The Acrylamide (S)-2 As a Positive and Negative Modulator of Kv7 Channels Expressed in Xenopus laevis Oocytes

Background

Activation of voltage-gated potassium channels of the Kv7 (KCNQ) family reduces cellular excitability. These channels are therefore attractive targets for treatment of diseases characterized by hyperexcitability, such as epilepsy, migraine and neuropathic pain. Retigabine, which opens Kv7.2-5, is now in clinical trial phase III for the treatment of partial onset seizures. One of the main obstacles in developing Kv7 channel active drugs has been to identify compounds that can discriminate between the neuronal subtypes, a feature that could help diminish side effects and increase the potential of drugs for particular indications.

Methodology/Principal Findings

In the present study we have made a thorough investigation of the Bristol-Myers Squibb compound (S)-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo[1], [4]oxazin-6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide [(S)-2] on human Kv7.1-5 channels expressed in Xenopus laevis oocytes. We found that the compound was a weak inhibitor of Kv7.1. In contrast, (S)-2 efficiently opened Kv7.2-5 by producing hyperpolarizing shifts in the voltage-dependence of activation and enhancing the maximal current amplitude. Further, it reduced inactivation, accelerated activation kinetics and slowed deactivation kinetics. The mechanisms of action varied between the subtypes. The enhancing effects of (S)-2 were critically dependent on a tryptophan residue in S5 also known to be crucial for the effects of retigabine, (S)-1 and BMS-204352. However, while (S)-2 did not at all affect a mutant Kv7.4 with a leucine in this position (Kv7.4-W242L), a Kv7.2 with the same mutation (Kv7.2-W236L) was inhibited by the compound, showing that (S)-2 displays a subtype-selective interaction with in the Kv7 family.

Conclusions/Significance

These results offer further insight into pharmacological activation of Kv7 channels, add to the understanding of small molecule interactions with the channels and may contribute to the design of subtype selective modulators.     

Differential Protein Kinase C-dependent Modulation of Kv7.4 and Kv7.5 Subunits of Vascular Kv7 Channels

The Kv7 family (Kv7.1–7.5) of voltage-activated potassium channels contributes to the maintenance of resting membrane potential in excitable cells. Previously, we provided pharmacological and electrophysiological evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels and that Kv7 activity is regulated by protein kinase C (PKC) in response to vasoconstrictors in vascular smooth muscle cells. Direct evidence for Kv7.4/7.5 heteromer formation, however, is lacking. Furthermore, it remains to be determined whether both subunits are regulated by PKC. Utilizing proximity ligation assays to visualize single molecule interactions, we now show that Kv7.4/Kv.7.5 heteromers are endogenously expressed in vascular smooth muscle cells. Introduction of dominant-negative Kv7.4 and Kv7.5 subunits in mesenteric artery myocytes reduced endogenous Kv7 currents by 84 and 76%, respectively. Expression of an inducible protein kinase Cα (PKCα) translocation system revealed that PKCα activation is sufficient to suppress endogenous Kv7 currents in A7r5 rat aortic and mesenteric artery smooth muscle cells. Arginine vasopressin (100 and 500 pm) and the PKC activator phorbol 12-myristate 13-acetate (1 nm) each inhibited human (h) Kv7.5 and hKv7.4/7.5, but not hKv7.4 channels expressed in A7r5 cells. A decrease in hKv7.5 and hKv7.4/7.5 current densities was associated with an increase in PKC-dependent phosphorylation of the channel proteins. These findings provide further evidence for a differential regulation of Kv7.4 and Kv7.5 channel subunits by PKC-dependent phosphorylation and new mechanistic insights into the role of heteromeric subunit assembly for regulation of vascular Kv7 channels.     

Interaction of the scorpion toxin discrepin with Kv4.3 channels and A-type K channels in cerebellum granular cells

Background

The peptide discrepin from the α-KTx15 subfamily of scorpion toxins preferentially affects transient A-type potassium currents, which regulate many aspects of neuronal function in the central nervous system. However, the specific Kv channel targeted by discrepin and the molecular mechanism of interaction are still unknown.

Methods

Different variant peptides of discrepin were chemically synthesized and their effects were studied using patch clamp technique on rat cerebellum granular cells (CGC) and HEK cells transiently expressing Kv4.3 channels.

Results

Functional analysis indicated that nanomolar concentrations of native discrepin blocked Kv4.3 expressed channels, as previously observed in CGC. Similarly, the apparent affinities of all mutated peptides for Kv4.3 expressed channels were analogous to those found in CGC. In particular, in the double variant [V6K, D20K] the apparent affinity increased about 10-fold, whereas in variants carrying a deletion (ΔK13) or substitution (K13A) at position K13, the blockage was removed and the apparent affinity decreased more than 20-fold.

Conclusion

These results indicate that Kv4.3 is likely the target of discrepin and highlight the importance of the basic residue K13, located in the α-helix of the toxin, for current blockage.

General significance

We report the first example of a Kv4 subfamily potassium channel blocked by discrepin and identify the amino acid residues responsible for the blockage. The availability of discrepin variant peptides stimulates further research on the functions and pharmacology of neuronal Kv4 channels and on their possible roles in neurodegenerative disorders.     

Inactivation as a new regulatory mechanism for neuronal Kv7 channels

Voltage-gated K(+) channels of the Kv7 (KCNQ) family have important physiological functions in both excitable and nonexcitable tissue. The family encompasses five genes encoding the channel subunits Kv7.1-5. Kv7.1 is found in epithelial and cardiac tissue. Kv7.2-5 channels are predominantly neuronal channels and are important for controlling excitability. Kv7.1 channels have been considered the only Kv7 channels to undergo inactivation upon depolarization. However, here we demonstrate that inactivation is also an intrinsic property of Kv7.4 and Kv7.5 channels, which inactivate to a larger extent than Kv7.1 channels at all potentials. We demonstrate that at least 30% of these channels are inactivated at physiologically relevant potentials. The onset of inactivation is voltage dependent and occurs on the order of seconds. Both time- and voltage-dependent recovery from inactivation was investigated for Kv7.4 channels. A time constant of 1.47 +/- 0.21 s and a voltage constant of 54.9 +/- 3.4 mV were determined. It was further demonstrated that heteromeric Kv7.3/Kv7.4 channels had inactivation properties different from homomeric Kv7.4 channels. Finally, the Kv7 channel activator BMS-204352 was in contrast to retigabine found to abolish inactivation of Kv7.4. In conclusion, this work demonstrates that inactivation is a key regulatory mechanism of Kv7.4 and Kv7.5 channels.     

Allosteric modulators of human A2B adenosine receptor

Background

Among adenosine receptors (ARs) the A_2B subtype exhibits low affinity for the endogenous agonist compared with the A₁, A_2A, and A₃ subtypes and is therefore activated when concentrations of adenosine increase to a large extent following tissue damages (e.g. ischemia, inflammation). For this reason, A_2B AR represents an important pharmacological target.

Methods

We evaluated seven 1-benzyl-3-ketoindole derivatives (7–9) for their ability to act as positive or negative allosteric modulators of human A_2B AR through binding and functional assays using CHO cells expressing human A₁, A_2A, A_2B, and A₃ ARs.

Results

The investigated compounds behaved as specific positive or negative allosteric modulators of human A_2B AR depending on small differences in their structures. The positive allosteric modulators 7a,b and 8a increased agonist efficacy without any effect on agonist potency. The negative allosteric modulators 8b,c and 9a,b reduced agonist potency and efficacy.

Conclusions

A number of 1-benzyl-3-ketoindole derivatives were pharmacologically characterized as selective positive (7a,b) or negative (8c, 9a,b) allosteric modulators of human A_2B AR.

General significance

The 1-benzyl-3-ketoindole derivatives 7–9 acting as positive or negative allosteric modulators of human A_2B AR represent new pharmacological tools useful for the development of therapeutic agents to treat pathological conditions related to an altered functionality of A_2B AR.     

N-Linked glycan site occupancy impacts the distribution of a potassium channel in the cell body and outgrowths of neuronal-derived cells

Background

Vacancy of occupied N-glycosylation sites of glycoproteins is quite disruptive to a multicellular organism, as underlined by congenital disorders of glycosylation. Since a neuronal component is typically associated with this disease, we evaluated the impact of N-glycosylation processing of a neuronal voltage gated potassium channel, Kv3.1b, expressed in a neuronal-derived cell line, B35 neuroblastoma cells.

Methods

Total internal reflection fluorescence and differential interference contrast microscopy measurements of live B35 cells expressing wild type and glycosylation mutant Kv3.1b proteins were used to evaluate the distribution of the various forms of the Kv3.1b protein in the cell body and outgrowths. Cell adhesion assays were also employed.

Results

Microscopy images revealed that occupancy of both N-glycosylation sites of Kv3.1b had relatively similar amounts of Kv3.1b in the outgrowth and cell body while vacancy of one or both sites led to increased accumulation of Kv3.1b in the cell body. Further both the fully glycosylated and partially glycosylated N229Q Kv3.1b proteins formed higher density particles in outgrowths compared to cell body. Cellular assays demonstrated that the distinct spatial arrangements altered cell adhesion properties.

Conclusions

Our findings provide direct evidence that occupancy of the N-glycosylation sites of Kv3.1b contributes significantly to its lateral heterogeneity in membranes of neuronal-derived cells, and in turn alters cellular properties.

General significance

Our study demonstrates that N-glycans of Kv3.1b contain information regarding the association, clustering, and distribution of Kv3.1b in the cell membrane, and furthermore that decreased occupancy caused by congenital disorders of glycosylation may alter the biological activity of Kv3.1b.     

GATA-4 induces changes in electrophysiological properties of rat mesenchymal stem cells

Background

Transplanted mesenchymal stem cells (MSC) can differentiate into cardiac cells that have the potential to contribute to heart repair following ischemic injury. Overexpression of GATA-4 can significantly increase differentiation of MSC into cardiomyocytes (CM). However, the specific impact of GATA-4 overexpression on the electrophysiological properties of MSC-derived CM has not been well documented.

Methods

Adult rat bone marrow MSC were retrovirally transduced with GATA-4 (MSC^GATA-4) and GFP (MSC^Null) and subsequently co-cultured with neonatal rat ventricular cardiomyocytes (CM). Electrophysiological properties and mRNA levels of ion channels were assessed in MSC using patch-clamp technology and real-time PCR.

Results

MSC^GATA-4 exhibited higher levels of the TTX-sensitive Na⁺ current (I_Na.TTX), L-type calcium current (I_Ca.L), transient outward K⁺ current (I_to), delayed rectifier K⁺ current (IK_DR) and inwardly rectifying K⁺ current (I_K1) channel activities reflective of electrophysiological characteristics of CM. Real-time PCR analyses showed that MSC^GATA-4 exhibited upregulated mRNA levels of Kv1.2, Kv2.1, SCN2a1, CCHL2a, KV1.4 and Kir1.1 channels versus MSC^Null. Interestingly, MSC^GATA-4 treated with IGF-1 neutralizing antibodies resulted in a significant decrease in Kir1.1, Kv2.1, KV1.4, CCHL2a and SCN2a1 channel mRNA expression. Similarly, MSC^GATA-4 treated with VEGF neutralizing antibodies also resulted in an attenuated expression of Kv2.1, Kv1.2, Kv1.4, Kir1.1, CCHL2a and SCN2a1 channel mRNAs.

Conclusions

GATA-4 overexpression increases I_to, IK_DR, I_K1, I_Na.TTX and I_Ca.L currents in MSC. Cytokine (VGEF and IGF-1) release from GATA-4 overexpressing MSC can partially account for the upregulated ion channel mRNA expression.

General significance

Our results highlight the ability of GATA4 to boost the cardiac electrophysiological potential of MSC.     

Oral administration of D-aspartate,but not L-aspartate,depresses rectal temperature and alters plasma metabolites in chicks

Aims

L-Aspartate (L-Asp) and D-aspartate (D-Asp) are physiologically important amino acids in mammals and birds. However, the functions of these amino acids have not yet been fully understood. In this study, we therefore examined the effects of L-Asp and D-Asp in terms of regulating body temperature, plasma metabolites and catecholamines in chicks.

Main methods

Chicks were first orally administered with different doses (0, 3.75, 7.5 and 15 mmol/kg body weight) of L- or D-Asp to monitor the effects of these amino acids on rectal temperature during 120 min of the experimental period.

Key findings

Oral administration of D-Asp, but not of L-Asp, linearly decreased the rectal temperature in chicks. Importantly, orally administered D-Asp led to a significant reduction in body temperature in chicks even under high ambient temperature (HT) conditions. However, centrally administered D-Asp did not significantly influence the body temperature in chicks. As for plasma metabolites and catecholamines, orally administered D-Asp led to decreased triacylglycerol and uric acid concentrations and increased glucose and chlorine concentrations but did not alter plasma catecholamines.

Significance

These results suggest that oral administration of D-Asp may play a potent role in reducing body temperature under both normal and HT conditions. The alteration of plasma metabolites further indicates that D-Asp may contribute to the regulation of metabolic activity in chicks.     

Role of TRPM2 and TRPV1 cation channels in cellular responses to radiation-induced DNA damage

Background

Radiation exposure causes DNA damage, and DNA repair systems are essential to rescue damaged cells. Although DNA damage or oxidative stress activates transient receptor potential melastatin 2 (TRPM2) and vanilloid 1 (TRPV1) cation channels, it has not been established whether these TRP channels are involved in cellular responses to radiation-induced DNA damage. Here, we investigated the contribution of TRPM2 and TRPV1 channels to γ-irradiation- and UVB-induced DNA damage responses in human lung cancer A549 cells.

Methods

A549 cells were irradiated with γ-rays (2.0 Gy) or UVB (5–10 mJ/cm²). γH2AX foci, ATM activation, 53BP1 accumulation and EGFR expression were evaluated by immunofluorescence staining. Extracellular ATP concentration was measured by luciferin–luciferase assay. Knockdown of TRPM2 and TRPV1 expression was done by siRNA transfection.

Results

γ-Irradiation-induced γH2AX focus formation, ATM activation, 53BP1 accumulation and EGFR nuclear translocation, which are all associated with DNA repair, were suppressed by knockdown of TRPM2 and TRPV1 channels in A549 cells. Release of ATP, which mediates DNA damage response-associated activation of P2Y receptors, was suppressed by pre-treatment with catalase or knockdown of TRPM2 channel, but not TRPV1 channel. Similarly, UVB-induced γH2AX focus formation was suppressed in TRPM2- and TRPV1-knockdown cells, while UVB-induced ATP release was blocked in TRPM2- but not TRPV1-knockdown cells.

Conclusion

Our results suggest that the activation of TRPM2 channel, which mediates ATP release, and TRPV1 channel plays significant roles in the cellular responses to DNA damage induced by γ-irradiation and UVB irradiation.

General significance

Our results provide a new insight into the function of TRP channels from the viewpoint of radiation biology.     

Specific and Slow Inhibition of the Kir2.1 K+ Channel by Gambogic Acid

Although Kir2.1 channels are important in the heart and other excitable cells, there are virtually no specific drugs for this K⁺ channel. In search of Kir2.1 modulators, we screened a library of 720 naturally occurring compounds using a yeast strain in which mammalian Kir2.1 enables growth at low [K⁺]. One of the identified compounds, gambogic acid (GA), potently (EC₅₀ ≤ 100 nm) inhibited Kir2.1 channels in mammalian cells when applied chronically for 3 h. This potent and slow inhibition was not seen with Kv2.1, HERG or Kir1.1 channels. However, acutely applied GA acted as a weak (EC₅₀ = ∼10 μm) non-selective K⁺ channel blocker. Intracellular delivery of GA via a patch pipette did not potentiate the acute effect of GA on Kir2.1, showing that slow uptake is not responsible for the delayed, potent effect. Immunoblots showed that total Kir2.1 protein expression was not altered by GA. Similarly, immunostaining of intact cells expressing Kir2.1 with an extracellular epitope tag demonstrated that GA does not affect Kir2.1 surface expression. However, the 3-h treatment with GA caused redistribution of Kir2.1 and Kv2.1 from the Triton X-100-insoluble to the Triton X-100-soluble membrane fraction. Thus, GA changes the K⁺ channel membrane microenvironment resulting in potent, specific, and slow acting inhibition of Kir2.1 channels.K⁺ channels of the inwardly rectifying family (Kir)4 play key roles in the electric activity of many cell types. Kir2.1 channels are particularly important in the heart where they set the resting potential and contribute to the terminal phase of action potential repolarization. Mutations in the Kir2.1 gene cause Andersen syndrome, a triad of periodic paralysis, arrhythmia, and dysmorphic features (1), as well as short QT syndrome (2). Kir channel activity is regulated by endogenous magnesium and polyamines (3–5), as well as by membrane lipids including phosphoinositides (6, 7), fatty acid acyl-CoA esters (8), and cholesterol (9). Kir channels are blocked nonspecifically by extracellular cations such as cesium and barium (10), but there are no known Kir2.1-specific pharmacologic modulators that could be used in physiological studies or as drugs.High throughput screening (HTS) methods have been used for identifying novel K⁺ channel modulators in organic compound libraries (11–14). This approach has led to identification of inhibitors that target the K⁺ channel pore directly (11–13). Usually, such inhibitors act rapidly and reversibly. However, a few inhibitors inhibit Kir2.1 current with chronic application for hours. Thus far, such inhibitors have been found to interfere with Kir2.1 channel trafficking to the cell surface (14).Here an assay utilizing Kir2.1-dependent growth of yeast is used to identify gambogic acid (GA) as a Kir2.1 inhibitor. Functional studies in mammalian cells showed that GA acts acutely in the micromolar range as a nonselective K⁺ channel blocker. However, GA also acts slowly at nanomolar concentrations to abolish Kir2.1, but not Kv2.1, HERG, or Kir1.1 channel activity. The time course of this specific high affinity action does not reflect limited penetration into the cell or a change in Kir2.1 surface expression. Instead, GA causes marked partitioning of Kir2.1 into the Triton X-100-soluble membrane fraction, consistent with a redistribution of Kir2.1 into plasma membrane microdomains whereby its activity is silenced. Thus, GA has revealed a novel regulatory mechanism that specifically abolishes the activity of Kir2.1 inwardly rectifying channels.     

Rearrangements in the Relative Orientation of Cytoplasmic Domains Induced by a Membrane-anchored Protein Mediate Modulations in Kv Channel Gating

Interdomain interactions between intracellular N and C termini have been described for various K⁺ channels, including the voltage-gated Kv2.1, and suggested to affect channel gating. However, no channel regulatory protein directly affecting N/C interactions has been demonstrated. Most Kv2.1 channel interactions with regulatory factors occur at its C terminus. The vesicular SNARE that is also present at a high concentration in the neuronal plasma membrane, VAMP2, is the only protein documented to affect Kv2.1 gating by binding to its N terminus. As its binding target has been mapped near a site implicated in Kv2.1 N/C interactions, we hypothesized that VAMP2 binding to the N terminus requires concomitant conformational changes in the C terminus, which wraps around the N terminus from the outside, to give VAMP2 access. Here, we first determined that the Kv2.1 N terminus, although crucial, is not sufficient to convey functional interaction with VAMP2, and that, concomitant to its binding to the “docking loop” at the Kv2.1 N terminus, VAMP2 binds to the proximal part of the Kv2.1 C terminus, C1a. Next, using computational biology approaches (ab initio modeling, docking, and molecular dynamics simulations) supported by molecular biology, biochemical, electrophysiological, and fluorescence resonance energy transfer analyses, we mapped the interaction sites on both VAMP2 and Kv2.1 and found that this interaction is accompanied by rearrangements in the relative orientation of Kv2.1 cytoplasmic domains. We propose that VAMP2 modulates Kv2.1 inactivation by interfering with the interaction between the docking loop and C1a, a mechanism for gating regulation that may pertain also to other Kv channels.Interdomain interactions between intracellularly located N and C termini have been described for various K⁺ channels, including inwardly rectifying Kir2.3 and Kir6.2 (1, 2), small conductance Ca²⁺-activated (hSK3) (3), and voltage-gated Kv2.1 (4) and Kv4.1 (5) channels. In the case of Kv2.1, two modes of interaction have been proposed: an association of the distal part of Kv2.1 C terminus (termed CTA domain; amino acids (aa) 741–853)⁴ with aa 67 and 75 of the Kv2.1 N terminus (4); or an association between the proximal part of the Kv2.1 C terminus (aa 444–477) and the predicted loop structure (aa 55–71) in the N-terminal T1 domain (6). In addition, involvement of the S4-S5 linker in this interaction has been suggested (7). Although these studies propose two different C-terminal sites, they indicate a specific loop in the N terminus of Kv2.1 (6, 8), which could be functionally related to the Shaker and Shal docking loops in the lateral part of their T1 domains (9, 10). These latter loops are responsible for the subfamily-specific association with β-subunits (Kvβ and KChIP, respectively). Further, the interaction between the N and C cytoplasmic termini (N/C interaction) of Kv2.1 has been shown to be dynamic and voltage-dependent and to involve structural rearrangements between these domains, which could affect both activation and inactivation gating of the channel (4, 6, 7). These rearrangements can be clearly detected with fluorescence resonance energy transfer (FRET) (11). A similar N/C interaction has been shown to affect gating of the closely related Kv4.1 channel (5, 12).It is conceivable that the specific packaging of Kv2.1 cytoplasmic termini (a relatively long C terminus (>400 aa) wrapping the N terminus (<190 aa) from the outside (4)) not only supports multiple interactions between the termini but also reflects the fact that most of the interactions of the channel with intracellular and membrane-bound regulatory factors occur at the C terminus, including channel phosphorylation (13–15), clustering through a unique proxinal restriction and clustering signal (16), and protein-protein interactions with both the plasma membrane SNAREs, syntaxin 1A and SNAP-25 (17–19), and the MiRP2 (KCNE3) peptide (20). For the Kv2.1 N terminus, on the other hand, there are only two examples of protein-protein interactions: a transient association with KChAP (21), which does not affect channel function; and an interaction with the vesicular SNARE partner VAMP2 (synaptobrevin 2), which is also present at a high concentration in the neuronal plasma membrane and enhances channel inactivation (8). Specifically, VAMP2 has been shown to associate with the extension of a docking loop in the lateral part of the T1 domain (8) near the site of interaction with the C terminus (4, 6). Thus, it is reasonable to hypothesize that interaction with VAMP2 will affect the N/C interaction, similar to proton-mediated Kir2.3 (1) and Kir1.1 (22) N/C interactions or the ATP-dependent Kir6.2 (2) N/C interaction. To date, no protein molecule that directly affects N/C interactions in a K⁺ channel has been demonstrated. Because VAMP2 was the first protein documented to affect Kv2.1 channel gating by binding to a specific N-terminal site, which is probably masked by the C terminus, we have put forward the idea that its interaction with the Kv2.1 N terminus requires conformational changes in the C terminus that will enable its access to the N terminus.Here we endeavored to gain a mechanistic and structural understanding of the Kv2.1-VAMP2 interaction. Based on our evidence, we propose that VAMP2 modulates Kv2.1 gating by interfering with the Kv2.1 cytoplasmic N/C interaction.     

Syntheses and characterization of non-bisphosphonate quinoline derivatives as new FPPS inhibitors

Background

Farnesyl pyrophosphate synthase (FPPS) is a key regulatory enzyme in the biosynthesis of cholesterol and in the post-translational modification of signaling proteins. It has been reported that non-bisphosphonate FPPS inhibitors targeting its allosteric binding pocket are potentially important for the development of promising anti-cancer drugs.

Methods

The following methods were used: organic syntheses of non-bisphosphonate quinoline derivatives, enzyme inhibition studies, fluorescence titration assays, synergistic effect studies of quinoline derivatives with zoledronate, ITC studies for the binding of FPPS with quinoline derivatives, NMR-based HAP binding assays, molecular modeling studies, fluorescence imaging assay and MTT assays.

Results

We report our syntheses of a series of quinoline derivatives as new FPPS inhibitors possibly targeting the allosteric site of the enzyme. Compound 6b showed potent inhibition to FPPS without significant hydroxyapatite binding affinity. The compound showed synergistic inhibitory effect with active-site inhibitor zoledronate. ITC experiment confirmed the good binding effect of compound 6b to FPPS, and further indicated the binding ratio of 1:1. Molecular modeling studies showed that 6b could possibly bind to the allosteric binding pocket of the enzyme. The fluorescence microscopy indicated that these compounds could get into cancer cells.

Conclusions

Our results showed that quinoline derivative 6b could become a new lead compound for further optimization for cancer treatment.

General significance

The traditional FPPS active-site inhibitors bisphosphonates show poor membrane permeability to tumor cells, due to their strong polarity. The development of new non-bisphosphonate FPPS inhibitors with good cell membrane permeability is potentially important.     

The allosteric modulation of thyroxine-binding globulin affinity is entropy driven

Background

Thyroxine-binding globulin (TBG) is a non-inhibitory member of the serpin family of proteins whose main structural element is the reactive center loop (RCL), that, upon cleavage by proteases, is inserted into the protein core adopting a β-strand conformation (stressed to relaxed transition, S-to-R). After S-to-R transition thyroxine (T4) affinity decreases. However, crystallographic studies in the presence or absence of the hormone in different states are unable to show significant differences in the structure and interactions of the binding site. Experimental results also suggest the existence of several S states (differing in the number of inserted RCL residues), associated with a differential affinity.

Methods

To shed light into the molecular basis that regulates T4 affinity according to the degree of RCL insertion in TBG, we performed extended molecular dynamics simulations combined with several thermodynamic analysis of the T4 binding to TBG in three different S states, and in the R state.

Results

Our results show that, despite T4 binding in the protein by similar interactions in all states, a good correlation between the degree of RCL insertion and the binding affinity, driven by a change in TBG conformational entropy, was observed.

Conclusion

TBG allosteric regulation is entropy driven. The presence of multiple S states may allow more efficient T4 release due to protease activity.

General significance

The presented results are clear examples of how computer simulation methods can reveal the thermodynamic basis of allosteric effects, and provide a general framework for understanding serpin allosteric affinity regulation.     

The effects of methyl palmitate,a putative regulator from perivascular fat,on the contractility of pregnant human myometrium

Aims

Methyl palmitate is thought to cause relaxation in vascular smooth muscle by opening voltage-activated potassium channels. We have tested the hypothesis that methyl palmitate, a putative regulator from perivascular fat, is an inhibitor of the contractility of human pregnant myometrium and that its effects might partially explain the higher incidence of dysfunctional labor in obese women compared to those with normal body mass indices.

Main methods

Strips of myometrium obtained with informed consent from women undergoing elective cesarean section at term were mounted in organ baths. Strips stimulated with oxytocin (1 nM) or KCl (30 mM) were exposed to cumulatively increasing concentrations of methyl palmitate up to 10 μM. Similar strips were exposed to cumulative addition of the potassium channel blockers 4-aminopyridine and tetraethylammonium. The contractility of the strips was monitored and analyzed using conventional methods.

Key findings

Methyl palmitate failed to inhibit oxytocin- or KCl-induced contractions over the concentration range tested. In fact, it exerted a slight excitatory effect in the presence of KCl, though not in the presence of oxytocin. The contractility of naïve strips was unaltered by exposure to 1 μM methyl palmitate. Both 4-aminopyridine and tetraethylammonium produced concentration-dependent contractions of human pregnant myometrium providing pharmacological evidence for the presence of voltage-activated potassium channels in this preparation.

Significance

Our findings do not support the hypothesis that methyl palmitate is an inhibitor of human pregnant myometrial contractility. Alternate hypotheses must be pursued to explain the higher incidence of dysfunctional labor in obese women.     

Aquaporin and brain diseases

Background

The presence of water channel proteins, aquaporins (AQPs), in the brain led to intense research in understanding the underlying roles of each of them under normal conditions and pathological conditions.

Scope of review

In this review, we summarize some of the recent knowledge on the 3 main AQPs (AQP1, AQP4 and AQP9), with a special focus on AQP4, the most abundant AQP in the central nervous system.

Major conclusions

AQP4 was most studied in several brain pathological conditions ranging from acute brain injuries (stroke, traumatic brain injury) to the chronic brain disease with autoimmune neurodegenerative diseases. To date, no specific therapeutic agents have been developed to either inhibit or enhance water flux through these channels. However, experimental results strongly underline the importance of this topic for future investigation. Early inhibition of water channels may have positive effects in prevention of edema formation in brain injuries but at later time points during the course of a disease, AQP is critical for clearance of water from the brain into blood vessels.

General significance

Thus, AQPs, and in particular AQP4, have important roles both in the formation and resolution of edema after brain injury. The dual, complex function of these water channel proteins makes them an excellent therapeutic target. This article is part of a Special Issue entitled Aquaporins.     

Sialic acids attached to N- and O-glycans within the Nav1.4 D1S5–S6 linker contribute to channel gating

Background

Voltage-gated Na⁺ channels (Na_v) are responsible for the initiation and conduction of neuronal and muscle action potentials. Na_v gating can be altered by sialic acids attached to channel N-glycans, typically through isoform-specific electrostatic mechanisms.

Methods

Using two sets of Chinese Hamster Ovary cell lines with varying abilities to glycosylate glycoproteins, we show for the first time that sialic acids attached to O-glycans and N-glycans within the Na_v1.4 D1S5–S6 linker modulate Na_v gating.

Results

All measured steady-state and kinetic parameters were shifted to more depolarized potentials under conditions of essentially no sialylation. When sialylation of only N-glycans or of only O-glycans was prevented, the observed voltage-dependent parameter values were intermediate between those observed under full versus no sialylation. Immunoblot gel shift analyses support the biophysical data.

Conclusions

The data indicate that sialic acids attached to both N- and O-glycans residing within the Na_v1.4 D1S5-S6 linker modulate channel gating through electrostatic mechanisms, with the relative contribution of sialic acids attached to N- versus O-glycans on channel gating being similar.

General significance

Protein N- and O-glycosylation can modulate ion channel gating simultaneously. These data also suggest that environmental, metabolic, and/or congenital changes in glycosylation that impact sugar substrate levels, could lead, potentially, to changes in Na_v sialylation and gating that would modulate AP waveforms and conduction.     

Protein mechanics: How force regulates molecular function

Background

Regulation of proteins is ubiquitous and vital for any organism. Protein activity can be altered chemically, by covalent modifications or non-covalent binding of co-factors. Mechanical forces are emerging as an additional way of regulating proteins, by inducing a conformational change or by partial unfolding.

Scope

We review some advances in experimental and theoretical techniques to study protein allostery driven by mechanical forces, as opposed to the more conventional ligand driven allostery. In this respect, we discuss recent single molecule pulling experiments as they have substantially augmented our view on the protein allostery by mechanical signals in recent years. Finally, we present a computational analysis technique, Force Distribution Analysis, that we developed to reveal allosteric pathways in proteins.

Major conclusions

Any kind of external perturbation, being it ligand binding or mechanical stretching, can be viewed as an external force acting on the macromolecule, rendering force-based experimental or computational techniques, a very general approach to the mechanics involved in protein allostery.

General significance

This unifying view might aid to decipher how complex allosteric protein machineries are regulated on the single molecular level.     

5-Hydroxydecanoate and coenzyme A are inhibitors of native sarcolemmal KATP channels in inside-out patches

Background

5-Hydroxydecanoate (5-HD) inhibits preconditioning, and it is assumed to be a selective inhibitor of mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels. However, 5-HD is a substrate for mitochondrial outer membrane acyl-CoA synthetase, which catalyzes the reaction: 5?HD + CoA + ATP → 5-HD-CoA (5-hydroxydecanoyl-CoA) + AMP + pyrophosphate. We aimed to determine whether the reactants or principal product of this reaction modulate sarcolemmal K_ATP (sarcK_ATP) channel activity.

Methods

Single sarcK_ATP channel currents were measured in inside-out patches excised from rat ventricular myocytes. In addition, sarcK_ATP channel activity was recorded in whole-cell configuration or in giant inside-out patches excised from oocytes expressing Kir6.2/SUR2A.

Results

5-HD inhibited (IC₅₀ ∼ 30 μM) K_ATP channel activity, albeit only in the presence of (non-inhibitory) concentrations of ATP. Similarly, when the inhibitory effect of 0.2 mM ATP was reversed by 1 μM oleoyl-CoA, subsequent application of 5-HD blocked channel activity, but no effect was seen in the absence of ATP. Furthermore, we found that 1 μM coenzyme A (CoA) inhibited sarcK_ATP channels. Using giant inside-out patches, which are weakly sensitive to “contaminating” CoA, we found that Kir6.2/SUR2A channels were insensitive to 5-HD-CoA. In intact myocytes, 5-HD failed to reverse sarcK_ATP channel activation by either metabolic inhibition or rilmakalim.

General significance

SarcK_ATP channels are inhibited by 5-HD (provided that ATP is present) and CoA but insensitive to 5-HD-CoA. 5-HD is equally potent at “directly” inhibiting sarcK_ATP and mitoK_ATP channels. However, in intact cells, 5-HD fails to inhibit sarcK_ATP channels, suggesting that mitochondria are the preconditioning-relevant targets of 5-HD.