Block of Voltage-Gated Sodium Channels by Aripiprazole in a State-Dependent Manner.

Aripiprazole is an atypical antipsychotic drug, which is prescribed for many psychiatric diseases such as schizophrenia and mania in bipolar disorder. It primarily acts as an agonist of dopaminergic and other G-protein coupled receptors. So far, an interaction with ligand- or voltage-gated ion channels has been classified as weak. Meanwhile, we identified aripiprazole in a preliminary test as a potent blocker of voltage-gated sodium channels. Here, we present a detailed analysis about the interaction of aripiprazole with the dominant voltage-gated sodium channel of heart muscle (hNav1.5). Electrophysiological experiments were performed by means of the patch clamp technique at human heart muscle sodium channels (hNav1.5), heterologously expressed in human TsA cells. Aripiprazole inhibits the hNav1.5 channel in a state- but not use-dependent manner. The affinity for the resting state is weak with an extrapolated Kr of about 55 µM. By contrast, the interaction with the inactivated state is strong. The affinities for the fast and slow inactivated state are in the low micromolar range (0.5-1 µM). Kinetic studies indicate that block development for the inactivated state must be described with a fast (ms) and a slow (s) time constant. Even though the time constants differ by a factor of about 50, the resulting affinity constants were nearly identical (in the range of 0.5 µM). Besides this, aripirazole also interacts with the open state of the channel. Using an inactivation deficit mutant, an affinity of about 1 µM was estimated. In summary, aripiprazole inhibits voltage-gated sodium channels at low micromolar concentrations. This property might add to its possible anticancer and neuroprotective properties.

Block of Voltage-Gated Sodium Channels as a Potential Novel Anti-cancer Mechanism of TIC10.

Background: Tumor therapeutics are aimed to affect tumor cells selectively while sparing healthy ones. For this purpose, a huge variety of different drugs are in use. Recently, also blockers of voltage-gated sodium channels (VGSCs) have been recognized to possess potentially beneficial effects in tumor therapy. As these channels are a frequent target of numerous drugs, we hypothesized that currently used tumor therapeutics might have the potential to block VGSCs in addition to their classical anti-cancer activity. In the present work, we have analyzed the imipridone TIC10, which belongs to a novel class of anti-cancer compounds, for its potency to interact with VGSCs. Methods: Electrophysiological experiments were performed by means of the patch-clamp technique using heterologously expressed human heart muscle sodium channels (hNav1.5), which are among the most common subtypes of VGSCs occurring in tumor cells. Results: TIC10 angular inhibited the hNav1.5 channel in a state- but not use-dependent manner. The affinity for the resting state was weak with an extrapolated Kr of about 600 µM. TIC10 most probably did not interact with fast inactivation. In protocols for slow inactivation, a half-maximal inhibition occurred around 2 µM. This observation was confirmed by kinetic studies indicating that the interaction occurred with a slow time constant. Furthermore, TIC10 also interacted with the open channel with an affinity of approximately 4 µM. The binding site for local anesthetics or a closely related site is suggested as a possible target as the affinity for the well-characterized F1760K mutant was reduced more than 20-fold compared to wild type. Among the analyzed derivatives, ONC212 was similarly effective as TIC10 angular, while TIC10 linear more selectively interacted with the different states. Conclusion: The inhibition of VGSCs at low micromolar concentrations might add to the anti-tumor properties of TIC10.

Thermodynamic profile of mutual subunit control in a heteromeric receptor.

Cyclic nucleotide-gated (CNG) ion channels of olfactory neurons are tetrameric membrane receptors that are composed of two A2 subunits, one A4 subunit, and one B1b subunit. Each subunit carries a cyclic nucleotide-binding domain in the carboxyl terminus, and the channels are activated by the binding of cyclic nucleotides. The mechanism of cooperative channel activation is still elusive. Using a complete set of engineered concatenated olfactory CNG channels, with all combinations of disabled binding sites and fit analyses with systems of allosteric models, the thermodynamics of microscopic cooperativity for ligand binding was subunit- and state-specifically quantified. We show, for the closed channel, that preoccupation of each of the single subunits increases the affinity of each other subunit with a Gibbs free energy (ΔΔG) of ∼-3.5 to ∼-5.5 kJ ⋅ mol-1, depending on the subunit type, with the only exception that a preoccupied opposite A2 subunit has no effect on the other A2 subunit. Preoccupation of two neighbor subunits of a given subunit causes the maximum affinity increase with ΔΔG of ∼-9.6 to ∼-9.9 kJ ⋅ mol-1 Surprisingly, triple preoccupation leads to fewer negative ΔΔG values for a given subunit as compared to double preoccupation. Channel opening increases the affinity of all subunits. The equilibrium constants of closed-open isomerizations systematically increase with progressive liganding. This work demonstrates, on the example of the heterotetrameric olfactory CNG channel, a strategy to derive detailed insights into the specific mutual control of the individual subunits in a multisubunit membrane receptor.

Terahertz refractive index-based morphological dilation for breast carcinoma delineation.

This paper reports investigations led on the combination of the refractive index and morphological dilation to enhance performances towards breast tumour margin delineation during conserving surgeries. The refractive index map of invasive ductal and lobular carcinomas were constructed from an inverse electromagnetic problem. Morphological dilation combined with refractive index thresholding was conducted to classify the tissue regions as malignant or benign. A histology routine was conducted to evaluate the performances of various dilation geometries associated with different thresholds. It was found that the combination of a wide structuring element and high refractive index was improving the correctness of tissue classification in comparison to other configurations or without dilation. The method reports a sensitivity of around 80% and a specificity of 82% for the best case. These results indicate that combining the fundamental optical properties of tissues denoted by their refractive index with morphological dilation may open routes to define supporting procedures during breast-conserving surgeries.

Pilot study of freshly excised breast tissue response in the 300-600 GHz range.

The failure to accurately define tumor margins during breast conserving surgery (BCS) results in a 20% re-excision rate. The present paper reports the investigation to evaluate the potential of terahertz imaging for breast tissue recognition within the under-explored 300 - 600 GHz range. Such a frequency window matches new BiCMOS technology capabilities and thus opens up the opportunity for near-field terahertz imaging using these devices. To assess the efficacy of this frequency band, data from 16 freshly excised breast tissue samples were collected and analyzed directly after excision. Complex refractive indices have been extracted over the as-mentioned frequency band, and amplitude frequency images show some contrast between tissue types. Principal component analysis (PCA) has also been applied to the data in an attempt to automate tissue classification. Our observations suggest that the dielectric response could potentially provide contrast for breast tissue recognition within the 300 - 600 GHz range. These results open the way for silicon-based terahertz subwavelength near field imager design, efficient up to 600 GHz to address ex vivo life-science applications.

Deciphering the function of the CNGB1b subunit in olfactory CNG channels.

Olfactory cyclic nucleotide-gated (CNG) ion channels are key players in the signal transduction cascade of olfactory sensory neurons. The second messengers cAMP and cGMP directly activate these channels, generating a depolarizing receptor potential. Olfactory CNG channels are composed of two CNGA2 subunits and two modulatory subunits, CNGA4, and CNGB1b. So far the exact role of the modulatory subunits for channel activation is not fully understood. By measuring ligand binding and channel activation simultaneously, we show that in functional heterotetrameric channels not only the CNGA2 subunits and the CNGA4 subunit but also the CNGB1b subunit binds cyclic nucleotides and, moreover, also alone translates this signal to open the pore. In addition, we show that the CNGB1b subunit is the most sensitive subunit in a heterotetrameric channel to cyclic nucleotides and that it accelerates deactivation to a similar extent as does the CNGA4 subunit. In conclusion, the CNGB1b subunit participates in ligand-gated activation of olfactory CNG channels and, particularly, contributes to rapid termination of odorant signal in an olfactory sensory neuron.

Protein arginine methyl transferases-3 and -5 increase cell surface expression of cardiac sodium channel.

The α-subunit of the cardiac voltage-gated sodium channel (NaV1.5) plays a central role in cardiomyocyte excitability. We have recently reported that NaV1.5 is post-translationally modified by arginine methylation. Here, we aimed to identify the enzymes that methylate NaV1.5, and to describe the role of arginine methylation on NaV1.5 function. Our results show that protein arginine methyl transferase (PRMT)-3 and -5 methylate NaV1.5 in vitro, interact with NaV1.5 in human embryonic kidney (HEK) cells, and increase NaV1.5 current density by enhancing NaV1.5 cell surface expression. Our observations are the first evidence of regulation of a voltage-gated ion channel, including calcium, potassium, sodium and TRP channels, by arginine methylation.

Differential regulation by cyclic nucleotides of the CNGA4 and CNGB1b subunits in olfactory cyclic nucleotide-gated channels.

Olfactory cyclic nucleotide-gated (CNG) ion channels are essential contributors to signal transduction of olfactory sensory neurons. The activity of the channels is controlled by the cyclic nucleotides guanosine 3',5'-monophosphate (cGMP) and adenosine 3',5'-monophosphate (cAMP). The olfactory CNG channels are composed of two CNGA2 subunits, one CNGA4 and one CNGB1b subunit, each containing a cyclic nucleotide-binding domain. Using patch-clamp fluorometry, we measured ligand binding and channel activation simultaneously and showed that cGMP activated olfactory CNG channels not only by binding to the two CNGA2 subunits but also by binding to the CNGA4 subunit. In a channel in which the CNGA2 subunits were compromised for ligand binding, cGMP binding to CNGA4 was sufficient to partly activate the channel. In contrast, in heterotetrameric channels, the CNGB1b subunit did not bind cGMP, but channels with this subunit showed activation by cAMP. Thus, the modulatory subunits participate actively in translating ligand binding to activation of heterotetrameric olfactory CNG channels and enable the channels to differentiate between cyclic nucleotides.

How subunits cooperate in cAMP-induced activation of homotetrameric HCN2 channels.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric membrane proteins that generate electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are primarily activated by voltage but are receptors as well, binding the intracellular ligand cyclic AMP. The molecular mechanism of channel activation is still unknown. Here we analyze the complex activation mechanism of homotetrameric HCN2 channels by confocal patch-clamp fluorometry and kinetically quantify all ligand binding steps and closed-open isomerizations of the intermediate states. For the binding affinity of the second, third and fourth ligand, our results suggest pronounced cooperativity in the sequence positive, negative and positive, respectively. This complex interaction of the subunits leads to a preferential stabilization of states with zero, two or four ligands and suggests a dimeric organization of the activation process: within the dimers the cooperativity is positive, whereas it is negative between the dimers.

Alternative splicing of the cardiac sodium channel creates multiple variants of mutant T1620K channels.

Alternative splicing creates several Na(v)1.5 transcripts in the mammalian myocardium and in various other tissues including brain, dorsal root ganglia, breast cancer cells as well as neuronal stem cell lines. In total nine Na(v)1.5 splice variants have been discovered. Four of them, namely Na(v)1.5a, Na(v)1.5c, Na(v)1.5d, and Na(v)1.5e, generate functional channels in heterologous expression systems. The significance of alternatively spliced transcripts for cardiac excitation, in particular their role in SCN5A channelopathies, is less well understood. In the present study, we systematically investigated electrophysiological properties of mutant T1620K channels in the background of all known functional Na(v)1.5 splice variants in HEK293 cells. This mutation has been previously associated with two distinct cardiac excitation disorders: with long QT syndrome type 3 (LQT3) and isolated cardiac conduction disease (CCD). When investigating the effect of the T1620K mutation, we noticed similar channel defects in the background of hNa(v)1.5, hNa(v)1.5a, and hNa(v)1.5c. In contrast, the hNa(v)1.5d background produced differential effects: In the mutant channel, some gain-of-function features did not emerge, whereas loss-of-function became more pronounced. In case of hNa(v)1.5e, the neonatal variant of hNa(v)1.5, both the splice variant itself as well as the corresponding mutant channel showed electrophysiological properties that were distinct from the wild-type and mutant reference channels, hNa(v)1.5 and T1620K, respectively. In conclusion, our data show that alternative splicing is a mechanism capable of generating a variety of functionally distinct wild-type and mutant hNa(v)1.5 channels. Thus, the cellular splicing machinery is a potential player affecting genotype-phenotype correlations in SCN5A channelopathies.

Interdependence of receptor activation and ligand binding in HCN2 pacemaker channels.

HCN pacemaker channels are tetramers mediating rhythmicity in neuronal and cardiac cells. The activity of these channels is controlled by both membrane voltage and the ligand cAMP, binding to each of the four channel subunits. The molecular mechanism underlying channel activation and the relationship between the two activation stimuli are still unknown. Using patch-clamp fluorometry and a fluorescent cAMP analog, we show that full ligand-induced activation appears already with only two ligands bound to the tetrameric channel. Kinetic analysis of channel activation and ligand binding suggests direct interaction between the voltage sensor and the cyclic nucleotide-binding domain, bypassing the pore. By exploiting the duality of activation in HCN2 channels by voltage and ligand binding, we quantify the increase of the binding affinity and overall free energy for binding upon channel activation, proving thus the principle of reciprocity between ligand binding and conformational change in a receptor protein.

Structure and function of splice variants of the cardiac voltage-gated sodium channel Na(v)1.5.

Voltage-gated sodium channels mediate the rapid upstroke of the action potential in excitable tissues. The tetrodotoxin (TTX) resistant isoform Na(v)1.5, encoded by the SCN5A gene, is the predominant isoform in the heart. This channel plays a key role for excitability of atrial and ventricular cardiomyocytes and for rapid impulse propagation through the specific conduction system. During recent years, strong evidence has been accumulated in support of the expression of several Na(v)1.5 splice variants in the heart, and in various other tissues and cell lines including brain, dorsal root ganglia, breast cancer cells and neuronal stem cell lines. This review summarizes our knowledge on the structure and putative function of nine Na(v)1.5 splice variants detected so far. Attention will be paid to the distinct biophysical properties of the four functional splice variants, to the pronounced tissue- and species-specific expression, and to the developmental regulation of Na(v)1.5 splicing. The implications of alternative splicing for SCN5A channelopathies, and for a better understanding of genotype-phenotype correlations, are discussed.

Region-specific alterations of global protein expression in the remodelled rat myocardium.

We applied the novel ProteinChip technology (SELDI-MS) to investigate and identify differentially regulated proteins upon myocardial remodelling in different heart regions. Tissue samples were isolated from the atria, the interventricular septum, and the right and left ventricles three months after surgically-induced myocardial infarction (MI) in rats. Corresponding protein extracts from control versus MI hearts were analysed on two different ProteinChip surfaces. In each of the functionally distinct cardiac regions, we obtained specific protein profile alterations upon myocardial remodelling. Most alterations were observed in the non-infarcted right ventricle, where down-regulation occurred more frequently than up-regulation of protein expression. Three of the differentially regulated proteins were identified: the metabolic enzyme triosephosphate isomerase (TIM), the cell signalling protein Raf-1 kinase inhibitory protein (RKIP), also known as phosphatidylethanolamine binding protein (PEBP), and the small heat shock protein alphaB-crystallin. These proteins showed a pronounced tissue-dependent regulation. TIM was down-regulated only in the atrium and in the left ventricle, RKIP/PEBP was down-regulated only in the right ventricle and in the interventricular septum, and alphaB-crystallin was up-regulated only in the right and in the left ventricle. A simple correlation of peak intensity changes using two of the identified peaks demonstrated the diagnostic potential of SELDI-MS. We conclude that this novel proteomic method is a powerful high-throughput tool for the fast detection of region-specific cardiac protein profiles in small biopsy samples, and that SELDI-MS may become a useful complementary technique for the diagnosis and prognosis of cardiac diseases.

Na(+) current through KATP channels: consequences for Na(+) and K(+) fluxes during early myocardial ischemia.

During early myocardial ischemia, the myocytes are loaded with Na(+), which in turn leads to Ca(2+) overload and cell death. The pathway of the Na(+) influx has not been fully elucidated. The aim of the study was to quantify the Na(+) inward current through sarcolemmal KATP channels (IKATP,Na) in anoxic isolated cardiomyocytes at the actual reversal potential (Vrev) and to estimate the contribution of this current to the Na(+) influx in the ischemic myocardium. IKATP,Na was determined in excised single channel patches of mouse ventricular myocytes and macropatches of Xenopus laevis oocytes expressing SUR2A/Kir6.2 channels. In the presence of K+ ions, the respective permeability ratios for Na(+) to K(+) ions, PNa/PK, were close to 0.01. Only in the presence of Na(+) ions on both sides of the membrane was IKATP,Na similarly large to that calculated from the permeability ratio PNa/PK, indicative of a Na(+) influx that is largely independent of the K+ efflux at Vrev. With the use of a peak KATP channel conductance in anoxic cardiomyocytes of 410 nS, model simulations for a myocyte within the ischemic myocardium showed that the amplitude of the Na(+) influx and K(+) efflux is even larger than the respective fluxes by the Na(+) - K(+) pump and all other background fluxes. These results suggest that during early ischemia the Na(+) influx through KATP channels essentially contributes to the total Na+ influx and that it also balances the K(+) efflux through KATP channels.