Differential Cav2.1 and Cav2.3 channel inhibition by baclofen and α-conotoxin Vc1.1 via GABAB receptor activation

Neuronal Ca(v)2.1 (P/Q-type), Ca(v)2.2 (N-type), and Ca(v)2.3 (R-type) calcium channels contribute to synaptic transmission and are modulated through G protein-coupled receptor pathways. The analgesic. alpha-conotoxin Vc1.1 acts through. gamma-aminobutyric acid type B (GABA(B)) receptors (GABA(B)Rs) to inhibit Cav2.2 channels. We investigated GABA(B)R-mediated modulation by Vc1.1, a cyclized form of Vc1.1 (c-Vc1.1), and the GABA(B)R agonist baclofen of human Cav2.1 or Cav2.3 channels heterologously expressed in human embryonic kidney cells. 50 mu M baclofen inhibited Cav2.1 and Cav2.3 channel Ba2+ currents by. similar to 40%, whereas c-Vc1.1 did not affect Cav2.1 but potently inhibited Cav2.3, with a half-maximal inhibitory concentration of. 300 pM. Depolarizing paired pulses revealed that. similar to 75% of the baclofen inhibition of Cav2.1 was voltage dependent and could be relieved by strong depolarization. In contrast, baclofen or Vc1.1 inhibition of Cav2.3 channels was solely mediated through voltage-independent pathways that could be disrupted by pertussis toxin, guanosine 5' -[beta-thio] diphosphate trilithium salt, or the GABABR antagonist CGP55845. Overexpression of the kinase c-Src significantly increased inhibition of Cav2.3 by c-Vc1.1. Conversely, coexpression of a catalytically inactive double mutant form of c-Src or pretreatment with a phosphorylated pp60c-Src peptide abolished the effect of c-Vc1.1. Site-directed mutational analyses of Cav2.3 demonstrated that tyrosines 1761 and 1765 within exon 37 are critical for inhibition of Cav2.3 by c-Vc1.1 and are involved in baclofen inhibition of these channels. Remarkably, point mutations introducing specific c-Src phosphorylation sites into human Cav2.1 channels conferred c-Vc1.1 sensitivity. Our findings show that Vc1.1 inhibition of Cav2.3, which defines Cav2.3 channels as potential targets for analgesic. alpha-conotoxins, is caused by specific c-Src phosphorylation sites in the C terminus

Real-time risk analysis for hybrid earthquake early warning systems

Earthquake Early Warning Systems (EEWS), based on real-time prediction of ground motion or structural response measures, may play a role in reducing vulnerability and/or exposition of buildings and lifelines. In fact, recently seismologists developed efficient methods for rapid estimation of event features by means of limited information of the P-waves. Then, when an event is occurring, probabilistic distributions of magnitude and source-to-site distance are available and the prediction of the ground motion at the site, conditioned to the seismic network measures, may be performed in analogy with the Probabilistic Seismic Hazard Analysis (PSHA). Consequently the structural performance may be obtained by the Probabilistic Seismic Demand Analysis (PSDA), and used for real-time risk management purposes. However, such prediction is performed in very uncertain conditions which have to be taken into proper account to limit false and missed alarms. In the present study, real-time risk analysis for early warning purposes is discussed. The magnitude estimation is performed via the Bayesian approach, while the earthquake localization is based on the Voronoi cells. To test the procedure it was applied, by simulation, to the EEWS under development in the Campanian region (southern Italy). The results lead to the conclusion that the PSHA, conditioned to the EEWS, correctly predicts the hazard at the site and that the false/missed alarm probabilities may be controlled by set up of an appropriate decisional rule and alarm threshold

Re-Evaluation of the Action Potential Upstroke Velocity as a Measure of the Na+ Current in Cardiac Myocytes at Physiological Conditions

Background: The SCN5A encoded sodium current (INa) generates the action potential (AP) upstroke and is a major determinant of AP characteristics and AP propagation in cardiac myocytes. Unfortunately, in cardiac myocytes, investigation of kinetic properties of INa with near-physiological ion concentrations and temperature is technically challenging due to the large amplitude and rapidly activating nature of INa, which may seriously hamper the quality of voltage control over the membrane. We hypothesized that the alternating voltage clamp-current clamp (VC/CC) technique might provide an alternative to traditional voltage clamp (VC) technique for the determination of INa properties under physiological conditions. Principal Findings: We studied INa under close-to-physiological conditions by VC technique in SCN5A cDNA-transfected HEK cells or by alternating VC/CC technique in both SCN5A cDNA-transfected HEK cells and rabbit left ventricular myocytes. In these experiments, peak INa during a depolarizing VC step or maximal upstroke velocity, dV/dtmax, during VC/CC served as an indicator of available INa. In HEK cells, biophysical properties of INa, including current density, voltage dependent (in)activation, development of inactivation, and recovery from inactivation, were highly similar in VC and VC/CC experiments. As an application of the VC/CC technique we studied INa in left ventricular myocytes isolated from control or failing rabbit hearts

Mechanism of direct Ca\u3csub\u3ev\u3c/sub\u3e2.2 channel block by the κ-opioid receptor agonist U50488H

U50488H is a benzeneacetamide κ-opioid receptor (κ-OR) agonist analgesic, widely used for investigating the pharmacology of G protein-coupled κ-ORs. However, U50488H is also known to directly block various voltage-gated ion channels in a G protein-independent manner. We investigated the direct actions of U50488H on various high voltage-activated (HVA) and low voltage-activated (LVA) neuronal Ca2+ channels heterologously expressed in human embryonic kidney (HEK293) cells. U50488H inhibited HVA rat Cav1.3 (rCav1.3), human Cav2.1 (hCav2.1), hCav2.2, hCav2.3, and LVA hCav3.1 and hCav3.2 channels in a concentration-dependent manner, with similar potencies characterised with half-maximal inhibitory concentration (IC50) values of ∼30 μM. U50488H concentrations causing direct Cav inhibition are typically \u3e100 times higher than those producing κ-OR activation. Investigation of the mechanism of U50488H block of the Cav2.2 channel revealed that U50488H interacted with all major kinetic states of the channel − resting, open, and inactivated. U50488H did not affect the voltage dependence of activation but shifted the steady-state inactivation curve by ∼11 mV to more hyperpolarized potentials. U50488H also increased the rate of Ba2+ current inactivation during a step depolarization and significantly delayed recovery from slow inactivation, compared with control. Cav2.2 current inhibition was frequency dependent during repetitive step depolarization at 1 Hz and 3 Hz, consistent with use-dependent block. In summary, our results suggest that preferential interaction of U50488H with inactivated Cav2.2 channels significantly contributes to reduced Cav2.2 channel availability and slow recovery form inactivation. We conclude that U50488H non-selectively blocks heterologously expressed neuronal HVA and LVA Cav channels in the absence of κ-ORs. This cross-reactivity also suggests potentially common U50488H binding motifs across Cav channel targets

Differential Cav2.1 and Cav2.3 channel inhibition by baclofen and α-conotoxin Vc1.1 via GABAB receptor activation

Neuronal Cav2.1 (P/Q-type), Cav2.2 (N-type), and Cav2.3 (R-type) calcium channels contribute to synaptic transmission and are modulated through G protein-coupled receptor pathways. The analgesic α-conotoxin Vc1.1 acts through γ-aminobutyric acid type B (GABAB) receptors (GABABRs) to inhibit Cav2.2 channels. We investigated GABABR-mediated modulation by Vc1.1, a cyclized form of Vc1.1 (c-Vc1.1), and the GABABR agonist baclofen of human Cav2.1 or Cav2.3 channels heterologously expressed in human embryonic kidney cells. 50 µM baclofen inhibited Cav2.1 and Cav2.3 channel Ba2+ currents by ∼40%, whereas c-Vc1.1 did not affect Cav2.1 but potently inhibited Cav2.3, with a half-maximal inhibitory concentration of ∼300 pM. Depolarizing paired pulses revealed that ∼75% of the baclofen inhibition of Cav2.1 was voltage dependent and could be relieved by strong depolarization. In contrast, baclofen or Vc1.1 inhibition of Cav2.3 channels was solely mediated through voltage-independent pathways that could be disrupted by pertussis toxin, guanosine 5′-[β-thio]diphosphate trilithium salt, or the GABABR antagonist CGP55845. Overexpression of the kinase c-Src significantly increased inhibition of Cav2.3 by c-Vc1.1. Conversely, coexpression of a catalytically inactive double mutant form of c-Src or pretreatment with a phosphorylated pp60c-Src peptide abolished the effect of c-Vc1.1. Site-directed mutational analyses of Cav2.3 demonstrated that tyrosines 1761 and 1765 within exon 37 are critical for inhibition of Cav2.3 by c-Vc1.1 and are involved in baclofen inhibition of these channels. Remarkably, point mutations introducing specific c-Src phosphorylation sites into human Cav2.1 channels conferred c-Vc1.1 sensitivity. Our findings show that Vc1.1 inhibition of Cav2.3, which defines Cav2.3 channels as potential targets for analgesic α-conotoxins, is caused by specific c-Src phosphorylation sites in the C terminus

Less is more: design of a highly stable disulfide-deleted mutant of analgesic cyclic α-conotoxin Vc1.1

Cyclic α-conotoxin Vc1.1 (cVc1.1) is an orally active peptide with analgesic activity in rat models of neuropathic pain. It has two disulfide bonds, which can have three different connectivities, one of which is the native and active form. In this study we used computational modeling and nuclear magnetic resonance to design a disulfide-deleted mutant of cVc1.1, [C2H,C8F]cVc1.1, which has a larger hydrophobic core than cVc1.1 and, potentially, additional surface salt bridge interactions. The new variant, hcVc1.1, has similar structure and serum stability to cVc1.1 and is highly stable at a wide range of pH and temperatures. Remarkably, hcVc1.1 also has similar selectivity to cVc1.1, as it inhibited recombinant human α9α10 nicotinic acetylcholine receptor-mediated currents with an IC50 of 13 μM and rat N-type (Cav2.2) and recombinant human Cav2.3 calcium channels via GABAB receptor activation, with an IC50 of ~900 pM. Compared to cVc1.1, the potency of hcVc1.1 is reduced three-fold at both analgesic targets, whereas previous attempts to replace Vc1.1 disulfide bonds by non-reducible dicarba linkages resulted in at least 30-fold decreased activity. Because it has only one disulfide bond, hcVc1.1 is not subject to disulfide bond shuffling and does not form multiple isomers during peptide synthesis

γ-Aminobutyric acid type B (GABAB) receptor expression is needed for inhibition of N-type (Cav2.2) calcium channels by analgesic α-conotoxins

α-Conotoxins Vc1.1 and RgIA are small peptides isolated from the venom of marine cone snails. They have effective anti-nociceptive actions in rat models of neuropathic pain. Pharmacological studies in rodent dorsal root ganglion (DRG) show their analgesic effect is mediated by inhibition of N-type (Cav2.2) calcium channels via a pathway involving γ-aminobutyric acid type B (GABAB) receptor. However, there is no direct demonstration that functional GABAB receptors are needed for inhibition of the Cav2.2 channel by analgesic α-conotoxins. This study examined the effect of the GABAB agonist baclofen and α-conotoxins Vc1.1 and RgIA on calcium channel currents after transient knockdown of the GABAB receptor using RNA interference. Isolated rat DRG neurons were transfected with small interfering RNAs (siRNA) targeting GABAB subunits R1 and R2. Efficient knockdown of GABAB receptor expression at mRNA and protein levels was confirmed by quantitative real time PCR (qRT-PCR) and immunocytochemical analysis, respectively. Whole-cell patch clamp recordings conducted 2-4 days after transfection showed that inhibition of N-type calcium channels in response to baclofen, Vc1.1 and RgIA was significantly reduced in GABAB receptor knockdown DRG neurons. In contrast, neurons transfected with a scrambled nontargeting siRNA were indistinguishable from untransfected neurons. In the HEK 293 cell heterologous expression system, Vc1.1 and RgIA inhibition of Cav2.2 channels needed functional expression of both human GABAB receptor subunits. Together, these results confirm that GABAB receptors must be activated for the modulation of N-type (Cav2.2) calcium channels by analgesic α-conotoxins Vc1.1 and RgIA

Pro- and antiarrhythmic properties of a diet rich in fish oil

Increased consumption of fish rich in omega-3 polyunsaturated fatty acids (omega3-PUFAs) is associated with decreased incidence of sudden cardiac death in post-myocardial infarction patients, but is also related to increased incidence of sudden death and arrhythmias in patients with acute myocardial ischemia. This review discusses the possible pro- and antiarrhythmic mechanisms of omega3-PUFAs in relation to various cardiac pathologies. Differences between circulating and incorporated omega3-PUFAs with respect to electrophysiology are emphasized. We conclude that omega3-PUFAs alter cardiac electrophysiology and thereby may be pro- or antiarrhythmic, dependent on the mechanism of arrhythmia. As omega3-PUFAs may be antiarrhythmic under conditions that favour triggered activity, they may facilitate re-entrant arrhythmias. This may explain the contradictory outcomes of increased intake of fish oil on sudden death and arrhythmias in clinical trials. Advice to increase intake of omega3-PUFA supplements or fatty fish should be tailored to individual patients with respect to the arrhythmogenic mechanisms associated with the underlying patholog