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PubMed2005

Firing pattern and rapid modulation of activity by estrogen in primate luteinizing hormone releasing hormone-1 neurons.

We have shown previously that cultured LHRH-1 neurons, derived from monkey olfactory placode region, exhibit pulsatile LHRH-1 release at hourly intervals and spontaneous intracellular calcium oscillations, which synchronize at a frequency similar to LHRH-1 release. Brief application of estrogen induced a rapid increase in the frequency of intracellular calcium oscillations and the frequency of synchronizations. The estrogen-induced frequency of intracellular calcium oscillations was mediated by estrogen receptors (ER), whereas the frequency of synchronizations was not mediated by ER. In the present study, we further examined the rapid action of estrogen using patch-clamp recording in primate LHRH-1 neurons. Cell-attached patch-clamp recording showed that LHRH-1 neurons exhibited monophasic or biphasic action currents that were sensitive to an increase in extracellular K+ and the sodium channel blocker tetrodotoxin. The majority (90%) of LHRH-1 neurons showed irregular firing patterns composed of bursts and irregular beatings of action currents, which further formed a "cluster" firing pattern. Brief application of 17beta-estradiol (1 nM) increased the firing frequency and burst duration of LHRH-1 neurons with a latency of 60-120 sec for up to 25 min. ICI182,780, an ER antagonist, blocked the 17beta-estradiol-induced increase in the firing activity of LHRH-1 neurons. These results suggest that 1) primate LHRH-1 neurons exhibit complex firing patterns composed of activities with different time domains, 2) estrogen causes rapid stimulatory action of firing activity, and 3) this estrogen action is mediated by ER in primate LHRH-1 neurons.

Animals↗

PubMed2001

Angiotensin II type 1 receptor modulation of L-type calcium currents in guinea-pig ventricular cells.

OBJECTIVE: To study the cellular mechanism of the effect of Ang II on ICa,L in single guinea-pig ventricular cells by using losartan and 1-(5-lsoquinolinylsulfonyl)-2-Methyl-Piperazine (H-7) as the Ang II type 1 receptor (AT1) inhibitor and protein kinase C inhibitor, respectively. METHODS: Patch clamp techniques were used to study the cellular mechanism of the effect of Ang II on ICa,L in single guinea-pig ventricular cells. RESULTS: In the whole cell patch clamp recording model, Ang II stimulated ICa,L in a concentration dependent manner; the maximal effect was obtained at 100 nmol/L (n = 9). At 30 nmol/L, Ang II stimulated peak ICa,L from 11.3 +/- 0.6 pA/pF to 15.3 +/- 0.6 pA/pF (at + 10 mV, n = 9, P < 0.05). 100 nmol/L Losartan, a specific AT1 receptor inhibitor, had no effect on ICa,L (n = 9), but the effect of Ang II on ICa,L was inhibited by 100 nmol/L Losartan. Ang II on ICa,L was also inhibited by 20 mumol/L H-7, a specific protein kinase C inhibitor, whereas H-7 alone has no effect on ICa,L (n = 9). CONCLUSION: Ang II stimulates ICa,L in guinea-pig ventricular cells by binding to AT1 through a transduction pathway involving protein kinase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1998

PKC regulation of cardiac CFTR Cl- channel function in guinea pig ventricular myocytes.

The role of protein kinase C (PKC) in regulating the protein kinase A (PKA)-activated Cl- current conducted by the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (cCFTR) was studied in guinea pig ventricular myocytes using the whole cell patch-clamp technique. Although stimulation of endogenous PKC with phorbol 12,13-dibutyrate (PDBu) alone did not activate this Cl- current, even when intracellular dialysis was limited with the perforated patch-clamp technique, activation of PKC did elicit a significant response in the presence of PKA-dependent activation of the current by the beta-adrenergic receptor agonist isoproterenol. PDBu increased the magnitude of the Cl- conductance activated by a supramaximally stimulating concentration of isoproterenol by 21 +/- 3.3% (n = 9) when added after isoproterenol and by 36 +/- 16% (n = 14) when introduced before isoproterenol. 4alpha-Phorbol 12, 13-didecanoate, a phorbol ester that does not activate PKC, did not mimic these effects. Preexposure to chelerythrine or bisindolylmaleimide, two highly selective inhibitors of PKC, significantly reduced the magnitude of the isoproterenol-activated Cl- current by 79 +/- 7.7% (n = 11) and 52 +/- 10% (n = 8), respectively. Our results suggest that although acute activation of endogenous PKC alone does not significantly regulate cCFTR Cl- channel activity in native myocytes, it does potentiate PKA-dependent responses, perhaps most dramatically demonstrated by basal PKC activity, which may play a pivotal role in modulating the function of these channels.

Adrenergic beta-Agonists↗

PubMed1997

Effects of volatile anaesthetics on the membrane potential and ion channels of cultured neocortical astrocytes.

Volatile anaesthetics cause changes in the membrane resting potential of central neurons. This effect probably arises from actions on neuronal ion channels, but may also involve alterations in the ion composition of the extracellular space. Since glial cells play a key role in regulating the extracellular ion composition in the brains of mammals, we analyzed the effects of halothane, isoflurane and enflurane on the membrane conductances and ion channels of cultured cortical astrocytes. Astrocytes were dissociated from the neocortex of 0-2-day old rats and grown in culture for 3-4 weeks. Anaesthetic-induced changes in the membrane potential were recorded in the whole cell current-clamp configuration of the patch-clamp technique. We further studied the effects of halothane and enflurane on single ion channels in excised membrane patches. At concentrations corresponding to 1-2 MAC (1 MAC induces general anaesthesia in 50% of the patients and rats), membrane potentials recorded in the presence of enflurane, isoflurane and halothane did not differ significantly from the control values. At higher concentrations, effects of enflurane and halothane, but not of isoflurane, were statistically significant. Single-channel recordings revealed that halothane and enflurane activated a high conductance anion channel, which possibly mediated the effects observed during whole cell recordings. In less than 10% of the membrane patches, volatile anaesthetics either increased or decreased the mean open time of K+-selective ion channels without altering single-channel conductances. In summary, it seems unlikely that the actions of volatile anaesthetics described here are involved in the state of general anaesthesia. Statistically significant effects occurred at concentrations ten times higher than those required to cause half-maximal depression of action potential firing of neocortical neurons in cultured brain slices. However, it cannot be excluded that the changes observed in the membrane conductance of cortical astrocytes disturb the physiological function of these cells, thereby influencing the membrane resting potential of neurons.

Anesthetics, Inhalation↗

PubMed1993

Mechanism of rapid desensitization of muscarinic K+ current in adult rat and guinea pig atrial cells.

Acetylcholine (Ach) activates the muscarinic K+ current in atrial cells via the inhibitory GTP binding protein. After activation, the whole-cell K+ current decreases rapidly (rapid desensitization) to approximately half of the initial current within approximately 20 seconds. The mechanism of this rapid desensitization was investigated in adult rat and guinea pig atrial cells. Whole-cell voltage-clamp and patch-clamp techniques were used to study the K+ current. In voltage-clamped whole cells, ACh activated a K+ current that desensitized rapidly during the initial approximately 20 seconds followed by a slower decrease over several minutes. The rapid K+ current desensitization (a rapid decrease in channel open probability) was also observed at the single-channel level in cell-attached patches and was associated with a progressive shortening of the channel open time and prolongation of the closed time. These changes in channel current and kinetics were abolished by removal of the cytoplasm (by forming inside-out patches) and were partially inhibited by phosphatase inhibitors, suggesting an involvement of cytosolic phosphatase(s) in K+ current desensitization. In inside-out patches with ACh in the pipette and GTP in the bath, the open time of muscarinic K+ channels and channel open probability were increased by 1 mM Mg(2+)-ATP (but not by the nonhydrolyzable analogue, adenylylimidodiphosphate) and decreased by alkaline phosphatase. These results suggest that the rapid K+ current desensitization in adult rat or guinea pig atrial cells is produced by changes in the gating kinetics of the K+ channel, possibly mediated via membrane-associated protein kinase and cytosolic phosphatase(s).

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1988

Conductance and gating of epithelial Na channels from rat cortical collecting tubule. Effects of luminal Na and Li.

The behavior of individual Na channels in the apical membrane of the rat cortical collecting tubule (CCT) was studied at different concentrations of the permeant ions Na and Li. Tubules were opened to expose their luminal surfaces and bathed in K-gluconate medium to minimize tubule-to-tubule variation in cell membrane potential and intracellular Na concentration. The patch-clamp technique was used to resolve currents through individual channels. The patch-clamp pipette was filled with solutions containing variable concentrations of either NaCl or LiCl. In one series of experiments, the concentrations were changed without substitutions. In another series, the ionic strength and Cl concentration were maintained constant by partial substitution of Li with N-methyl-D-glucamine (NMDG). In cell-attached patches, both the single-channel conductance (g) and the single-channel current (i) saturated as functions of the Na or Li activity in the pipette. Without NMDG, the saturation of i was well described by Michaelis-Menten kinetics with an apparent Km of approximately 20 mM activity for Na and approximately 50 mM activity for Li. Km was independent of voltage for both ions. With substitution for Li by NMDG, the apparent Km value for Li transport through the channels increased. The values of the probability of a channel's being open (Po) varied from patch to patch, but no effect of pipette ion activity on Po could be demonstrated. A weak dependence of Po on membrane voltage was observed, with hyperpolarization increasing Po by an average of 2.3%/mV.

Animals↗

PubMed2004

Rb+ flux through hERG channels affects the potency of channel blocking drugs: correlation with data obtained using a high-throughput Rb+ efflux assay.

The nonradioactive Rb+ efflux assay has become a reliable and efficient high-throughput hERG screening method, but it is limited by its low sensitivity for potent hERG blockers. Using the patch clamp technique, the authors found that the low sensitivity is due in part to the use of Rb+ as the permeating cation in the assay. The affinities of the drugs measured by patch clamp technique in the presence of Rb+ were 3- to 10-fold lower than when measured by the same method in the presence of K+ ions. The apparent affinity of the drugs decreased even further when monitored by the Rb+ efflux assay. It was also observed that Rb+ had minimal effects on the activation properties of channels while there was a significant change in the half-inactivation potential. This voltage shift reduces hERG channel inactivation at efflux assay potentials, and will reduce the affinity of hERG-blocking drugs that bind to inactivated states of the channel. In combination with the effects of elevated extracellular ion concentrations, it is likely that Rb+ modulation of hERG channel inactivation is largely responsible for the reduced drug potencies observed in the Rb+ efflux assay.

Biological Assay↗

PubMed1996

Expression of glycine receptor subunits in glial cells of the rat spinal cord.

We previously demonstrated that the inhibitory neurotransmitter glycine induced membrane currents in glial cells from rat spinal cord. In this present study, the patch-clamp technique was combined with the reverse transcription-mediated PCR to analyze the glycine receptor-subunit expression in individual glial cells of rats age 3-18 days. Using the patch-clamp technique in the whole-cell configuration, glial cells were identified by their membrane current pattern and tested for responsiveness to glycine. Subsequently, the cytoplasm was harvested followed by reverse transcription of total cytoplasmic RNA. Subunit-specific cDNA fragments were amplified and analyzed by agarose gel electrophoresis, Southern blotting, and sequencing. In all cell types investigated, transcripts of the alpha1 subunit, but not of alpha 2 or alpha 3 subunits, were detected. In addition, about one-half the glial cells analyzed contained beta-subunit mRNA. These results illustrate that glial cells of rat spinal cord express functional glycine receptors in contrast to cultured glial cells. Glial cells are in intimate contact with synaptic regions making it likely that these nonneuronal receptors may be activated during glycinergic transmission and may trigger yet unknown responses in the glial cells.

Age Factors↗

PubMed2003

L-type Ca2+ channel and Ca2+-permeable nonselective cation channel as a Ca2+ conducting pathway in myocytes isolated from the cricket lateral oviduct.

The Ca2+-conducting pathway of myocytes isolated from the cricket lateral oviduct was investigated by means of the whole-cell patch clamp technique. In voltage-clamp configuration, two types of whole cell inward currents were identified. One was voltage-dependent, initially activated at -40 mV and reaching a maximum at 10 mV with the use of 140 mM Cs2+-aspartate in the patch pipette and normal saline in the bath solution. Replacement of the external Ca2+ with Ba2+ slowed the current decay. Increasing the external Ca2+ or Ba2+ concentration increased the amplitude of the inward current and the current-voltage (I-V) relationship was shifted as expected from a screening effect on negative surface charges. The inward current could be carried by Na+ in the absence of extracellular Ca2+. Current carried by Na+ (INa) was almost completely blocked by the dihydropyridine Ca2+ channel antagonist, nifedipine, suggesting that the INa is through voltage-dependent L-type Ca2+ channels. The other inward current is voltage-independent and its I-V relationship was linear between -100 mV to 0 mV with a slight inward rectification at more hyperpolarizing membrane potentials when 140 mM Cs+-aspartate and 140 mM Na+-gluconate were used in the patch pipette and in the bath solution, respectively. A similar current was observed even when the external Na+ was replaced with an equimolar amount of K+ or Cs+, or 50 mM Ca2+ or Ba2+. When the osmolarity of the bath solution was reduced by removing mannitol from the bath solution, the inward current became larger at negative potentials. The I-V relationship for the current evoked by the hypotonic solution also showed a linear relationship between -100 mV to 0 mV. Bath application of Gd3+ (10 microM) decreased the inward current activated by membrane hyperpolarization. These results clearly indicate that the majority of current activated by a membrane hyperpolarization is through a stretch-activated Ca2+-permeable nonselective cation channel (NSCC). Here, for the first time, we have identified voltage-dependent L-type Ca2+ channel and stretch-activated Ca2+-permeable NSCCs from enzymatically isolated muscle cells of the cricket using the whole-cell patch clamp recording technique.

Animals↗

PubMed2003

Optical single-channel recording: imaging Ca2+ flux through individual N-type voltage-gated channels expressed in Xenopus oocytes.

Functional studies of single membrane ion channels were made possible by the introduction of the patch-clamp technique, which allows single-channel currents to be measured with unprecedented resolution. Nevertheless, patch clamping has some limitations: including the need for physical access of the patch pipette, possible disruption of local cellular architecture, inability to monitor multiple channels, and lack of spatial information. Here, we demonstrate the use of confocal fluorescence microscopy as a non-invasive technique to optically monitor the gating of individual Ca2+ channels. Near-membrane fluorescence signals track the gating of N-type Ca2+ channels with a kinetic resolution of about 10ms, provide a simultaneous and independent readout from several channels, and allow their locations to be mapped with sub-micrometer spatial resolution. Optical single-channel recording should be applicable to diverse voltage- and ligand-gated Ca2+-permeable channels, and has the potential for high-throughput functional analysis of single channels.

Animals↗

PubMed2003

Electrophysiological analysis of neuronal chemokine receptors.

Several studies have shown that neurons in the central and peripheral nervous systems express a variety of chemokine receptors (CKRs). Activation of these receptors can influence neuronal signaling by regulating synaptic transmission and neuronal excitability. This article presents electrophysiological methods that are currently used to study the normal and pathophysiological role for CKRs in the nervous system. Conventional electrophysiological methods such as patch-clamp recording of isolated neurons, brain slices, and heterologous expression systems are described. In addition, single-cell reverse transcription-polymerase chain reaction is discussed as a technique that can be used in conjunction with patch-clamp recording to further investigate the molecular basis of neuronal CKR activation.

Animals↗

PubMed1997

Chloride- and voltage-dependent Ca2+ transient in cultured human aortic endothelial cells.

The Cl(-)-sensitive nature of histamine-induced Ca2+ entry was investigated in cultured human aortic endothelial cells by the patch-clamp method, combined with the fluorescence measurement of intracellular Ca2+ concentration ([Ca2+]i). The sustained increase in [Ca2+]i, caused by histamine, was decreased by lowering the external concentration of Cl-. The patch-clamp experiment revealed that [Ca2+]i was dependent on the membrane potential in the presence of histamine, but not in the absence of histamine. The sustained increase in [Ca2+]i was accompanied by activation of the Cl- current, of which the reversal potential was measured to be around -28 mV with physiological internal and external Cl- concentrations. The membrane potential of the endothelial cells varied from -90 to 0 mV in the resting condition and approached the equilibrium potential for Cl- during histamine application. The results indicate that the histamine-induced Cl- current plays a functional role in the sustained increase of intracellular Ca2+ during histamine application by providing a constant driving force for Ca2+ entry across the plasma membrane.

Aorta↗

PubMed2006

Increased sensitivity of desensitized TRPV1 by PMA occurs through PKCepsilon-mediated phosphorylation at S800.

Important mechanisms that regulate inhibitory and facilitatory effects on TRPV1-mediated nociception are desensitization and phosphorylation, respectively. Using Ca2+-imaging, we have previously shown that desensitization of TRPV1 upon successive capsaicin applications was reversed by protein kinase C activation in dorsal root ganglion neurons and CHO cells. Here, using both Ca2+-imaging and patch-clamp methods, we show that PMA-induced activation of PKCepsilon is essential for increased sensitivity of desensitized TRPV1. TRPV1 has two putative substrates S502 and S800 for PKCepsilon-mediated phosphorylation. Patch-clamp analysis showed that contribution of single mutant S502A or S800A towards increased sensitivity of desensitized TRPV1 is indistinguishable from that observed in a double mutant S502A/S800A. Since S502 is a non-specific substrate for TRPV1 phosphorylation by kinases like PKC, PKA or CAMKII, evidence for a role of PKC specific substrate S800 was investigated. Evidence for in vivo phosphorylation of TRPV1 at S800 was demonstrated for the first time. We also show that the expression level of PKCepsilon paralleled the amount of phosphorylated TRPV1 protein using an antibody specific for phosphorylated TRPV1 at S800. Furthermore, the anti-phosphoTRPV1 antibody detected phosphorylation of TRPV1 in mouse and rat DRG neurons and may be useful for research regarding nociception in native tissues. This study, therefore, identifies PKCepsilon and S800 as important therapeutic targets that may help regulate inhibitory effects on TRPV1 and hence its desensitization.

Amino Acid Sequence↗

PubMed2001

The voltage-dependent Cl(-) channel ClC-5 and plasma membrane Cl(-) conductances of mouse renal collecting duct cells (mIMCD-3).

1. We have tested the hypothesis that the voltage-dependent Cl(-) channel, ClC-5 functions as a plasma membrane Cl(-) conductance in renal inner medullary collecting duct cells. 2. Full-length mouse kidney ClC-5 (mClC-5) was cloned and transiently expressed in CHO-K1 cells. Fast whole-cell patch-clamp recordings confirmed that mClC-5 expression produces a voltage-dependent, strongly outwardly rectifying Cl(-) conductance that was unaffected by external DIDS. 3. Slow whole-cell recordings, using nystatin-perforated patches from transfected CHO-K1 cells, also produced voltage-dependent Cl(-) currents consistent with ClC-5 expression. However, under this recording configuration an endogenous DIDS-sensitive Ca(2+)-activated Cl(-) conductance was also evident, which appeared to be activated by green fluorescent protein (GFP) transfection. 4. A mClC-5-GFP fusion protein was transiently expressed in CHO-K1 cells; confocal laser scanning microscopy (CLSM) showed localization at the plasma membrane, consistent with patch-clamp experiments. 5. Endogenous expression of mClC-5 was demonstrated in mouse renal collecting duct cells (mIMCD-3) by RT-PCR and by immunocytochemistry. 6. Using slow whole-cell current recordings, mIMCD-3 cells displayed three biophysically distinct Cl(-)-selective currents, which were all inhibited by DIDS. However, no cells exhibited whole-cell currents that had mClC-5 characteristics. 7. Transient transfection of mIMCD-3 cells with antisense mClC-5 had no effect on the endogenous Cl(-) conductances. Transient transfection with sense mClC-5 failed to induce the Cl(-) conductance seen in CHO-K1 cells but stimulated levels of the endogenous Ca(2+)-activated Cl(-) conductance 24 h post-transfection. 8. Confocal laser scanning microscopy of mIMCD-3 cells transfected with mClC-5-GFP showed that the protein was absent from the plasma membrane and was instead localized to acidic endosomal compartments. 9. These data discount a major role for ClC-5 as a plasma membrane Cl(-) conductance in mIMCD-3 cells but suggest a role in endosomal function.

Algorithms↗

PubMed2000

A novel method to isolate odontoblasts from rat incisor.

Historically, odontoblasts have been isolated from rat incisor using a surgical curette to separate these cells from the dentin. Isolation of odontoblasts using this approach typically resulted in cells with membrane properties that made the application of patch-clamp electrophysiological techniques prohibitive. The studies here describe a new procedure for isolating mature odontoblasts from adult rat incisor to obtain enriched populations of intact, viable odontoblasts that can be readily studied using patch-clamp methodologies. Identification of isolated cells as odontoblasts was confirmed using in situ mRNA hybridization for expression of dentin sialoprotein, osteocalcin, bone sialoprotein, and type I collagen, and calcium flux was monitored in these cells by means of fura-2 microfluorometry. We suggest that either single odontoblasts or clusters of these cells isolated by this new method would be an ideal preparation for the study of odontoblast properties using electrophysiological techniques, in situ hybridization and/or microfluorometry.

Animals↗

PubMed2002

The physiology and biophysics of an aluminum tolerance mechanism based on root citrate exudation in maize.

Al-induced release of Al-chelating ligands (primarily organic acids) into the rhizosphere from the root apex has been identified as a major Al tolerance mechanism in a number of plant species. In the present study, we conducted physiological investigations to study the spatial and temporal characteristics of Al-activated root organic acid exudation, as well as changes in root organic acid content and Al accumulation, in an Al-tolerant maize (Zea mays) single cross (SLP 181/71 x Cateto Colombia 96/71). These investigations were integrated with biophysical studies using the patch-clamp technique to examine Al-activated anion channel activity in protoplasts isolated from different regions of the maize root. Exposure to Al nearly instantaneously activated a concentration-dependent citrate release, which saturated at rates close to 0.5 nmol citrate h(-1) root(-1), with the half-maximal rates of citrate release occurring at about 20 microM Al(3+) activity. Comparison of citrate exudation rates between decapped and capped roots indicated the root cap does not play a major role in perceiving the Al signal or in the exudation process. Spatial analysis indicated that the predominant citrate exudation is not confined to the root apex, but could be found as far as 5 cm beyond the root cap, involving cortex and stelar cells. Patch clamp recordings obtained in whole-cell and outside-out patches confirmed the presence of an Al-inducible plasma membrane anion channel in protoplasts isolated from stelar or cortical tissues. The unitary conductance of this channel was 23 to 55 pS. Our results suggest that this transporter mediates the Al-induced citrate release observed in the intact tissue. In addition to the rapid Al activation of citrate release, a slower, Al-inducible increase in root citrate content was also observed. These findings led us to speculate that in addition to the Al exclusion mechanism based on root citrate exudation, a second internal Al tolerance mechanism may be operating based on Al-inducible changes in organic acid synthesis and compartmentation. We discuss our findings in terms of recent genetic studies of Al tolerance in maize, which suggest that Al tolerance in maize is a complex trait.

Adaptation, Physiological↗

PubMed2006

Synaptically-silent immature neurons show gaba and glutamate receptor-mediated currents in adult rat dentate gyrus.

The fate of adult-generated neurons in dentate gyrus is mainly determined early, before they receive synapses. In developing brain, classical neurotransmitters such as GABA and glutamate exert trophic effects before synaptogenesis. In order for this to occur in adult brain as well, immature non-contacted cells must express functional receptors to GABA and glutamate. In this investigation, patch-clamp recordings were used in adult rat dentate gyrus slices to assess the presence and analyze the characteristics of GABA- and glutamate-evoked currents in highly immature, synaptically-silent granule cells. Whole-cell patch-clamp recordings showed that all the analyzed cells responded to puff application of GABA and most of them responded to glutamate. Currents evoked by GABA were mediated exclusively by GABAA receptors and those elicited by glutamate were mediated by NMDA and AMPA/Kainate receptors. GABAA receptor-mediated currents were reduced by furosemide, which suggests that synaptically-silent immature neurons express high-affinity, alpha4-subunit-containing GABAA receptors. Gramicidin-perforated-patch recordings showed that GABAA receptor-mediated currents exerted a depolarizing effect due to high intracellular chloride concentration. Synaptically-silent immature cells shared morphological and electrophysiological properties with GFP-expressing, 7-day-old adult-generated granule layer cells, indicating that they could be in the first week of life, the period of maximal newborn cell death. Moreover, the presence of functional GABA and glutamate receptors was confirmed in these GFP-expressing cells. Present findings are mostly consistent with previous data obtained in female mice undergoing spontaneous activity and in transgenic mice, except for some inconsistencies about the presence of functional glutamatergic receptors. We speculate that adult-generated, non-contacted granule cells may be able to sense activity-related variations of GABA and glutamate extracellular levels. This condition is necessary, even if not sufficient, for these neurotransmitters to have a direct role in addressing cell survival.

Animals↗

PubMed1996

A cation non-selective channel induced by extracellular ATP in macrophages and phagocytic cells of the thymic reticulum.

Extracellular ATP4- can bind to P2Z purinergic receptors including depolarization and cytoplasmic membrane permeabilization to small molecular weight solutes in macrophages, thymocytes, mast cells, phagocytic cells of the thymic reticulum and other cell types. An ATP(4-)-induced cation current has been described in whole-cell records of some of these cells but it is currently not clear whether these currents and the phenomenon of membrane permeabilization are a consequence of only one type of P2Z-associated channel/pore or two different phenomena triggered by one or more receptors. Here we use the outside-out patch-clamp technique to describe a single channel associated with this cation current in two murine phagocytic cells: intraperitoneal macrophages and phagocytic cells of the thymic reticulum. Multi channel currents could be readily observed in 77% of the outside-out patches of macrophages. Single channels of 7.8 pS could usually be resolved only in tail currents. Reversal potential measurements and ion replacement experiments indicated a lack of cation selectivity, similarly to what has already been described for the ATP(4-)-induced whole-cell inward current. No large-conductance channels that could explain the permeabilization to small molecular weight studies solutes was observed under our experimental conditions. A single channel of approx. 5 pS was also observed in phagocytic cells of the thymic reticulum under similar conditions. We conclude that the channel here described is the main carrier of cation current usually associated with the binding of ATP4- to P2Z receptors in whole-cell and outside-out patch-clamp experiments.

Adenosine Triphosphate↗