Small Molecules as Modulators of Voltage-Gated Calcium Channels in Neurological Disorders: State of the Art and Perspectives.

Voltage-gated calcium channels (VGCCs) are widely expressed in the brain, heart and vessels, smooth and skeletal muscle, as well as in endocrine cells. VGCCs mediate gene transcription, synaptic and neuronal structural plasticity, muscle contraction, the release of hormones and neurotransmitters, and membrane excitability. Therefore, it is not surprising that VGCC dysfunction results in severe pathologies, such as cardiovascular conditions, neurological and psychiatric disorders, altered glycemic levels, and abnormal smooth muscle tone. The latest research findings and clinical evidence increasingly show the critical role played by VGCCs in autism spectrum disorders, Parkinson's disease, drug addiction, pain, and epilepsy. These findings outline the importance of developing selective calcium channel inhibitors and modulators to treat such prevailing conditions of the central nervous system. Several small molecules inhibiting calcium channels are currently used in clinical practice to successfully treat pain and cardiovascular conditions. However, the limited palette of molecules available and the emerging extent of VGCC pathophysiology require the development of additional drugs targeting these channels. Here, we provide an overview of the role of calcium channels in neurological disorders and discuss possible strategies to generate novel therapeutics.

Structural basis for Ca2+ activation of the heteromeric PKD1L3/PKD2L1 channel.

The heteromeric complex between PKD1L3, a member of the polycystic kidney disease (PKD) protein family, and PKD2L1, also known as TRPP2 or TRPP3, has been a prototype for mechanistic characterization of heterotetrametric TRP-like channels. Here we show that a truncated PKD1L3/PKD2L1 complex with the C-terminal TRP-fold fragment of PKD1L3 retains both Ca2+ and acid-induced channel activities. Cryo-EM structures of this core heterocomplex with or without supplemented Ca2+ were determined at resolutions of 3.1 Å and 3.4 Å, respectively. The heterotetramer, with a pseudo-symmetric TRP architecture of 1:3 stoichiometry, has an asymmetric selectivity filter (SF) guarded by Lys2069 from PKD1L3 and Asp523 from the three PKD2L1 subunits. Ca2+-entrance to the SF vestibule is accompanied by a swing motion of Lys2069 on PKD1L3. The S6 of PKD1L3 is pushed inward by the S4-S5 linker of the nearby PKD2L1 (PKD2L1-III), resulting in an elongated intracellular gate which seals the pore domain. Comparison of the apo and Ca2+-loaded complexes unveils an unprecedented Ca2+ binding site in the extracellular cleft of the voltage-sensing domain (VSD) of PKD2L1-III, but not the other three VSDs. Structure-guided mutagenic studies support this unconventional site to be responsible for Ca2+-induced channel activation through an allosteric mechanism.

Effects of mitochondria-associated Ca2+ transporters suppression on oocyte activation.

Oocyte activation deficiency leads to female infertility. [Ca2+ ]i oscillations are required for mitochondrial energy supplement transition from the resting to the excited state, but the underlying mechanisms are still very little known. Three mitochondrial Ca2+ channels, Mitochondria Calcium Uniporter (MCU), Na+ /Ca2+ Exchanger (NCLX) and Voltage-dependent Ca2+ Channel (VDAC), were deactivated by inhibitors RU360, CGP37157 and Erastin, respectively. Both Erastin and CGP37157 inhibited mitochondrial activity significantly while attenuating [Ca2+ ]i and [Ca2+ ]m oscillations, which caused developmental block of pronuclear formation. Thus, NCLX and VDAC are two mitochondria-associated Ca2+ transporter proteins regulating oocyte activation, which may be used as potential targets to treat female infertility. SIGNIFICANCE OF THE STUDY: NCLX and VDAC are two mitochondria-associated Ca2+ transporter proteins regulating oocyte activation.

Subnanometer-resolution structure determination in situ by hybrid subtomogram averaging - single particle cryo-EM.

Cryo-electron tomography combined with subtomogram averaging (StA) has yielded high-resolution structures of macromolecules in their native context. However, high-resolution StA is not commonplace due to beam-induced sample drift, images with poor signal-to-noise ratios (SNR), challenges in CTF correction, and limited particle number. Here we address these issues by collecting tilt series with a higher electron dose at the zero-degree tilt. Particles of interest are then located within reconstructed tomograms, processed by conventional StA, and then re-extracted from the high-dose images in 2D. Single particle analysis tools are then applied to refine the 2D particle alignment and generate a reconstruction. Use of our hybrid StA (hStA) workflow improved the resolution for tobacco mosaic virus from 7.2 to 4.4 Å and for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. These resolution gains make hStA a promising approach for other StA projects aimed at achieving subnanometer resolution.

Ex vivo and in vivo studies of Viola tricolor Linn. as potential cardio protective and hypotensive agent: Inhibition of voltage-gated Ca++ ion channels.

Viola tricolor Linn. is used as cardio-protective and anti-hypertensive agent in traditional medicine. Current study objective was to evaluate cardio-protective and hypotensive effects of Viola tricolor L. in vitro and in vivo studies. Viola tricolor L. crude extract (Vt.Cr) and its fractions (Aqueous and organic) were tested at rabbit atria and aorta coupled to Power Lab Data Acquisition System for cardio depressant and vasorelaxant effects in vitro whereas in vivo Blood Pressure was checked by invasive method in normotensive ketamine-diazepam anesthetized rats. Isoproterenol was employed for acute myocardial infarction (AMI) and left ventricular hypertrophy (LVH) development and cardioprotective effects of Vt.Cr were evaluated hemodynamically and histopathologically. Vt.Cr and its fractions decreased heart rate and contractile force in paired atria and relaxed Phenylephrine (1 µM) and K+ (80 mM) stimulated contractions in aorta possibly mediated through Voltage dependent L-type calcium channels blockage supported by in vivo hypotensive action. In LVH, Vt.Cr lowered Angiotensin Converting Enzymes and renin, increased cyclic Guanosine Monophosphate and nitric oxide levels, decreased cardiomyocytes size and fibrosis attributed to Gallic acid as detected by High Performance Liquid Chromatography. Partial positive results were seen hemodynamically and histologically in AMI Viola tricolor L. showed vasorelaxant, cardio-relaxant, hypotensive, and cardio protective effect validating traditional practice in cardiovascular disorders.

Influence of Echeveria gibbiflora DC aqueous crude extract on mouse sperm energy metabolism and calcium-dependent channels.

ETHNOPHARMACOLOGY RELEVANCE: In traditional Mexican medicine, Echeveria gibbiflora DC has been used as a vaginal post-coital rinse to prevent pregnancy. The aqueous crude extract (OBACE) induces sperm immobilization/agglutination and a hypotonic-like effect, likely attributed to the high concentration of calcium bis-(hydrogen-1-malate) hexahydrate [Ca2+ (C4H5O5)2•6H2O]. Likewise, OBACE impedes the increase of [Ca2+]i during capacitation. AIM OF THE STUDY: Evaluate the effect of OBACE on sperm energy metabolism and the underlying mechanism of action on sperm-specific channel. MATERIAL AND METHODS: In vitro, we quantified the mouse sperm immobilization effect and the antifertility potential of OBACE. The energetic metabolism status was also evaluated by assessing the ATP levels, general mitochondrial activity, mitochondrial membrane potential, and enzymatic activity of three key enzymes of energy metabolism. Furthermore, the effect of the ion efflux of Cl- and K+, as well as the pHi, were investigated in order to elucidate which channel is suitable to perform an in silico study. RESULTS: Total and progressive motility notably decreased, as did fertility rates. ATP levels, mitochondrial activity and membrane potential were reduced. Furthermore, the activities of the three enzymes decreased. Neither Cl- or K+ channels activities were affected at low concentrations of OBACE; nevertheless, pHi did not alkalinize. Finally, an in silico analysis was performed between the Catsper channel and calcium bis-(hydrogen-1-malate) hexahydrate, which showed a possible blockade of this sperm cation channel. CONCLUSION: The results were useful to elucidate the effect of OBACE and to propose it as a future male contraceptive.

Multi-target 1,4-dihydropyridines showing calcium channel blockade and antioxidant capacity for Alzheimer's disease therapy.

In this work we describe the synthesis, Ca+2 channel blockade capacity and antioxidant power of N3,N5-bis(2-(5-methoxy-1H-indol-3-yl)ethyl)-2,6-dimethyl-4-aryl-1,4-dihydropyridine-3,5-dicarboxamides 1-9, a number of multi-target small 1,4-dihydropyridines (DHP), designed by juxtaposition of melatonin and nimodipine. As a result, we have identified antioxidant DHP 7 (Ca2+ channel blockade: 55%, and 8.78 Trolox/Equivalents), the most balanced DHP analyzed here, for potential Alzheimer's disease therapy.

(-)-Epicatechin-induced relaxation of isolated human saphenous vein: Roles of K+ and Ca2+ channels.

In this study, we aimed to investigate relaxant effect of flavanol (-)-epicatechin on the isolated human saphenous vein (HSV), as a part of its cardioprotective action, and to define the mechanisms underlying this vasorelaxation. (-)-Epicatechin induced a concentration-dependent relaxation of HSV pre-contracted by phenylephrine. Among K+ channel blockers, 4-aminopyridine, margatoxin, and iberiotoxin significantly inhibited relaxation of HSV, while glibenclamide considerably reduced effects of the high concentrations of (-)-epicatechin. Additionally, (-)-epicatechin relaxed contraction induced by 80 mM K+ , whereas in the presence of nifedipine produced partial relaxation of HSV rings pre-contracted by phenylephrine. In Ca2+ -free solution, (-)-epicatechin relaxed contraction induced by phenylephrine, but had no effect on contraction induced by caffeine. A sarcoplasmic reticulum Ca2+ -ATPase inhibitor, thapsigargin, significantly reduced relaxation of HSV produced by (-)-epicatechin. These results demonstrate that (-)-epicatechin produces endothelium-independent relaxation of isolated HSV rings. Vasorelaxation to (-)-epicatechin probably involves activation of 4-aminopyridine- and margatoxin-sensitive KV channels, BKCa channels, and at least partly, KATP channels. In addition, not only the inhibition of extracellular Ca2+ influx, but regulation of the intracellular Ca2+ release, via inositol-trisphosphate receptors and reuptake into sarcoplasmic reticulum, via stimulation of Ca2+ -ATPase, as well, most likely participate in (-)-epicatechin-induced relaxation of HSV.

Selenoprotein W redox-regulated Ca2+ channels correlate with selenium deficiency-induced muscles Ca2+ leak.

Selenium (Se) deficiency induces Ca2+ leak and calcification in mammal skeletal muscles; however, the exact mechanism is still unclear. In the present study, both Se-deficient chicken muscle models and selenoprotein W (SelW) gene knockdown myoblast and embryo models were used to study the mechanism. The results showed that Se deficiency-induced typical muscular injuries accompanied with Ca2+ leak and oxidative stress (P < 0.05) injured the ultrastructure of the sarcoplasmic reticulum (SR) and mitochondria; decreased the levels of the Ca2+ channels, SERCA, SLC8A, CACNA1S, ORAI1, STIM1, TRPC1, and TRPC3 (P < 0.05); and increased the levels of Ca2+ channel PMCA (P < 0.05). Similarly, SelW knockdown also induced Ca2+ leak from the SR and cytoplasm; increased mitochondrial Ca2+ levels and oxidative stress; injured SR and mitochondrial ultrastructure; decreased levels of SLC8A, CACNA1S, ORA1, TRPC1, and TRPC3; and caused abnormal activities of Ca2+ channels in response to inhibitors in myoblasts and chicken embryos. Thus, both Se deficiency and SelW knockdown induced Ca2+ leak, oxidative stress, and Ca2+ channel reduction. In addition, Ca2+ levels and the expression of the Ca2+ channels, RyR1, SERCA, CACNA1S, TRPC1, and TRPC3 were recovered to normal levels by N-acetyl-L-cysteine (NAC) treatment compared with SelW knockdown cells. Thus, with regard to the decreased Ca2+ channels, SelW knockdown closely correlated Se deficiency with Ca2+ leak in muscles. The redox regulation role of SelW is crucial in Se deficiency-induced Ca2+ leak in muscles.

Advanced cell therapies: targeting, tracking and actuation of cells with magnetic particles.

Regenerative medicine would greatly benefit from a new platform technology that enabled measurable, controllable and targeting of stem cells to a site of disease or injury in the body. Superparamagnetic iron-oxide nanoparticles offer attractive possibilities in biomedicine and can be incorporated into cells, affording a safe and reliable means of tagging. This review describes three current and emerging methods to enhance regenerative medicine using magnetic particles to guide therapeutic cells to a target organ; track the cells using MRI and assess their spatial localization with high precision and influence the behavior of the cell using magnetic actuation. This approach is complementary to the systemic injection of cell therapies, thus expanding the horizon of stem cell therapeutics.

Pharmacological justification of use of Solena heterophylla Lour. in gastrointestinal, respiratory and vascular disorders.

BACKGROUND: Solena heterophylla Lour. has traditionally been used in the management of diseases pertaining to gastrointestinal, respiratory and vascular system and present study was undertaken to validate its traditional uses. METHODS: The aqueous ethanolic extract of Solena heterophylla Lour (Sh.Cr) was tested in-vitro on isolated rabbit jejunum, tracheal and aorta preparations. The responses of tissues were recorded using isotonic transducers coupled with PowerLab data acquisition system. RESULTS: The aqueous ethanolic extract of Solena heterophylla Lour (Sh.Cr) (0.03-1.0 mg/ml) on application to spontaneous contractions in isolated rabbit jejunum preparation exerted relaxant effect through decrease in magnitude and frequency of contractions, caused relaxation of K(+)(80 mM)-induced contractions and shifted the Ca(2+) concentration response curves toward right in isolated rabbit jejunum preparations in a manner similar to verapamil (a standard Ca(2+) channel blocker), thus confirming its Ca(2+) channel blocking activity. The Sh.Cr also caused relaxation of carbachol (1 µM)- and K(+)(80 mM)-induced contractions in isolated rabbit tracheal preparations in a manner comparable to dicyclomine. CONCLUSIONS: The observed relaxant effect may be outcome of anti-muscarinic and Ca(2+) channel blocking activities. The Sh.Cr (0.03-1.0 mg/ml) against phenyephrine (1 µM)- and K(+)(80 mM)-induced contractions in isolated rabbit aortic preparations exerted a relaxant effect, possibly through Ca(2+) channel blocking activity. These findings provide a rationale for the folkloric uses of the plant in the management of ailments pertaining to gastrointestinal, respiratory and vascular system.

Discovery of 7-azaindole derivatives as potent Orai inhibitors showing efficacy in a preclinical model of asthma.

Synthesis and SAR of a series of 7-azaindoles as Orai channel inhibitors showing good potency inhibiting IL-2 production in Jurkat cells is described. Compound 14d displaying best pharmacokinetic properties was further characterized in a model of allergen induced asthma showing inhibition in the number of eosinophils in BALF. High lipophilicity remains as one of the main challenges for this class of compounds.

The extended transmembrane Orai1 N-terminal (ETON) region combines binding interface and gate for Orai1 activation by STIM1.

STIM1 and Orai1 represent the two molecular key components of the Ca(2+) release-activated Ca(2+) channels. Their activation involves STIM1 C terminus coupling to both the N terminus and the C terminus of Orai. Here we focused on the extended transmembrane Orai1 N-terminal (ETON, aa73-90) region, conserved among the Orai family forming an elongated helix of TM1 as recently shown by x-ray crystallography. To identify "hot spot" residues in the ETON binding interface for STIM1 interaction, numerous Orai1 constructs with N-terminal truncations or point mutations within the ETON region were generated. N-terminal truncations of the first four residues of the ETON region or beyond completely abolished STIM1-dependent Orai1 function. Loss of Orai1 function resulted from neither an impairment of plasma membrane targeting nor pore damage, but from a disruption of STIM1 interaction. In a complementary approach, we monitored STIM1-Orai interaction via Orai1 V102A by determining restored Ca(2+) selectivity as a consequence of STIM1 coupling. Orai1 N-terminal truncations that led to a loss of function consistently failed to restore Ca(2+) selectivity of Orai1 V102A in the presence of STIM1, demonstrating impairment of STIM1 binding. Hence, the major portion of the ETON region (aa76-90) is essential for STIM1 binding and Orai1 activation. Mutagenesis within the ETON region revealed several hydrophobic and basic hot spot residues that appear to control STIM1 coupling to Orai1 in a concerted manner. Moreover, we identified two basic residues, which protrude into the elongated pore to redound to Orai1 gating. We suggest that several hot spot residues in the ETON region contribute in aggregate to the binding of STIM1, which in turn is coupled to a conformational reorientation of the gate.

The calcium channel blocking and phosphodiesterase inhibitory activities of the extract of Andropogon muricatus explains its medicinal use in airways disorders.

The present work was carried out to provide a pharmacological base for the medicinal use of Andropogon muricatus in airways disorders, such as asthma. In isolated guinea-pig tracheal strips, the crude extract of Andropogon muricatus exhibited a non-specific relaxant effect against carbachol (1 µM) and high K⁺ precontractions, with EC50 values of 0.10 (0.07-0.11) and 0.15 mg/mL (0.11-0.18), respectively, similar to papaverine, while verapamil was more potent against high K⁺. This suggests the involvement of a non-specific relaxant effect, mediated possibly through Ca⁺⁺ channel blockade and phosphodiesterase inhibition. The functional nature of the relaxant effect was further confirmed through indirect evidence when pretreatment of the tissues with the plant extract caused potentiation of the isoprenaline inhibitory response curves, similar to papaverine, while the effect of verapamil remained unchanged. These data indicate that the crude extract of Andropogon muricatus contains constituent(s) that mediate the tracheal relaxant effect, possibly through dual inhibition of Ca⁺⁺ channels and phosphodiesterase and provide pharmacological evidence for its medicinal use in airways disorders, particularly asthma.

Mutant TRPV4-mediated toxicity is linked to increased constitutive function in axonal neuropathies.

Mutations in TRPV4 have been linked to three distinct axonal neuropathies. However, the pathogenic mechanism underlying these disorders remains unclear. Both gain and loss of calcium channel activity of the mutant TRPV4 have been suggested. Here, we show that the three previously reported TRPV4 mutant channels have a physiological localization and display an increased calcium channel activity, leading to increased cytotoxicity in three different cell types. Patch clamp experiments showed that cells expressing mutant TRPV4 have much larger whole-cell currents than those expressing the wild-type TRPV4 channel. Single channel recordings showed that the mutant channels have higher open probability, due to a modification of gating, and no change in single-channel conductance. These data support the hypothesis that a "gain of function" mechanism, possibly leading to increased intracellular calcium influx, underlies the pathogenesis of the TRPV4-linked axonal neuropathies, and may have immediate implications for designing rational therapies.

Selectivity sequences in a model calcium channel: role of electrostatic field strength.

The energetics that give rise to selectivity sequences of ionic binding selectivity of Li(+), Na(+), K(+), Rb(+), and Cs(+) in a model of a calcium channel are considered. This work generalizes Eisenman's classic treatment (Biophys J 2(Suppl. 2):259, 1962) by including multiple, mobile binding site oxygens that coordinate many permeating ions (all modeled as charged, hard spheres). The selectivity filter of the model calcium channel allows the carboxyl terminal groups of glutamate and aspartate side chains to directly interact with and coordinate the permeating ions. Ion dehydration effects are represented with a Born energy between the dielectric coefficients of the selectivity filter and the bath. High oxygen concentration creates a high field strength site that prefers small ions, as in Eisenman's model. On the other hand, a low filter dielectric constant also creates a high field strength site, but this site prefers large ions, contrary to Eisenman's model. These results indicate that field strength does not have a unique effect on ionic binding selectivity sequences once entropic, electrostatic, and dehydration forces are included in the model. Thus, Eisenman's classic relationship between field strength and selectivity sequences must be supplemented with additional information about selectivity filters such as the calcium channel that has amino acid side chains mixing with ions to make a crowded permeation pathway.

Costus pictus extracts stimulate insulin secretion from mouse and human islets of Langerhans in vitro.

Plant-derived extracts have been used as folk remedies for Type 2 diabetes mellitus (T2DM) for many centuries, and offer the potential of cheap and readily available alternatives to conventional pharmaceuticals in developing countries. Extracts of Costus pictus (CP), a plant belonging to the Costaceae family, are reported to have antidiabetic activity in vivo. The exact molecular mode of action(s) of CP is unclear but the antihyperglycemic effect seen in animal studies was associated with dramatic increases in insulin secretion so in our study we have measured the effect of aqueous CP extract on insulin secretion in vitro from the MIN6 ß-cell line and isolated mouse and human islets. Our data demonstrate that CP has a direct stimulatory effect on insulin secretion at basal but not stimulatory glucose concentrations which was not associated with compromised membrane integrity or decrease ß-cell viability. Single cell calcium microfluorimetry measurements showed that CP caused elevations in ß-cell intracellular Ca(2+) concentrations ([Ca(2+)](i)), an effect which was completely abolished by the removal of extracellular Ca(2+) or blockade of voltage-gated Ca(2+) channels (VGCC). These in vitro observations suggest that one mode of action of CP is through stimulating insulin secretion which may be mediated, in part, by the ability of CP to increase [Ca(2+)](i) levels through VGCC. CP extracts may provide an affordable and inexpensive alternative for treating patients with T2DM.

Endogenous transport systems in the Xenopus laevis oocyte plasma membrane.

Oocytes of the South African clawed frog Xenopus laevis are widely used as a heterologous expression system for the characterization of transport systems such as passive and active membrane transporters, receptors and a whole plethora of other membrane proteins originally derived from animal or plant tissues. The large size of the oocytes and the high degree of expression of exogenous mRNA or cDNA makes them an optimal tool, when compared with other expression systems such as yeast, Escherichia coli or eukaryotic cell lines, for the expression and functional characterization of membrane proteins. This easy to handle expression system is becoming increasingly attractive for pharmacological research. Commercially available automated systems that microinject mRNA into the oocytes and perform electrophysiological measurements fully automatically allow for a mass screening of new computer designed drugs to target membrane transport proteins. Yet, the oocytes possess a large variety of endogenous membrane transporters and it is absolutely mandatory to distinguish the endogenous transporters from the heterologous, expressed transport systems. Here, we review briefly the endogenous membrane transport systems of the oocytes.

Complementary conductance changes by IKx and Ih contribute to membrane impedance stability during the rod light response.

In addition to the HCN1 channels that mediate the h current, the Kx current also performs signal filtering in rod photoreceptors. This current is known to be mediated by potassium channels and has similarities to the neuronal M current and EAG potassium channels. Although it is known that in filtering the light response of rods, I(h) and I(Kx) undergo complementary conductance changes, the qualities and significance of these changes are not clear. Here we present an analysis demonstrating the filtering effect of HCN1 channels in salamander rods when I(Kx) is blocked, and a simulation of the rod light response showing the magnitude and time course of the conductance changes by both currents. From this analysis, we propose that the purpose of opposing conductance changes by I(h) and I(Kx) may be to optimize the lateral propagation of signals through gap junctions in the rod network.

Alanine-scanning mutagenesis defines a conserved energetic hotspot in the CaValpha1 AID-CaVbeta interaction site that is critical for channel modulation.

Voltage-gated calcium channels (CaVs) are large, multisubunit complexes that control cellular calcium entry. CaV pore-forming (CaValpha1) and cytoplasmic (CaVbeta) subunits associate through a high-affinity interaction between the CaValpha1 alpha interaction domain (AID) and CaVbeta alpha binding pocket (ABP). Here we analyze AID-ABP interaction thermodynamics using isothermal titration calorimetry. We find that commensurate with their strong sequence similarity, all CaV1 and CaV2 AID peptides bind CaVbeta with similar nanomolar affinities. Although the AID-ABP interface encompasses 24 side chains, alanine-scanning mutagenesis reveals that the binding energy is focused in two complementary hotspots comprising four deeply conserved residues. Electrophysiological experiments show that hotspot interaction disruption prevents trafficking and functional modulation of CaV1.2 by CaVbeta. Together, the data support the primacy of the AID-ABP interface for CaValpha1-CaVbeta association, underscore the idea that hotspots dominate protein-protein interaction affinities, and uncover a target for strategies to control cellular excitability by blocking CaValpha1-CaVbeta complex formation.