Géza Berecki

Analgesic α-Conotoxins Vc1.1 and Rg1A Inhibit N-Type Calcium Channels in Rat Sensory Neurons via GABAB Receptor Activation

α-Conotoxins Vc1.1 and Rg1A are peptides from the venom of marine Conus snails that are currently in development as a treatment for neuropathic pain. Here we report that the α9α10 nicotinic acetylcholine receptor-selective conotoxins Vc1.1 and Rg1A potently and selectively inhibit high-voltage-activated (HVA) calcium channel currents in dissociated DRG neurons in a concentration-dependent manner. The post-translationally modified peptides vc1a and [P6O]Vc1.1 were inactive, as were all other α-conotoxins tested. Vc1.1 inhibited the ω-conotoxin-sensitive HVA currents in DRG neurons but not those recorded from Xenopus oocytes expressing CaV2.2, CaV2.1, CaV2.3, or CaV1.2 channels. Inhibition of HVA currents by Vc1.1 was not reversed by depolarizing prepulses but was abolished by pertussis toxin (PTX), intracellular GDPβS, or a selective inhibitor of pp60c-src tyrosine kinase. These data indicate that Vc1.1 does not interact with N-type calcium channels directly but inhibits them via a voltage-independent mechanism involving a PTX-sensitive, G-protein-coupled receptor. Preincubation with a variety of selective receptor antagonists demonstrated that only the GABAB receptor antagonists, [S-(R*,R*)][-3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxy propyl]([3,4]-cyclohexylmethyl) phosphinic acid hydrochloride (2S)-3[[(1S)-1-(3,4-dichlorophenyl)-ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid and phaclofen, blocked the effect of Vc1.1 and Rg1A on Ca2+ channel currents. Together, the results identify CaV2.2 as a target of Vc1.1 and Rg1A, potentially mediating their analgesic actions. We propose a novel mechanism by which α-conotoxins Vc1.1 and Rg1A modulate native N-type (CaV2.2) Ca2+ channel currents, namely acting as agonists via G-protein-coupled GABAB receptors.

The presence and subcellular localization of caspase 3‐like proteinases in plant cells

Caspases play a very important role in initiating and executing apoptotic processes in animal cells. In this study we show that plant mitochondria were able to initiate the activation of caspase 3 in a Xenopus cell free system. Caspase 3‐like activity was found to be present in plant cells and could only be inhibited by the specific caspase 3 inhibitor N‐acetyl‐Asp‐Glu‐Val‐Asp‐fluoromethylketone (Ac‐DEVD‐fmk) and not by cysteine protease inhibitors. By micro‐injection of the caspase 3 substrate in living Chara cells we showed that caspase 3‐like activity was mainly present in the cytosol rather than in the vacuole. This is the first time that in vivo caspase 3‐like activity has been demonstrated in plants.

Acute administration of fish oil inhibits triggered activity in isolated myocytes from rabbits and patients with heart failure

Background— Fish oil reduces sudden death in patients with prior myocardial infarction. Sudden death in heart failure may be due to triggered activity based on disturbed calcium handling. We hypothesized that superfusion with &ohgr;3-polyunsaturated fatty acids (&ohgr;3-PUFAs) from fish inhibits triggered activity in heart failure. Methods and Results— Ventricular myocytes were isolated from explanted hearts of rabbits with volume- and pressure-overload–induced heart failure and of patients with end-stage heart failure. Membrane potentials (patch-clamp technique) and intracellular calcium (indo-1 fluorescence) were recorded after 5 minutes of superfusion with Tyrode’s solution (control), &ohgr;-9 monounsaturated fatty acid oleic acid (20 &mgr;mol/L), or &ohgr;3-PUFAs (docosahexaenoic acid or eicosapentaenoic acid 20 &mgr;mol/L). &ohgr;3-PUFAs shortened the action potential at low stimulation frequencies and caused an ≈25% decrease in diastolic and systolic calcium (all P<0.05). Subsequently, noradrenalin and rapid pacing were used to evoke triggered activity, delayed afterdepolarizations, and calcium aftertransients. &ohgr;3-PUFAs abolished triggered activity and reduced the number of delayed afterdepolarizations and calcium aftertransients compared with control and oleic acid. &ohgr;3-PUFAs reduced action potential shortening and intracellular calcium elevation in response to noradrenalin. Results from human myocytes were in accordance with the findings obtained in rabbit myocytes. Conclusion— Superfusion with &ohgr;3-PUFAs from fish inhibits triggered arrhythmias in myocytes from rabbits and patients with heart failure by lowering intracellular calcium and reducing the response to noradrenalin.

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. Conclusions Our results demonstrate that the alternating VC/CC technique is a valuable experimental tool for INa measurements under close-to-physiological conditions in cardiac myocytes.

Analgesic omega-conotoxins CVIE and CVIF selectively and voltage dependently block recombinant and native N-type calcium channels

Neuronal (N)-type Ca2+ channel-selective ω-conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new ω-conotoxins, CVIE and CVIF, were discovered from a Conus catus cDNA library. Both conopeptides potently displaced 125I-GVIA binding to rat brain membranes. In Xenopus laevis oocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba2+ currents through recombinant N-type (α1B-b/α2δ1/β3) Ca2+ channels. Recovery from block increased with membrane hyperpolarization, indicating that CVIE and CVIF have a higher affinity for channels in the inactivated state. The link between inactivation and the reversibility of ω-conotoxin action was investigated by creating molecular diversity in β subunits: N-type channels with β2a subunits almost completely recovered from CVIE or CVIF block, whereas those with β3 subunits exhibited weak recovery, suggesting that reversibility of the ω-conotoxin block may depend on the type of β-subunit isoform. In rat dorsal root ganglion sensory neurons, neither peptide had an effect on low-voltage-activated T-type channels but potently and selectively inhibited high voltage-activated N-type Ca2+ channels in a voltage-dependent manner. In rat spinal cord slices, both peptides reversibly inhibited excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. Homology models of CVIE and CVIF suggest that ω-conotoxin/voltage-gated Ca2+ channel interaction is dominated by ionic/electrostatic interactions. In the rat partial sciatic nerve ligation model of neuropathic pain, CVIE and CVIF (1 nM) significantly reduced allodynic behavior. These N-type Ca2+ channel-selective ω-conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.

γ-Aminobutyric Acid Type B (GABAB) Receptor Expression Is Needed for Inhibition of N-type (Cav2.2) Calcium Channels by Analgesic α-Conotoxins

Background: A class of analgesic α-conotoxins potently inhibits N-type calcium channels. Results: The activity of α-conotoxins Vc1.1 and RgIA was reduced following knockdown of GABAB receptor expression in sensory neurons and could be reconstituted in HEK 293 cells expressing human GABAB receptors and Cav2.2. Conclusion: GABAB receptors are needed for inhibition of Cav2.2 by Vc1.1 and RgIA. Significance: These analgesic α-conotoxins activate human GABAB receptors. 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.

Anion Channels in Chara corallina Tonoplast Membrane: Calcium Dependence and Rectification

Abstract. Tonoplast K+ channels of Chara corallina are well characterized but only a few reports mention anion channels, which are likely to play an important role in the tonoplast action potential and osmoregulation of this plant. For experiments internodal cells were isolated. Cytoplasmic droplets were formed in an iso-osmotic bath solution according to a modified procedure. Ion channels with conductances of 48 pS and 170 pS were detected by the patch-clamp technique. In the absence of K+ in the bath solution the 170 pS channel was not observed at negative pipette potential values. When Cl− on either the vacuolar side or the cytoplasmic side was partly replaced with F−, the reversal potential of the 48 pS channel shifted conform to the Cl− equilibrium potential with similar behavior in droplet-attached and excised patch mode. These results showed that the 48 pS channel was a Cl− channel. In droplet-attached mode the channel rectified outward current flow, and the slope conductance was smaller. When Chara droplets were formed in a bath solution containing low (10−8m) Ca2+, then no Cl− channels could be detected either in droplet-attached or in inside-out patch mode. Channel activity was restored if Ca2+ was applied to the cytoplasmic side of inside-out patches. Rectification properties in the inside-out patch configuration could be controlled by the holding pipette potential. Holding potential values negative or positive to the calculated reversal potential for Cl− ions induced opposite rectification properties. Our results show Ca2+-activated Cl− channels in the tonoplast of Chara with holding potential dependent rectification.

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 13u2009μM and rat N-type (Cav2.2) and recombinant human Cav2.3 calcium channels via GABAB receptor activation, with an IC50 of ~900u2009pM. 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.

Cardiac Channelopathies Studied With the Dynamic Action Potential-Clamp Technique

The cardiac long QT syndrome (LQTS) is characterized by a delayed repolarization of the ventricular myocytes, resulting in prolongation of the QT interval on the electrocardiogram and increased propensity to cardiac arrhythmias. Congenital LQTS has been linked to mutations in genes encoding ion channel subunits. For a better understanding of LQTS and associated arrhythmias, insight into the nature of ion channel (dys)function is indispensable. Conventionally, voltage-clamp analysis and subsequent mathematical modeling are used to study cardiac channelopathies and to link a certain genetic defect to its cellular phenotype. The recently introduced dynamic action potential clamp (dAPC) technique represents an alternative approach, in which a selected native ionic current of the ventricular myocyte can effectively be replaced with wild-type (WT) or mutant current recorded from a human embryonic kidney (HEK)-293 cell that is voltage clamped by the free-running action potential (AP) of the myocyte. Both a computed model of the human ventricular cell and a freshly isolated myocyte can effectively be used in dAPC experiments, resulting in rapid and unambiguous determination of the effect(s) of an ion channel mutation on the ventricular AP. The dAPC technique represents a promising new tool to study various cardiac ion channels and may also prove useful in related fields of research, for example, in neurophysiology.

Tonoplast anion channel activity modulation by pH in Chara corallina

Abstract. The patch-clamp technique was used to investigate regulation of anion channel activity in the tonoplast of Chara corallina in response to changing proton and calcium concentrations on both sides of the membrane. These channels are known to be Ca2+-dependent, with conductances in the range of 37 to 48 pS at pH 7.4. By using low pH at the vacuolar side (either pHvac 5.3 or 6.0) and a cytosolic pH (pHcyt) varying in a range of 4.3 to 9.0, anion channel activity and single-channel conductance could be reversibly modulated. In addition, Ca2+-sensitivity of the channels was markedly influenced by pH changes. At pHcyt values of 7.2 and 7.4 the half-maximal concentration (EC50) for calcium activation was 100–200 μm, whereas an EC50 of about 5 μm was found at a pHcyt of 6.0. This suggests an improved binding of Ca2+ ions to the channel protein at more acidic cytoplasm. At low pHcyt, anion channel activity and mean open times were voltage-dependent. At pipette potentials (Vp) of +100 mV, channel activity was approximately 15-fold higher than activity at negative pipette potentials and the mean open time of the channel increased. In contrast, at pHcyt 7.2, anion channel activity and the opening behavior seemed to be independent of the applied Vp. The kinetics of the channel could be further controlled by the Ca2+ concentration at the cytosolic membrane side: the mean open time significantly increased in the presence of a high cytosolic Ca2+ concentration. These results show that tonoplast anion channels are maintained in a highly active state in a narrow pH range, below the resting pHcyt. A putative physiological role of the pH-dependent modulation of these anion channels is discussed.