Exploration of the Parameter Space of an Ion Channel Kinetic Model by a Markov-Chain-Based Methodology.

In this work, we propose a methodology based on Monte Carlo Markov chains to explore the parameter space of kinetic models for ion channels. The methodology allows the detection of potential parameter sets of a model that are compatible with experimentally obtained whole-cell currents, which could remain hidden when methods focus on obtaining the parameters that provide the best fit. To show its implementation and utility, we considered a four-state kinetic model proposed in the literature to describe the activation of the voltage-gated proton channel (Hv1), Biophysical Journal, 2014, 107, 1564. In that work, a set of values for the rate transitions that describe the channel kinetics at different intracellular H+ concentration (pHi) were obtained by the Simplex method. With our approach, we find that, in fact, there is more than one parameter set for each pHi, which renders the same open probability temporal course within the experimental error margin for all of the considered voltages. The large differences that we obtained for the values of some rate constants among the different solutions show that there is more than one possible interpretation of this channel behavior as a function of pHi. We also simulated a proposed new experimental condition where it is possible to observe that different sets of parameters yield different results. Our study highlights the importance of a comprehensive analysis of parameter space in kinetic models and the utility of the proposed methodology for finding potential solutions.

A Combined Ligand- and Structure-Based Virtual Screening To Identify Novel NaV1.2 Blockers: In Vitro Patch Clamp Validation and In Vivo Anticonvulsant Activity.

Epilepsy is a neurological disorder characterized by recurrent seizures that arise from abnormal electrical activity in the brain. Voltage-gated sodium channels (NaVs), responsible for the initiation and propagation of action potentials in neurons, play a critical role in the pathogenesis of epilepsy. This study sought to discover potential anticonvulsant compounds that interact with NaVs, specifically, the brain subtype hNaV1.2. A ligand-based QSAR model and a docking model were constructed, validated, and applied in a parallel virtual screening over the DrugBank database. Montelukast, Novobiocin, and Cinnarizine were selected for in vitro testing, using the patch-clamp technique, and all of them proved to inhibit hNaV1.2 channels heterologously expressed in HEK293 cells. Two hits were evaluated in the GASH/Sal model of audiogenic seizures and demonstrated promising activity, reducing the severity of sound-induced seizures at the doses tested. The combination of ligand- and structure-based models presents a valuable approach for identifying potential NaV inhibitors. These findings may provide a basis for further research into the development of new antiseizure drugs for the treatment of epilepsy.

Novel Dimeric hHv1 Model and Structural Bioinformatic Analysis Reveal an ATP-Binding Site Resulting in a Channel Activating Effect.

The human voltage-gated proton channel (hHv1) is a highly selective ion channel codified by the HVCN1 gene. It plays a fundamental role in several physiological processes such as innate and adaptive immunity, insulin secretion, and sperm capacitation. Moreover, in humans, a higher hHv1 expression/function has been reported in several types of cancer cells. Here we report a multitemplate homology model of the hHv1 channel, built and refined as a dimer in Rosetta. The model was then subjected to extensive Gaussian accelerated molecular dynamics (GaMD) for enhanced conformational sampling, and representative snapshots were extracted by clustering analysis. Combining different structure- and sequence-based methodologies, we predicted a putative ATP-binding site located on the intracellular portion of the channel. Furthermore, GaMD simulations of the ATP-bound dimeric hHv1 model showed that ATP interacts with a cluster of positively charged residues from the cytoplasmic N and C terminal segments. According to the in silico predictions, we found that 3 mM intracellular ATP significantly increases the H+ current mediated by the hHv1 channel expressed in HEK293 cells and measured by the patch-clamp technique in an inside-out configuration (2.86 ± 0.63 fold over control at +40 mV). When ATP was added on the extracellular side, it was not able to activate the channel supporting the idea that the ATP-binding site resides in the intracellular face of the hHV1 channel. In a physiological and pathophysiological context, this ATP-mediated modulation could integrate the cell metabolic state with the H+ efflux, especially in cells where hHv1 channels are relevant for pH regulation, such as pancreatic ß-cells, immune cells, and cancer cells.

VDAC Modulation of Cancer Metabolism: Advances and Therapeutic Challenges.

Most anionic metabolites including respiratory substrates, glycolytic adenosine triphosphate (ATP), and small cations that enter mitochondria, and mitochondrial ATP moving to the cytosol, cross the outer mitochondrial membrane (OMM) through voltage dependent anion channels (VDAC). The closed states of VDAC block the passage of anionic metabolites, and increase the flux of small cations, including calcium. Consequently, physiological or pharmacological regulation of VDAC opening, by conditioning the magnitude of both anion and cation fluxes, is a major contributor to mitochondrial metabolism. Tumor cells display a pro-proliferative Warburg phenotype characterized by enhanced aerobic glycolysis in the presence of partial suppression of mitochondrial metabolism. The heterogeneous and flexible metabolic traits of most human tumors render cells able to adapt to the constantly changing energetic and biosynthetic demands by switching between predominantly glycolytic or oxidative phenotypes. Here, we describe the biological consequences of changes in the conformational state of VDAC for cancer metabolism, the mechanisms by which VDAC-openers promote cancer cell death, and the advantages of VDAC opening as a valuable pharmacological target. Particular emphasis is given to the endogenous regulation of VDAC by free tubulin and the effects of VDAC-tubulin antagonists in cancer cells. Because of its function and location, VDAC operates as a switch to turn-off mitochondrial metabolism (closed state) and increase aerobic glycolysis (pro-Warburg), or to turn-on mitochondrial metabolism (open state) and decrease glycolysis (anti-Warburg). A better understanding of the role of VDAC regulation in tumor progression is relevant both for cancer biology and for developing novel cancer chemotherapies.

Arachidonic acid effect on the allosteric gating mechanism of BK (Slo1) channels associated with the ß1 subunit.

Arachidonic acid (AA) is a fatty acid involved in the modulation of several ion channels. Previously, we reported that AA activates the high conductance Ca2+- and voltage-dependent K+ channel (BK) in vascular smooth muscle depending on the expression of the auxiliary ß1 subunit. Here, using the patch-clamp technique on BK channel co-expressed with ß1 subunit in a heterologous cell expression system, we analyzed whether AA modifies the three functional modules involved in the channel gating: the voltage sensor domain (VSD), the pore domain (PD), and the intracellular calcium sensor domain (CSD). We present evidence that AA activates BK channel in a direct way, inducing VSD stabilization on its active configuration observed as a significant left shift in the Q-V curve obtained from gating currents recordings. Moreover, AA facilitates the channel opening transitions when VSD are at rest, and the CSD are unoccupied. Furthermore, the activation was independent of the intracellular Ca2+ concentration and reduced when the BK channel was co-expressed with the Y74A mutant of the ß1 subunit. These results allow us to present new insigths in the mechanism by which AA modulates BK channels co-expressed with its auxiliary ß1 subunit.

Parabens inhibit hNaV 1.2 channels.

Propylparaben, a commonly used antimicrobial preservative, has been reported as an anticonvulsant agent targeting neuronal Na+ channels (NaV). However, the specific features of the NaV channel inhibition by this agent have so far not been extensively studied. Moreover, it is still unclear if it shares this pharmacological activity with other parabens. Here, we fully characterized the mechanism of action of the inhibitory effect that propylparaben and benzylparaben induce on human NaV 1.2 channel isoform (hNaV1.2). We established a first approach to know the parabens structural determinants for this channel inhibition. The parabens effects on hNaV1.2 channel mediated currents were recorded using the patch-clamp whole-cell configuration on hNaV1.2 stably transfected HEK293 cells. Propylparaben induced a typical state-dependent inhibition on hNaV1.2 channel carried current, characterized by a left-shift in the steady-state inactivation curve, a prolongation in the time needed for recovery from fast inactivation and a frequency-dependent blocking behavior. The state-dependent inhibition is increased for butylparaben and benzylparaben and diminished for methylparaben, ethylparaben and p-hydroxybenzoic acid (the major metabolite of parabens hydrolysis). Particularly, butylparaben and benzylparaben shift the steady-state inactivation curve 2- and 3-times more than propylparaben, respectively. Parabens are blockers of hNaV1.2 channels, sharing the mechanism of action of most of sodium channel blocking antiseizure drugs. The potency of this inhibition increases with the size of the lipophilic alcoholic residue of the ester group. These results provide a basis for rational drug design directed to generate new potential anticonvulsant agents.

Differential expression of the long and truncated Hv1 isoforms in breast-cancer cells.

Metabolic reprogramming of cancer cells results in a high production of acidic substances that must be extruded to maintain tumor-cell viability. The voltage-gated proton channel (Hv1) mediates highly selective effluxes of hydronium-ion (H+ ) that prevent deleterious cytoplasmic acidification. In the work described here, we demonstrated for the first time that the amino-terminal-truncated isoform of Hv1 is more highly expressed in tumorigenic breast-cancer-cell lines than in nontumorigenic breast cells. With respect to Hv1 function, we observed that pharmacologic inhibition of that channel, mediated by the specific blocker 5-chloro-2-guanidinobenzimidazole, produced a drop in intracellular pH and a decrease in cell viability, both in monolayer and in three-dimensional cultures, and adversely affected the cell-cycle in tumorigenic breast cells without altering the cycling of nontumorigenic cells. In conclusion, our results demonstrated that the Hv1 channel could be a potential tool both as a biomarker and as a therapeutic target in breast-cancer disease.

Searching for New Leads To Treat Epilepsy: Target-Based Virtual Screening for the Discovery of Anticonvulsant Agents.

The purpose of this investigation is to contribute to the development of new anticonvulsant drugs to treat patients with refractory epilepsy. We applied a virtual screening protocol that involved the search into molecular databases of new compounds and known drugs to find small molecules that interact with the open conformation of the Nav1.2 pore. As the 3D structure of human Nav1.2 is not available, we first assembled 3D models of the target, in closed and open conformations. After the virtual screening, the resulting candidates were submitted to a second virtual filter, to find compounds with better chances of being effective for the treatment of P-glycoprotein (P-gp) mediated resistant epilepsy. Again, we built a model of the 3D structure of human P-gp, and we validated the docking methodology selected to propose the best candidates, which were experimentally tested on Nav1.2 channels by patch clamp techniques and in vivo by the maximal electroshock seizure (MES) test. Patch clamp studies allowed us to corroborate that our candidates, drugs used for the treatment of other pathologies like Ciprofloxacin, Losartan, and Valsartan, exhibit inhibitory effects on Nav1.2 channel activity. Additionally, a compound synthesized in our lab, N, N'-diphenethylsulfamide, interacts with the target and also triggers significant Na1.2 channel inhibitory action. Finally, in vivo studies confirmed the anticonvulsant action of Valsartan, Ciprofloxacin, and N, N'-diphenethylsulfamide.

Environmental pollutant hexachlorobenzene induces hypertension in a rat model.

Hexachlorobenzene (HCB) is a dioxin-like environmental pollutant, widely distributed in the environment. New research links exposure to high levels of persistent organic environmental toxicants to cardiovascular disease, however little is known about the effect of HCB on vascular function and on blood pressure. The purpose of the present study was to evaluate biochemical and cardiovascular changes resulting from subchronic HCB exposure. Adult female Sprague-Dawley rats were treated with vehicle or HCB (5 or 500 mg/kg b.w) for 45 days. Systolic blood pressure (BP), recorded by tail cuff plethysmography, was significantly increased at 35, 40 and 45 days of 500 mg/kg HCB-treatment. HCB (500 mg/kg) increased arterial thickness, while both 5 and 500 mg/kg HCB decreased proliferating cell nuclear antigen (PCNA) protein levels and cellular nuclei in abdominal aortas indicating a hypertrophic process. Also, aortas from both groups of HCB-treated rats presented higher sensitivity to noradrenalin (NA) and a significant decrease in maximum contractile response. Arteries from 500 mg/kg HCB-treated rats showed a significant increase in the levels of transforming growth factor-ß1 (TGF-ß1) mRNA and angiotensin II type1 receptor (AT1), and a significant decrease in estrogen receptor alpha (ERα), endothelial nitric oxidide synthase (eNOS) protein expression and deiodinase II (DII) mRNA levels. In conclusion, we have demonstrated for the first time that subchronic HCB administration significantly increases BP and alters associated cardiovascular parameters in rats. In addition, HCB alters the expression of key vascular tissue molecules involved in BP regulation, such as TGF-ß1, AT1, ERα, eNOS and DII.

N-propyl-2,2-diphenyl-2-hydroxyacetamide, a novel α-hydroxyamide with anticonvulsant, anxiolytic and antidepressant-like effects that inhibits voltage-gated sodium channels.

In patients with epilepsy, anxiety and depression are the most frequent psychiatric comorbidities but they often remain unrecognized and untreated. We report herein the antidepressant-like activity in two animal models, tail suspension and forced swimming tests, of six anticonvulsants α-hydroxyamides. From these, N-propyl-2,2-diphenyl-2-hydroxyacetamide (compound 5) emerged not only as the most active as anticonvulsant (ED50 = 2.5mg/kg, MES test), but it showed the most remarkable antidepressant-like effect in the tail suspension and forced swimming tests (0.3-30mg/kg, i.p.); and, also, anxiolytic-like action in the plus maze test (3-10mg/kg, i.p.) in mice. Studies of its mechanism of action, by means of its capacity to act via the GABAA receptor ([3H]-flunitrazepam binding assay); the 5-HT1A receptor ([3H]-8-OH-DPAT binding assay) and the voltage-gated sodium channels (either using the patch clamp technique in hNav 1.2 expressed in HEK293 cell line or using veratrine, in vivo) were attempted. The results demonstrated that its effects are not likely related to 5-HT1A or GABAAergic receptors and that its anticonvulsant and antidepressant-like effect could be due to its voltage-gated sodium channel blocking properties.

Activation of human smooth muscle BK channels by hydrochlorothiazide requires cell integrity and the presence of BK ß1 subunit.

Thiazide-like diuretics are the most commonly used drugs to treat arterial hypertension, with their efficacy being linked to their chronic vasodilatory effect. Previous studies suggest that activation of the large conductance voltage- and Ca2+-dependent K+ (BK) channel (Slo 1, MaxiK channel) is responsible for the thiazide-induced vasodilatory effect. But the direct electrophysiological evidence supporting this claim is lacking. BK channels can be associated with one small accessory ß-subunit (ß1-ß4) that confers specific biophysical and pharmacological characteristics to the current phenotype. The ß1-subunit is primarily expressed in smooth muscle cells (SMCs). In this study we investigated the effect of hydrochlorothiazide (HCTZ) on BK channel activity in native SMCs from human umbilical artery (HUASMCs) and HEK293T cells expressing the BK channel (with and without the ß1-subunit). Bath application of HCTZ (10 µmol/L) significantly augmented the BK current in HUASMCs when recorded using the whole-cell configurations, but it did not affect the unitary conductance and open probability of the BK channel in HUASMCs evaluated in the inside-out configuration, suggesting an indirect mechanism requiring cell integrity. In HEK293T cells expressing BK channels, HCTZ-augmented BK channel activity was only observed when the ß1-subunit was co-expressed, being concentration-dependent with an EC50 of 28.4 µmol/L, whereas membrane potential did not influence the concentration relationship. Moreover, HCTZ did not affect the BK channel current in HEK293T cells evaluated in the inside-out configuration, but significantly increases the open probability in the cell-attached configuration. Our data demonstrate that a ß1-subunit-dependent mechanism that requires SMC integrity leads to HCTZ-induced BK channel activation.

Diphenhydramine inhibits voltage-gated proton channels (Hv1) and induces acidification in leukemic Jurkat T cells- New insights into the pro-apoptotic effects of antihistaminic drugs.

An established characteristic of neoplastic cells is their metabolic reprogramming, known as the Warburg effect, with greater reliance on energetically less efficient pathways (such as glycolysis and pentose phosphate shunt) compared with oxidative phosphorylation. This results in an overproduction of acidic species that must be extruded to maintain intracellular homeostasis. We recently described that blocking the proton currents in leukemic cells mediated by Hv1 ion channels triggers a marked intracellular acidification and apoptosis induction. Moreover, histamine H1-receptor antagonists were found to induce apoptosis in tumoral cells but the mechanism is still unclear. By using Jurkat T cells, we now show how diphenhydramine inhibits Hv1 mediated currents, inducing a drop in intracellular pH and cellular viability. This provides evidence of a new target structure responsible of the known pro-apoptotic action of antihistaminic drugs.

Gut Permeability and Glucose Absorption Are Affected at Early Stages of Graft Rejection in a Small Bowel Transplant Rat Model.

BACKGROUND: Intestinal transplantation (ITx) faces many challenges due to the complexity of surgery and to the multiple immunological reactions that lead to the necessity of rigorous follow-up for early detection of acute cellular rejection (ACR). Our aim was to determine the kinetics of ACR using an experimental ITx model, with emphasis in the characterization of the process using different approaches, including the use of functional assays of absorptive and barrier function. METHODS: ITx in rats conducting serial sampling was performed. Clinical monitoring, graft histology, proinflammatory gene expression, and nitrosative stress determination were performed. Also, glucose absorption, barrier function using ovalbumin translocation, and contractile function were analyzed. RESULTS: The model used reproduced the different stages of ACR. Allogeneic ITx recipients showed signs of rejection from postoperative day (POD) 5, with increasing severity until 12 POD. Histological evaluation showed mild rejection in early sampling and severe rejection at late stages, with alterations in all graft layers. IL-6, CXCL 10, IFNg, and nitrite plasmas levels showed behavior coincident with histopathology. Remarkably, allogeneic grafts showed a marked alteration of glucose absorptive capacity from POD 5 that was sustained until endpoint. Coincidently, barrier function alteration was evidenced by luminal ovalbumin translocation to serum. Contractile function was progressively impaired along ACR. CONCLUSIONS: Glucose absorption and barrier function are altered at early stages of ACR when histological alterations or gene expression changes were much subtle. This observation may provide simple evaluation tools that could be eventually translated to the clinics to contribute to early ACR diagnosis.

An allosteric gating model recapitulates the biophysical properties of IK,L expressed in mouse vestibular type I hair cells.

KEY POINTS: Vestibular type I and type II hair cells and their afferent fibres send information to the brain regarding the position and movement of the head. The characteristic feature of type I hair cells is the expression of a low-voltage-activated outward rectifying K+ current, IK,L , whose biophysical properties and molecular identity are still largely unknown. In vitro, the afferent nerve calyx surrounding type I hair cells causes unstable intercellular K+ concentrations, altering the biophysical properties of IK,L . We found that in the absence of the calyx, IK,L in type I hair cells exhibited unique biophysical activation properties, which were faithfully reproduced by an allosteric channel gating scheme. These results form the basis for a molecular and pharmacological identification of IK,L . ABSTRACT: Type I and type II hair cells are the sensory receptors of the mammalian vestibular epithelia. Type I hair cells are characterized by their basolateral membrane being enveloped in a single large afferent nerve terminal, named the calyx, and by the expression of a low-voltage-activated outward rectifying K+ current, IK,L . The biophysical properties and molecular profile of IK,L are still largely unknown. By using the patch-clamp whole-cell technique, we examined the voltage- and time-dependent properties of IK,L in type I hair cells of the mouse semicircular canal. We found that the biophysical properties of IK,L were affected by an unstable K+ equilibrium potential (Veq K+ ). Both the outward and inward K+ currents shifted Veq K+ consistent with K+ accumulation or depletion, respectively, in the extracellular space, which we attributed to a residual calyx attached to the basolateral membrane of the hair cells. We therefore optimized the hair cell dissociation protocol in order to isolate mature type I hair cells without their calyx. In these cells, the uncontaminated IK,L showed a half-activation at -79.6 mV and a steep voltage dependence (2.8 mV). IK,L also showed complex activation and deactivation kinetics, which we faithfully reproduced by an allosteric channel gating scheme where the channel is able to open from all (five) closed states. The 'early' open states substantially contribute to IK,L activation at negative voltages. This study provides the first complete description of the 'native' biophysical properties of IK,L in adult mouse vestibular type I hair cells.

Dynamic regulation of extracellular ATP in Escherichia coli.

We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-32P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1-1 µM). Exposure to MST7 and MEL enhanced ATP release by 3-7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6-7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

The inhibition of voltage-gated H+ channel (HVCN1) induces acidification of leukemic Jurkat T cells promoting cell death by apoptosis.

Cellular energetic deregulation is widely known to produce an overproduction of acidic species in cancer cells. This acid overload must be counterbalanced with a high rate of H+ extrusion to maintain cell viability. In this sense, many H+ transporters have been reported to be crucial for cell survival and proposed as antineoplastic target. By the way, voltage-gated proton channels (Hv1) mediate highly selective H+ outward currents, capable to compensate acid burden in brief periods of time. This structure is canonically described acting as NADPH oxidase counterbalance in reactive oxygen species production. In this work, we show, for the first time in a oncohematologic cell line, that inhibition of Hv1 channels by Zn2+ and the more selective blocker 2-(6-chloro-1H-benzimidazol-2-yl)guanidine (ClGBI) progressively decreases intracellular pH in resting conditions. This acidification is evident minutes after blockade and progresses under prolonged exposure (2, 17, and 48 h), and we firstly demonstrate that this is followed by cell death through apoptosis (annexin V binding). Altogether, these results contribute strong evidence that this channel might be a new therapeutic target in cancer.

Diversity of potassium channels in human umbilical artery smooth muscle cells: a review of their roles in human umbilical artery contraction.

Through their control of cell membrane potential, potassium (K(+)) channels are among the best known regulators of vascular tone. This article discusses the expression and function of K(+) channels in human umbilical artery smooth muscle cells (HUASMCs). We review the bibliographic reports and also present single-channel data recorded in freshly isolated cells. Electrophysiological properties of big conductance, voltage- and Ca(2+)-sensitive K(+) channel and voltage-dependent K(+) channels are clearly established in this vessel, where they are involved in contractile state regulation. Their role in the maintenance of membrane potential is an important control mechanism in the determination of the vessel diameter. Additionally, small conductance Ca(2+)-sensitive K(+) channels, 2-pore domains K(+) channels and inward rectifier K(+) channels also appear to be present in HUASMCs, while intermediate conductance Ca(2+)-sensitive K(+) channels and ATP-sensitive K(+) channels could not be identified. In both cases, additional investigation is necessary to reach conclusive evidence of their expression and/or functional role in HUASMCs. Finally, we discuss the role of K(+) channels in pregnancy-related pathologies like gestational diabetes and preeclampsia.

Arachidonic acid activation of BKCa (Slo1) channels associated to the ß1-subunit in human vascular smooth muscle cells.

Arachidonic acid (AA) is a polyunsaturated fatty acid involved in a complex network of cell signaling. It is well known that this fatty acid can directly modulate several cellular target structures, among them, ion channels. We explored the effects of AA on high conductance Ca(2+)- and voltage-dependent K(+) channel (BKCa) in vascular smooth muscle cells (VSMCs) where the presence of ß1-subunit was functionally demonstrated by lithocholic acid activation. Using patch-clamp technique, we show at the single channel level that 10 µM AA increases the open probability (Po) of BKCa channels tenfold, mainly by a reduction of closed dwell times. AA also induces a left-shift in Po versus voltage curves without modifying their steepness. Furthermore, AA accelerates the kinetics of the voltage channel activation by a fourfold reduction in latencies to first channel opening. When AA was tested on BKCa channel expressed in HEK cells with or without the ß1-subunit, activation only occurs in presence of the modulatory subunit. These results contribute to highlight the molecular mechanism of AA-dependent BKCa activation. We conclude that AA itself selectively activates the ß1-associated BKCa channel, destabilizing its closed state probably by interacting with the ß1-subunit, without modifying the channel voltage sensitivity. Since BKCa channels physiologically contribute to regulation of VSMCs contractility and blood pressure, we used the whole-cell configuration to show that AA is able to activate these channels, inducing significant cell hyperpolarization that can lead to VSMCs relaxation.

A BK (Slo1) channel journey from molecule to physiology.

Calcium and voltage-activated potassium (BK) channels are key actors in cell physiology, both in neuronal and non-neuronal cells and tissues. Through negative feedback between intracellular Ca (2+) and membrane voltage, BK channels provide a damping mechanism for excitatory signals. Molecular modulation of these channels by alternative splicing, auxiliary subunits and post-translational modifications showed that these channels are subjected to many mechanisms that add diversity to the BK channel α subunit gene. This complexity of interactions modulates BK channel gating, modifying the energetic barrier of voltage sensor domain activation and channel opening. Regions for voltage as well as Ca (2+) sensitivity have been identified, and the crystal structure generated by the 2 RCK domains contained in the C-terminal of the channel has been described. The linkage of these channels to many intracellular metabolites and pathways, as well as their modulation by extracellular natural agents, has been found to be relevant in many physiological processes. This review includes the hallmarks of BK channel biophysics and its physiological impact on specific cells and tissues, highlighting its relationship with auxiliary subunit expression.

Human umbilical artery smooth muscle exhibits a 2-APB-sensitive capacitative contractile response evoked by vasoactive substances and expresses mRNAs for STIM, Orai and TRPC channels.

After depletion of intracellular Ca2+ stores the capacitative response triggers an extracellular Ca2+ influx through store-operated channels (SOCs) which refills these stores. Our objective was to explore if human umbilical artery smooth muscle presented this response and if it was involved in the mechanism of serotonin- and histamine-induced contractions. Intracellular Ca2+ depletion by a Ca(2+)-free extracellular solution followed by Ca2+ readdition produced a contraction in artery rings which was inhibited by the blocker of Orai and TRPC channels 2-aminoethoxydiphenyl borate (2-APB), suggesting a capacitative response. In presence of 2-APB the magnitude of a second paired contraction by serotonin or histamine was significantly less than a first one, likely because 2-APB inhibited store refilling by capacitative Ca2+ entry. 2-APB inhibition of sarcoplasmic reticulum Ca2+ release was excluded because this blocker did not affect serotonin force development in a Ca(2+)-free solution. The PCR technique showed the presence of mRNAs for STIM proteins (1 and 2), for Orai proteins (1, 2 and 3) and for TRPC channels (subtypes 1, 3, 4 and 6) in the smooth muscle of the human umbilical artery. Hence, this artery presents a capacitative contractile response triggered by stimulation with physiological vasoconstrictors and expresses mRNAs for proteins and channels previously identified as SOCs.