Results of phase 2 randomized multi-center study to evaluate the safety and efficacy of infusion of memory T cells as adoptive therapy in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia and/or lymphopenia (RELEASE NCT04578210).

BACKGROUND AIMS: There are currently no effective anti-viral treatments for coronavirus disease 2019 (COVID-19)-hospitalized patients with hypoxemia. Lymphopenia is a biomarker of disease severity usually present in patients who are hospitalized. Approaches to increasing lymphocytes exerting an anti-viral effect must be considered to treat these patients. Following our phase 1 study, we performed a phase 2 randomized multicenter clinical trial in which we evaluated the efficacy of the infusion of allogeneic off-the-shelf CD45RA- memory T cells containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells from convalescent donors plus the standard of care (SoC) versus just the SoC treatment. METHODS: Eighty-four patients were enrolled in three Spanish centers. The patients were randomized into the infusion of 1 × 106/kg CD45RA- memory T cells or the SoC. We selected four unvaccinated donors based on the expression of interferon gamma SARS-CoV-2-specific response within the CD45RA- memory T cells and the most frequent human leukocyte antigen typing in the Spanish population. RESULTS: We analyzed data from 81 patients. The primary outcome for recovery, defined as the proportion of participants in each group with normalization of fever, oxygen saturation sustained for at least 24 hours and lymphopenia recovery through day 14 or at discharge, was met for the experimental arm. We also observed faster lymphocyte recovery in the experimental group. We did not observe any treatment-related adverse events. CONCLUSIONS: Adoptive cell therapy with off-the-shelf CD45RA- memory T cells containing SAR-CoV-2-specific T cells is safe, effective and accelerates lymphocyte recovery of patients with COVID-19 pneumonia and/or lymphopenia. TRIAL REGISTRATION: NCT04578210.

G-protein tonic inhibition of calcium channels in pancreatic ß-cells.

Voltage-gated calcium channels (CaV) conduct Ca2+ influx promoting neurotransmitters and hormone release. CaV are finely regulated by voltage-dependent and independent pathways either by G-protein-coupled receptors (GPCRs) or intramembrane lipids, respectively, in neurons and glands. Interestingly, pancreatic ß-cells are abundantly innervated by both sympathetic and parasympathetic neurons, while a variety of high-voltage activated (HVA) Ca2+ channels are present in these cells. Thus, autonomic system seems to exert a tonic inhibition on HVA Ca2+ channels throughout GPCRs, constitutively preventing hormone secretion. Therefore, this work aimed to investigate noradrenergic and cholinergic inhibition of HVA Ca2+ channels in pancreatic ß-cells. Experiments were conducted in pancreatic ß-cells of rat by using patch-clamping methods, immunocytochemistry, pharmacological probes, and biochemical reagents. A voltage-clamp protocol with a strong depolarizing prepulse was used to unmask tonic inhibition. Herein, we consistently find a basal tonic inhibition of HVA Ca2+ channels according to a GPCRs regulation. Facilitation ratio is enhanced by noradrenaline (NA) according to a voltage-dependent regulation and a membrane-delimited mechanism, while no facilitation changes are observed with carbachol or phosphatidylinositol 4,5-bisphosphate (PIP2). Furthermore, carbachol or intramembrane lipids, such as PIP2, do not change facilitation ratio according to a voltage-independent regulation. Together, HVA Ca2+ channels of pancreatic ß-cells are constitutively inhibited by GPCRs, suggesting a natural brake preventing cells from exhaustive insulin secretion.NEW & NOTEWORTHY Our results support the hypothesis that GPCRs tonically inhibit HVA Ca2+ channels in pancreatic ß-cells. A voltage-clamp protocol with a strong depolarizing prepulse was used to unmask voltage-dependent inhibition of Ca2+ channels. The novelty of these results strengthens the critical role of Gßγ's in Ca2+ channel regulation, highlighting kinetic slowing and increased facilitation ratio. Together, HVA Ca2+ channels of pancreatic ß-cells are constitutively inhibited by GPCRs underlying fine-tuning modulation of insulin secretion.

Inactivation of potassium channels by ceramide in rat pancreatic ß-cells.

Lipid regulation of ion channels is a fundamental mechanism in physiological processes as of neurotransmitter release and hormone secretion. Ceramide is a bioactive lipid proposed as a regulator of several voltage-gated ion channels including potassium channels (Kv). It is generated either de novo or by sphingomyelin (SM) hydrolysis in membranes of mammalian cells. In pancreatic ß-cells, ceramide is the main sphingolipid associated with lipotoxicity and likely involved in cell dysfunction. Despite of the wealth of information regarding regulation of potassium channels by ceramides, the regulation of Kv channels by accumulated ceramide in native pancreatic ß-cells has not been investigated. To do so, we used either the C2-ceramide, a cell-permeable short-chain analogue, or a sphingomyelinase (SMase C), a hydrolase causing ceramide to elevate from an endogenous production, in pancreatic ß-cells of rat. C2-ceramide markedly accelerates steady-state current inactivation according to kinetic changes in the channel machinery. Interestingly, only C2-ceramide accelerates current inactivation while SMase C decreases both, peak-current and step-current amplitude supporting differential effects of ceramide derivatives. A specific inhibitor of the Kv2.1 channel (GxTX-1E), readily inhibits a fraction of the Kv channel current while no further inhibition by C2-ceramide superfusion can be observed supporting Kv2.1 channel involvement in the ceramide inhibition. Thus, intramembrane ceramide accumulation, as a lipidic metabolite released under cell-stress conditions, may alter pancreatic ß-cell repolarization and secretion. These results may provide a new insight regarding lipid-protein regulation and advance our understanding in ceramide actions on Kv channels in pancreatic ß-cells.

A Sea Anemone Lebrunia neglecta Venom Fraction Decreases Boar Sperm Cells Capacitation: Possible Involvement of HVA Calcium Channels.

Sea anemones produce venoms characterized by a complex mixture of low molecular weight compounds, proteins and peptides acting on voltage-gated ion channels. Mammal sperm cells, like neurons, are characterized by their ion channels. Calcium channels seem to be implicated in pivotal roles such as motility and capacitation. In this study, we evaluated the effect of a low molecular weight fraction from the venom of the sea anemone Lebrunia neglecta on boar sperm cells and in HVA calcium channels from rat chromaffin cells. Spermatozoa viability seemed unaffected by the fraction whereas motility and sperm capacitation were notoriously impaired. The sea anemone fraction inhibited the HVA calcium current with partial recovery and no changes in chromaffin cells' current kinetics and current-voltage relationship. These findings might be relevant to the pharmacological characterization of cnidarian venoms and toxins on voltage-gated calcium channels.

Potential Therapeutic Applications of Synthetic Conotoxin s-cal14.2b, Derived from Californiconus californicus, for Treating Type 2 Diabetes.

The FDA's approval of peptide drugs such as Ziconotide or Exendin for pain relief and diabetes treatment, respectively, enhanced the interest to explore novel conotoxins from Conus species venom. In general, conotoxins can be used in pathologies where voltage-gated channels, membrane receptors, or ligands alter normal physiological functions, as in metabolic diseases such as Type 2 diabetes. In this study, the synthetic cal14.2b (s-cal14.2b) from the unusual Californiconus californicus demonstrated bioactivity on NIT-1 insulinoma cell lines stimulating insulin secretion detecting by high performance liquid chromatography (HPLC). Accordingly, s-cal14.2b increased the CaV1.2/1.3 channel-current by 35 ± 4% with a recovery τ of 10.3 ± 4 s in primary cell culture of rat pancreatic ß-cells. The in vivo results indicated a similar effect of insulin secretion on mice in the glucose tolerance curve model by reducing the glucose from 500 mg/dL to 106 mg/dL in 60 min, compared to the negative control of 325 mg/dL at the same time. The PET-SCAN with radiolabeling 99mTc-s-cal14.2b demonstrated biodistribution and accumulation in rat pancreas with complete depuration in 24 h. These findings show the potential therapeutic use of s-cal14.2b in endocrinal pathologies such as early stages of Type 2 Diabetes where the pancreas's capability to produce insulin is still effective.

Motor learning impairment in rats under a high sucrose diet.

Motor learning skills are reliable indicators of behavioral acquisition and cognitive disorders. The ease with which learning skills are measured disparities the complexity of the interpretation concerning neural plasticity. Conversely, a wealth of information regarding metabolic derangements has long been reported with direct connection to high sucrose diets. However, the impact of excessive sucrose consumption on undergoing cognitive processes has been only scarcely addressed up to now. Therefore, the goal of this work was to describe the associative relationship between high sucrose consumption and changes in motor learning skills acquisition. Motor learning impairments conditioned by central alterations are hypothesized. Rotarod, elevated plus-maze and open field trials, along with metabolic and pro-inflammatory biomarkers tests in Wistar rats under a high sucrose treatment, were performed. Motor learning impairment in high sucrose diet-treated rats was found while spontaneous locomotor activity remained unchanged. Even though, no anxiety-like behavior under high sucrose diet-treatment was observed. Consistently, the worst outcome in the glucose tolerance test was developed, the worst motor learning performance was observed. Furthermore, insulin resistance correlated positively with a pro-inflammatory state and a decreased latency to fall in the rotarod test. Indeed, C-reactive protein and tumor necrosis factor-α serum levels, along with the homeostasis model assessment of insulin resistance (HOMA-IR), significantly increased in motor learning impairment. Together, these results support behavioral, metabolic and pro-inflammatory changes associated with deleterious changes in central nervous system likely involving crucial motor learning structures. Underlying pro-inflammatory-triggered processes may explain cognitive disorders in advanced states of metabolic derangements.

Maintenance of CaV2.2 channel-current by PIP2 unveiled by neomycin in sympathetic neurons of the rat.

Membrane lipids are key determinants in the regulation of voltage-gated ion channels. Phosphatidylinositol 4,5-bisphosphate (PIP2), a native membrane phospholipid, has been involved in the maintenance of the current amplitude and in the voltage-independent regulation of voltage-gated calcium channels (VGCC). However, the nature of the PIP2 regulation on VGCC has not been fully elucidated. This work aimed to investigate whether the interacting PIP2 electrostatic charges may account for maintaining the current amplitude of CaV2.2 channels. Furthermore, we tested whether charge shielding of PIP2 mimics the voltage-independent inhibition induced by M1 muscarinic acetylcholine receptor (M1R) activation. Therefore, neomycin, a polycation that has been shown to block electrostatic interactions of PIP2, was intracellularly dialyzed in superior cervical ganglion (SCG) neurons of the rat. Consistently, neomycin time-dependently diminished the calcium current amplitude letting the channel exhibit the hallmarks of the voltage-independent regulation. These results support that interacting PIP2 charges not only underly the maintenance of the channel-current but also that charge screening of PIP2 by itself unveils the voltage-independent features of CaV2.2 channels in SCG neurons.

Dynamical phase transition in spike neuronal firing patterns of hippocampal cells.

There is increasing evidence that the brain resides in a state of criticality. The purpose of the present work is to characterize the dynamics of individual hippocampal CA1 pyramidal cells and to investigate how it is influenced by changes in Kv7.2/7.3 (M-channel) ion channel modulation, which is known to be key in determining the neuronal excitability. We show that the resting activity of CA1 neurons exhibit random dynamics with low information content, while changes in M-channel modulation move the neuronal activity near a phase transition to richer non-trivial dynamics. We interpret these results as the basis upon which the state of self-organized criticality is built.

Electrophysiological characterization of glucose sensing neurons in the hypothalamic arcuate nucleus of male rats.

The arcuate nucleus (ARC), located at the base of hypothalamus, contains two main populations of neurons involved in the regulation of food intake and energy expenditure. The NPY neurons are orexigenic and their activation stimulates food intake while the activation of POMC neurons promote the opposite effect. Several works have tried to identify these neurons based on their electrophysiological and pharmacological characteristics. However, the classification of ARC neurons is still inconclusive. In this work, glucose concentrations were changed within at physiological range, and the response of rat ARC neurons to this stimulus was used to identify them. Subsequently, the cells were classified on the basis of their passive and active electrophysiological properties. Finally, calcium imaging experiments were done to study the response of ARC neurons populations changing glucose concentrations. We found that NPY and putative POMC neurons can be distinguished based on their electrophysiological properties such as input resistance and firing pattern. Calcium imaging experiments confirmed the diversity of ARC neurons.

Fast Inactivation of CaV2.2 Channels Is Prevented by the Gß1 Subunit in Rat Sympathetic Neurons.

Voltage-dependent regulation of CaV2.2 channels by G-proteins is performed by the ß (Gß) subunit. Most studies of regulation by G-proteins have focused on channel activation; however, little is known regarding channel inactivation. This study investigated inactivation of CaV2.2 channels in superior cervical ganglion neurons that overexpressed Gß subunits. CaV2.2 currents were recorded by whole-cell patch clamping configuration. We found that the Gß1 subunit reduced inactivation, while Gß5 subunit did not alter at all inactivation kinetics compared to control recordings. CaV2.2 current decay in control neurons consisted of both fast and slow inactivation; however, Gß1-overexpressing neurons displayed only the slow inactivation. Fast inactivation was restored by a strong depolarization of Gß1-overexpressing neurons, therefore, through a voltage-dependent mechanism. The Gß1 subunit shifted the voltage dependence of inactivation to more positive voltages and reduced the fraction of CaV2.2 channels resting in the inactivated state. These results support that the Gß1 subunit inhibits the fast inactivation of CaV2.2 channels in SCG neurons. They explain the long-observed sustained Ca2+ current under G-protein modulation.

PIP2 in pancreatic ß-cells regulates voltage-gated calcium channels by a voltage-independent pathway.

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a membrane phosphoinositide that regulates the activity of many ion channels. Influx of calcium primarily through voltage-gated calcium (CaV) channels promotes insulin secretion in pancreatic ß-cells. However, whether CaV channels are regulated by PIP2, as is the case for some non-insulin-secreting cells, is unknown. The purpose of this study was to investigate whether CaV channels are regulated by PIP2 depletion in pancreatic ß-cells through activation of a muscarinic pathway induced by oxotremorine methiodide (Oxo-M). CaV channel currents were recorded by the patch-clamp technique. The CaV current amplitude was reduced by activation of the muscarinic receptor 1 (M1R) in the absence of kinetic changes. The Oxo-M-induced inhibition exhibited the hallmarks of voltage-independent regulation and did not involve PKC activation. A small fraction of the Oxo-M-induced CaV inhibition was diminished by a high concentration of Ca2+ chelator, whereas ≥50% of this inhibition was prevented by diC8-PIP2 dialysis. Localization of PIP2 in the plasma membrane was examined by transfecting INS-1 cells with PH-PLCδ1, which revealed a close temporal association between PIP2 hydrolysis and CaV channel inhibition. Furthermore, the depletion of PIP2 by a voltage-sensitive phosphatase reduced CaV currents in a way similar to that observed following M1R activation. These results indicate that activation of the M1R pathway inhibits the CaV channel via PIP2 depletion by a Ca2+-dependent mechanism in pancreatic ß- and INS-1 cells and thereby support the hypothesis that membrane phospholipids regulate ion channel activity by interacting with ion channels.

Activity of Palythoa caribaeorum Venom on Voltage-Gated Ion Channels in Mammalian Superior Cervical Ganglion Neurons.

The Zoanthids are an order of cnidarians whose venoms and toxins have been poorly studied. Palythoa caribaeorum is a zoanthid commonly found around the Mexican coastline. In this study, we tested the activity of P. caribaeorum venom on voltage-gated sodium channel (NaV1.7), voltage-gated calcium channel (CaV2.2), the A-type transient outward (IA) and delayed rectifier (IDR) currents of KV channels of the superior cervical ganglion (SCG) neurons of the rat. These results showed that the venom reversibly delays the inactivation process of voltage-gated sodium channels and inhibits voltage-gated calcium and potassium channels in this mammalian model. The compounds responsible for these effects seem to be low molecular weight peptides. Together, these results provide evidence for the potential use of zoanthids as a novel source of cnidarian toxins active on voltage-gated ion channels.

Voltage-Independent Inhibition of the Tetrodotoxin-Sensitive Sodium Currents by Oxotremorine and Angiotensin II in Rat Sympathetic Neurons.

Tetrodotoxin-sensitive Na(+) currents have been extensively studied because they play a major role in neuronal firing and bursting. In this study, we showed that voltage-dependent Na(+) currents are regulated in a slow manner by oxotremorine (oxo-M) and angiotensin II in rat sympathetic neurons. We found that these currents can be readily inhibited through a signaling pathway mediated by G proteins and phospholipase C (PLC) ß1. This inhibition is slowly established, pertussis toxin-insensitive, partially reversed within tens of seconds after oxo-M washout, and not relieved by a strong depolarization, suggesting a voltage-insensitive mechanism of inhibition. Specificity of the M1 receptor was tested by the MT-7 toxin. Activation and inactivation curves showed no shift in the voltage dependency under the inhibition by oxo-M. This inhibition is blocked by a PLC inhibitor (U73122, 1-(6-{[(17ß)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione), and recovery from inhibition is prevented by wortmannin, a PI3/4 kinase inhibitor. Hence, the pathway involves Gq/11 and is mediated by a diffusible second messenger. Oxo-M inhibition is occluded by screening phosphatidylinositol 4,5-bisphosphate (PIP2)-negative charges with poly-l-lysine and prevented by intracellular dialysis with a PIP2 analog. In addition, bisindolylmaleimide I, a specific ATP-competitive protein kinase C (PKC) inhibitor, rules out that this inhibition may be mediated by this protein kinase. Furthermore, oxo-M-induced suppression of Na(+) currents remains unchanged when neurons are treated with calphostin C, a PKC inhibitor that targets the diacylglycerol-binding site of the kinase. These results support a general mechanism of Na(+) current inhibition that is widely present in excitable cells through modulation of ion channels by specific G protein-coupled receptors.

A purified Palythoa venom fraction delays sodium current inactivation in sympathetic neurons.

Palythoa caribaeorum is a zoanthid (Phylum Cnidaria, class Anthozoa) commonly found in shallow waters of coral reefs along the Mexican Atlantic coast. Little is known on the pharmacological and biochemical properties of the venom components of this animal group. Toxin peptides from other cnidarian venoms, like sea anemones, target sodium and potassium voltage-gated channels. In this study, we tested the activity of a low molecular weight fraction from the venom of P. caribaeorum on voltage-gated sodium channels of the superior cervical ganglion (SCG) neurons of the rat. Our results showed that this fraction delays tetrodotoxin (TTX)-sensitive sodium channel inactivation indicated by a reversible 2-fold increase of the current at the decay. A peptide responsible for this activity was isolated and characterized. Its sequence showed that it does not resemble any previously reported toxin. Together, these results evidence the presence of neurotoxins in P. caribaeorum that act on sodium channels.

Gß2 mimics activation kinetic slowing of CaV2.2 channels by noradrenaline in rat sympathetic neurons.

Several neurotransmitters and hormones acting through G protein-coupled receptors elicit a voltage-dependent regulation of CaV2.2 channels, having profound effects on cell function and the organism. It has been hypothesized that protein-protein interactions define specificity in signal transduction. Yet it is unknown how the molecular interactions in an intracellular signaling cascade determine the specificity of the voltage-dependent regulation induced by a specific neurotransmitter. It has been suspected that specific effector regions on the Gß subunits of the G proteins are responsible for voltage-dependent regulation. The present study examines whether a neurotransmitter's specificity can be revealed by simple ion-current kinetic analysis likely resulting from interactions between Gß subunits and the channel-molecule. Noradrenaline is a neurotransmitter that induces voltage-dependent regulation. By using biochemical and patch-clamp methods in rat sympathetic neurons we examined calcium current modulation induced by each of the five Gß subunits and found that Gß2 mimics activation kinetic slowing of CaV2.2 channels by noradrenaline. Furthermore, overexpression of the Gß2 isoform reproduces the effect of noradrenaline in the willing-reluctant model. These results advance our understanding on the mechanisms by which signals conveying from a variety of membrane receptors are able to display precise homeostatic responses.

A Conus regularis conotoxin with a novel eight-cysteine framework inhibits CaV2.2 channels and displays an anti-nociceptive activity.

A novel peptide, RsXXIVA, was isolated from the venom duct of Conus regularis, a worm-hunting species collected in the Sea of Cortez, México. Its primary structure was determined by mass spectrometry and confirmed by automated Edman degradation. This conotoxin contains 40 amino acids and exhibits a novel arrangement of eight cysteine residues (C-C-C-C-CC-CC). Surprisingly, two loops of the novel peptide are highly identical to the amino acids sequence of ω-MVIIA. The total length and disulfide pairing of both peptides are quite different, although the two most important residues for the described function of ω-MVIIA (Lys2 and Tyr13) are also present in the peptide reported here. Electrophysiological analysis using superior cervical ganglion (SCG) neurons indicates that RsXXIVA inhibits CaV2.2 channel current in a dose-dependent manner with an EC50 of 2.8 µM, whose effect is partially reversed after washing. Furthermore, RsXXIVA was tested in hot-plate assays to measure the potential anti-nociceptive effect to an acute thermal stimulus, showing an analgesic effect in acute thermal pain at 30 and 45 min post-injection. Also, the toxin shows an anti-nociceptive effect in a formalin chronic pain test. However, the low affinity for CaV2.2 suggests that the primary target of the peptide could be different from that of ω-MVIIA.

PIP2 hydrolysis is responsible for voltage independent inhibition of CaV2.2 channels in sympathetic neurons.

GPCRs regulate Ca(V)2.2 channels through both voltage dependent and independent inhibition pathways. The aim of the present work was to assess the phosphatidylinositol-4,5-bisphosphate (PIP2) as the molecule underlying the voltage independent inhibition of Ca(V)2.2 channels in SCG neurons. We used a double pulse protocol to study the voltage independent inhibition and changed the PIP(2) concentration by means of blocking the enzyme PLC, filling the cell with a PIP(2) analogue and preventing the PIP(2) resynthesis with wortmannin. We found that voltage independent inhibition requires the activation of PLC and can be hampered by internal dialysis of exogenous PIP(2). In addition, the recovery from voltage independent inhibition is blocked by inhibition of the enzymes involved in the resynthesis of PIP(2). These results support that the hydrolysis of PIP(2) is responsible for the voltage independent inhibition of Ca(V)2.2 channels.

A novel CaV2.2 channel inhibition by piracetam in peripheral and central neurons.

No mechanistic actions for piracetam have been documented to support its nootropic effects. Voltage-gated calcium channels have been proposed as a promising pharmacological target of nootropic drugs. In this study, we investigated the effect of piracetam on Ca(V)2.2 channels in peripheral neurons, using patch-clamp recordings from cultured superior cervical ganglion neurons. In addition, we tested if Ca(V)2.2 channel inhibition could be related with the effects of piracetam on central neurons. We found that piracetam inhibited native Ca(V)2.2 channels in superior cervical ganglion neurons in a dose-dependent manner, with an IC(50) of 3.4 µmol/L and a Hill coefficient of 1.1. GDPßS dialysis did not prevent piracetam-induced inhibition of Ca(V)2.2 channels and G-protein-coupled receptor activation by noradrenaline did not occlude the piracetam effect. Piracetam altered the biophysical characteristics of Ca(V)2.2 channel such as facilitation ratio. In hippocampal slices, piracetam and ω-conotoxin GVIA diminished the frequency of excitatory postsynaptic potentials and action potentials. Our results provide evidence of piracetam's actions on Ca(V)2.2 channels in peripheral neurons, which might explain some of its nootropic effects in central neurons.

Voltage-independent inhibition of Ca(V)2.2 channels is delimited to a specific region of the membrane potential in rat SCG neurons.

Neurotransmitters and hormones regulate Ca(V)2.2 channels through a voltage-independent pathway which is not well understood. It has been suggested that this voltage-independent inhibition is constant at all membrane voltages. However, changes in the percent of voltage-independent inhibition of Ca(V)2.2 have not been tested within a physiological voltage range. Here, we used a double-pulse protocol to isolate the voltage-independent inhibition of Ca(V)2.2 channels induced by noradrenaline in rat superior cervical ganglion neurons. To assess changes in the percent of the voltage-independent inhibition, the activation voltage of the channels was tested between -40 and +40 mV. We found that the percent of voltage-independent inhibition induced by noradrenaline changed with the activation voltage used. In addition, voltage-independent inhibition induced by oxo-M, a muscarinic agonist, exhibited the same dependence on activation voltage, which supports that this pattern is not exclusive for adrenergic activation. Our results suggested that voltage-independent inhibition of Ca(V)2.2 channels depends on the activation voltage of the channel in a physiological voltage range. This may have relevant implications in the understanding of the mechanism involved in voltage-independent inhibition.

Gating charges per channel of Ca(V)2.2 channels are modified by G protein activation in rat sympathetic neurons.

It has been suggested that voltage-dependent G protein modulation of Ca(V)2.2 channels is carried out at closed states of the channel. Our purpose was to estimate the number of gating charges of Ca(V)2.2 channel in control and G protein-modulated conditions. By using a Cole-Moore protocol we observed a significant delay in Ca(V)2.2 channel activation according to a transit of the channel through a series of closed states before channel opening. If G protein voltage-dependent modulation were carried out at these closed states, then we would have expected a greater Cole-Moore lag in the presence of a neurotransmitter. This prediction was confirmed for noradrenaline, while no change was observed in the presence of angiotensin II, a voltage-insensitive G protein modulator. We used the limiting slope method for calculation of the gating charge per channel. Effective charge z was 6.32+/-0.65 for Ca(V)2.2 channels in unregulated conditions, while GTPgammaS reduced elementary charge by approximately 4 e(0). Accordingly, increased concentration of noradrenaline induced a gradual decrease on z, indicating that this decrement was due to a G protein voltage-sensitive modulation. This paper shows for the first time a significant and reversible decrease in charge transfer of Ca(V)2.2 channels under G protein modulation, which might depend on the activated G protein inhibitory pathway.