neuronal membranes. This study clarifies the electrophysiological basis of the complex symptoms induced by scorpion depressant toxins in insects, and highlights for the first time molecular features involved in their activity.

An 'Old World' scorpion beta-toxin that recognizes both insect and mammalian sodium channels.

Scorpion toxins that affect sodium channel (NaCh) gating in excitable cells are divided into alpha- and beta-classes. Whereas alpha-toxins have been found in scorpions throughout the world, anti-mammalian beta-toxins have been assigned, thus far, to 'New World' scorpions while anti-insect selective beta-toxins (depressant and excitatory) have been described only in the 'Old World'. This distribution suggested that diversification of beta-toxins into distinct pharmacological groups occurred after the separation of the continents, 150 million years ago. We have characterized a unique toxin, Lqhbeta1, from the 'Old World' scorpion, Leiurus quinquestriatus hebraeus, that resembles in sequence and activity both 'New World'beta-toxins as well as 'Old World' depressant toxins. Lqhbeta1 competes, with apparent high affinity, with anti-insect and anti-mammalian beta-toxins for binding to cockroach and rat brain synaptosomes, respectively. Surprisingly, Lqhbeta1 also competes with an anti-mammalian alpha-toxin on binding to rat brain NaChs. Analysis of Lqhbeta1 effects on rat brain and Drosophila Para NaChs expressed in Xenopus oocytes revealed a shift in the voltage-dependence of activation to more negative membrane potentials and a reduction in sodium peak currents in a manner typifying beta-toxin activity. Moreover, Lqhbeta1 resembles beta-toxins by having a weak effect on cardiac NaChs and a marked effect on rat brain and skeletal muscle NaChs. These multifaceted features suggest that Lqhbeta1 may represent an ancestral beta-toxin group in 'Old World' scorpions that gave rise, after the separation of the continents, to depressant toxins in 'Old World' scorpions and to various beta-toxin subgroups in 'New World' scorpions.

Forward transport. 14-3-3 binding overcomes retention in endoplasmic reticulum by dibasic signals.

Proteins with dibasic retention motifs are subject to retrograde transport to endoplasmic reticulum (ER) by COPI-coated vesicles. As forward transport requires escape from ER retention, general release mechanisms have been expected. Here, KCNK3 potassium channels are shown to bear two cytoplasmic trafficking motifs: an N-terminal dibasic site that binds beta-COP to hold channels in ER and a C-terminal "release" site that binds the ubiquitous intracellular regulator 14-3-3beta on a nonclassical motif in a phosphorylation-dependent fashion to suppress beta-COP binding and allow forward transport. The strategy appears to be common. The major histocompatibility antigen class II-associated invariant chain Iip35 exhibits dibasic retention, carries a release motif, and shows mutually exclusive binding of beta-COP and 14-3-3beta on adjacent N-terminal sites. Other retained proteins are demonstrated to carry functional 14-3-3beta release motifs.