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Patch-clamp Experiments (patch-clamp + experiment)
Kinds of Patch-clamp Experiments Selected AbstractsLow-threshold heat response antagonized by capsazepine in chick sensory neurons, which are capsaicin-insensitiveEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2000Antonia Marín-Burgin Abstract The heat-transducing receptor VR1 cloned from rat sensory neurons can be activated by both noxious heat and capsaicin. As the response of sensory neurons to capsaicin is species dependent, it is conceivable that the responses to noxious heat and to capsaicin are transduced by distinct receptors across different species. Therefore, we investigated responses to noxious heat from a capsaicin-insensitive (chick) and a capsaicin-sensitive (rat) species. In chick, whole-cell patch-clamp experiments in isolated dorsal root ganglion neurons revealed two populations of neurons with different thresholds to noxious heat, activated at ,,43 °C and ,,53 °C. In cobalt uptake experiments, the proportion of neurons showing a heat-induced response increased with increasing heat stimuli. Application of capsaicin (1,10 ,m) did not result in inward currents or cobalt uptake. Rat neurons yielded comparable results in heat experiments, but were capsaicin-sensitive. Although chick neurons are insensitive to capsaicin, the competitive capsaicin antagonist capsazepine (1,10 ,m) was effective in blocking heat-induced responses, verified by patch-clamp and cobalt uptake methods. The noncompetitive capsaicin antagonist ruthenium red (10 ,m) reduced to almost nil the proportion of heat-responsive neurons identified with the cobalt uptake method. These findings suggest that chick DRG neurons express a low-threshold heat-transducing receptor with a pharmacological profile distinct from the low-threshold heat receptor VR1 cloned from rat DRG neurons. The data support the idea that there might be heat receptor subtypes with differences in the capsaicin binding site. [source] Involvement of Calmodulin in Glucagon-Like Peptide 1(7-36) Amide-Induced Inhibition of the ATP-Sensitive K+ Channel in Mouse Pancreatic ,-CellsEXPERIMENTAL PHYSIOLOGY, Issue 3 2001W. G. Ding The present investigation was designed to examine whether calmodulin is involved in the inhibition of the ATP-sensitive K+ (KATP) channel by glucagon-like peptide 1(7-36) amide (GLP-1) in mouse pancreatic ,-cells. Membrane potential, single channel and whole-cell currents through the KATP channels, and intracellular free Ca2+ concentration ([Ca2+]i) were measured in single mouse pancreatic ,-cells. Whole-cell patch-clamp experiments with amphotericin-perforated patches revealed that membrane conductance at around the resting potential is predominantly supplied by the KATP channels in mouse pancreatic ,-cells. The addition of 20 nM GLP-1 in the presence of 5 mM glucose significantly reduced the membrane KATP conductance, accompanied by membrane depolarization and the generation of electrical activity. A calmodulin inhibitor N -(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7, 20 ,M) completely reversed the inhibitory actions of GLP-1 on the membrane KATP conductance and resultant membrane depolarization. Cell-attached patch recordings confirmed the inhibition of the KATP channel activity by 20 nM GLP-1 and its restoration by 20 ,M W-7 or 10 ,M calmidazolium at the single channel level. Bath application of 20 ,M W-7 also consistently abolished the GLP-1-evoked increase in [Ca2+]i in the presence of 5 mM glucose. These results strongly suggest that the mechanisms by which GLP-1 inhibits the KATP channel activity accompanied by the initiation of electrical activity in mouse pancreatic ,-cells include a calmodulin-dependent mechanism in addition to the well-documented activation of the cyclic AMP-protein kinase A system. [source] Cerebellar granule cells cultured from adolescent rats express functional NMDA receptors: an in vitro model for studying the developing cerebellumJOURNAL OF NEUROCHEMISTRY, Issue 2 2008R. Lisa Popp Abstract In the developing rat cerebellum functional NMDA receptors (NMDARs) expressing the NR2C subunit have been identified on or after postnatal day 19. We obtained primary cultured cells from 19- to 35-day-old rat cerebellum that expressed few oligodendrocytes or astrocytes. Cultured cells were immunoreactive for neuron-specific proteins thus indicating a neuronal population. The primary neuron present was the granule cell as indicated by immunofluorescence for the GABAA alpha 6 subunit. Whole-cell patch-clamp experiments indicated that functional NMDARs were present. Functional characteristics of NMDARs expressed in cerebellar granule cells (CGCs) obtained from adolescent animals were similar to those previously reported for NMDARs expressed in CGCs obtained from neonatal rats. Cultured CGCs obtained from older animals contained NMDARs that were inhibited by EtOH and were less sensitive to the NR2B subunit-specific antagonist Ro 25-6981. Furthermore, NMDA-induced currents were smaller than those observed in CGCs. Western blot analysis indicated the presence of the NMDA NR2A and NR2C subunits, but not the NR2B in cultures obtained from the adolescent rats. CGCs obtained from adolescent rats express functional NMDARs consistent with a developmental profile observed in vivo. [source] Melanotrope Cells of Xenopus laevis Express Multiple Types of High-Voltage-Activated Ca2+ ChannelsJOURNAL OF NEUROENDOCRINOLOGY, Issue 1 2005H.-Y. Zhang Abstract Pituitary melanotrope cells are neuroendocrine signal transducing cells that translate physiological stimuli into adaptive hormonal responses. In this translation process, Ca2+ channels play essential roles. We have characterised which types of Ca2+ current are present in melanotropes of the amphibian Xenopus laevis, using whole-cell, voltage-clamp, patch-clamp experiments and specific blockers of the various current types. Running an activation current,voltage relationship protocol from a holding potential (HP) of ,80 mV/or ,110 mV, shows that Xenopus melanotropes possess only high-voltage activated (HVA) Ca2+ currents. Steady-state inactivation protocols reveal that no inactivation occurs at ,80 mV, whereas 30% of the current is inactivated at ,30 mV. We determined the contribution of individual channel types to the total HVA Ca2+ current, examining the effect of each channel blocker at an HP of ,80 mV and ,30 mV. At ,80 mV, ,-conotoxin GVIA, ,-agatoxin IVA, nifedipine and SNX-482 inhibit Ca2+ currents by 21.8 ± 4.1%, 26.1 ± 3.1%, 24.2 ± 2.4% and 17.9 ± 4.7%, respectively. At ,30 mV, ,-conotoxin GVIA, nifedipine and ,-agatoxin IVA inhibit Ca2+ currents by 33.8 ± 3.0, 24.2 ± 2.6 and 16.0 ± 2.8%, respectively, demonstrating that these blockers substantially inhibit part of the Ca2+ current, independently from the HP. We have previously demonstrated that ,-conotoxin GVIA can block Ca2+ oscillations and steps. We now show that nifedipine and ,-agatoxin IVA do not affect the intracellular Ca2+ dynamics, whereas SNX-482 reduces the Ca2+ step amplitude. We conclude that Xenopus melanotrope cells express all four major types of HVA Ca2+ channel, as well as the resulting currents, but no low-voltage activated channels. The results provide the basis for future studies on the complex regulation of channel-mediated Ca2+ influxes into this neuroendocrine cell type as a function of its role in the animal's adaptation to external challenges. [source] Surface protein patterns govern morphology, proliferation, and expression of cellular markers but have no effect on physiological properties of cortical precursor cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 11 2008Anna K. Magnusson Abstract The ability to differentiate and give rise to neurons, astrocytes, and oligodendrocytes is an inherent feature of neural stem cells, which raises hopes for cell-based therapies of neurodegenerative diseases. However, there are many hurdles to cross before such regimens can be applied clinically. A considerable challenge is to elucidate the factors that contribute to neural differentiation. In this study, we evaluated the possibility of steering neuronal maturation by growing cortical precursor cells on microscale surface patterns of extracellular matrix (ECM) proteins. When the cells were encouraged to extend processes along lines of ECM proteins, they displayed a much more mature morphology, less proliferation capacity, and greater expression of a neuronal marker in comparison with cells grown in clusters on ECM dots. This implied that the growth pattern alone could play a crucial role for neural differentiation. However, in spite of the strikingly different morphology, when performing whole-cell patch-clamp experiments, we never observed any differences in the functional properties between cells grown on the two patterns. These results clearly demonstrate that morphological appearances are not representative measures of the functional phenotype or grade of neuronal maturation, stressing the importance of complementary electrophysiological evidence. To develop successful transplantation therapies, increased cell survival is critical. Because process-bearing neurons are sensitive and break easily, it would be of clinical interest to explore further the differentiating capacity of the cells cultured on the ECM dot pattern, described in this article, which are devoid of processes but display the same functional properties as neurons with mature morphology. © 2008 Wiley-Liss, Inc. [source] Membrane Hyperpolarization Is Not Required for Sustained Muscarinic Agonist-Induced Increases in Intracellular Ca2+ in Arteriolar Endothelial CellsMICROCIRCULATION, Issue 2 2005KENNETH D. COHEN ABSTRACT Objective: Hyperpolarization modulates Ca2+ influx during agonist stimulation in many endothelial cells, but the effects of hyperpolarization on Ca2+ influx in freshly isolated arteriolar endothelial cells are unknown. Therefore, the purpose of the present study was to characterize agonist-induced Ca2+ transients in freshly isolated arteriolar endothelial cells and to test the hypothesis that membrane hyperpolarization augments agonist-induced Ca2+ influx into these cells. Methods: Arterioles were removed from hamster cremaster muscles and arteriolar endothelial cells were enzymatically isolated. Endothelial cells were loaded with Fura 2-AM and the Fura 2 ratio measured photometrically as an index of intracellular Ca2+. The cells were then stimulated with the muscarinic, cholinergic agonist, methacholine, and the resulting Ca2+ transients were measured. Results: Methacholine (1 , M) increased the endothelial cell Fura 2 ratio from a baseline of 0.81 ± 0.02 to an initial peak of 1.17 ± 0.05 (n = 17) followed by a sustained plateau of 1.12 ± 0.07. The plateau phase of the Ca2+ transient was inhibited by removal of extracellular Ca2+ (n = 12, p < .05), or the nonselective cation channel blockers Gd3+ (30 , M; n = 7, p < .05) or La3+ (50 , M; n = 7, p < .05) without significant effect on the baseline or peak (p > .05). The initial peak of methacholine-induced Ca2+ transients was inhibited by the IP3 -receptor antagonist xestospongin D (10 , M, n = 5, p < .05). The methacholine-induced Ca2+ transients were accompanied by endothelial cell hyperpolarization of approximately 14,18 mV, as assessed by experiments using the potentiometric dye, di-8-ANEPPS as well as by patch-clamp experiments. However, inhibition of hyperpolarization by blockade of Ca2+ -activated K+ channels with charybdotoxin (100 nM) and apamin (100 nM) (n = 5), or exposure of endothelial cells to 80 or 145 mM KCl (both n = 7) had no effect on the plateau phase of methacholine-induced Ca2+ transients (p > .05). Conclusions: Freshly isolated arteriolar endothelial cells display agonist-induced Ca2+ transients. For the muscarinic agonist, methacholine, these Ca2+ transients result from release of Ca2+ from intracellular stores through IP3 receptors, followed by sustained influx of extracellular Ca2+. While these changes in intracellular Ca2+ are associated with endothelial cell hyperpolarization, the methacholine-induced, sustained increase in intracellular Ca2+ appears to be independent from this change in membrane potential. These data suggest that arteriolar endothelial cells may possess a novel Ca2+ influx pathway, or that the relationship between intracellular Ca2+ and Ca2+ influx is more complex than that observed in other endothelial cells. [source] Engineering physiologically controlled pacemaker cells with lentiviral HCN4 gene transferTHE JOURNAL OF GENE MEDICINE, Issue 5 2008Gerard J. J. Boink Abstract Background Research on biological pacemakers for the heart has so far mainly focused on short-term gene and cell therapies. To develop a clinically relevant biological pacemaker, long-term function and incorporation of autonomic modulation are crucial. Lentiviral vectors can mediate long-term gene expression, while isoform 4 of the Hyperpolarization-activated Cyclic Nucleotide-gated channel (encoded by HCN4) contributes to pacemaker function and responds maximally to cAMP, the second messenger in autonomic modulation. Material and Methods Action potential (AP) properties and pacemaker current (If) were studied in single neonatal rat ventricular myocytes that overexpressed HCN4 after lentiviral gene transduction. Autonomic responsiveness and cycle length stability were studied using extracellular electrograms of confluent cultured monolayers. Results Perforated patch-clamp experiments demonstrated that HCN4-transduced single cardiac myocytes exhibited a 10-fold higher If than non-transduced single myocytes, along with slow diastolic depolarization, comparable to pacemaker cells of the sinoatrial node, the dominant native pacemaker. HCN4-transduced monolayers exhibited a 47% increase in beating rate, compared to controls. Upon addition of DBcAMP, HCN4-transduced monolayers had beating rates which were 54% faster than baseline and significantly more regular than controls. Conclusions Lentiviral vectors efficiently transduce cardiac myocytes and mediate functional gene expression. Because HCN4-transduced myocytes demonstrate an increase in spontaneous beating rate and responsiveness to autonomic modulation, this approach may be useful to create a biological pacemaker. Copyright © 2008 John Wiley & Sons, Ltd. [source] Kinetic and functional analysis of transient, persistent and resurgent sodium currents in rat cerebellar granule cells in situ: an electrophysiological and modelling studyTHE JOURNAL OF PHYSIOLOGY, Issue 1 2006Jacopo Magistretti Cerebellar neurones show complex and differentiated mechanisms of action potential generation that have been proposed to depend on peculiar properties of their voltage-dependent Na+ currents. In this study we analysed voltage-dependent Na+ currents of rat cerebellar granule cells (GCs) by performing whole-cell, patch-clamp experiments in acute rat cerebellar slices. A transient Na+ current (INaT) was always present and had the properties of a typical fast-activating/inactivating Na+ current. In addition to INaT, robust persistent (INaP) and resurgent (INaR) Na+ currents were observed. INaP peaked at ,,40 mV, showed half-maximal activation at ,,55 mV, and its maximal amplitude was about 1.5% of that of INaT. INaR was elicited by repolarizing pulses applied following step depolarizations able to activate/inactivate INaT, and showed voltage- and time-dependent activation and voltage-dependent decay kinetics. The conductance underlying INaR showed a bell-shaped voltage dependence, with peak at ,35 mV. A significant correlation was found between GC INaR and INaT peak amplitudes; however, GCs expressing INaT of similar size showed marked variability in terms of INaR amplitude, and in a fraction of cells INaR was undetectable. INaT, INaP and INaR could be accounted for by a 13-state kinetic scheme comprising closed, open, inactivated and blocked states. Current-clamp experiments carried out to identify possible functional correlates of INaP and/or INaR revealed that in GCs single action potentials were followed by depolarizing afterpotentials (DAPs). In a majority of cells, DAPs showed properties consistent with INaR playing a role in their generation. Computer modelling showed that INaR promotes DAP generation and enhances high-frequency firing, whereas INaP boosts near-threshold firing activity. Our findings suggest that special properties of voltage-dependent Na+ currents provides GCs with mechanisms suitable for shaping activity patterns, with potentially important consequences for cerebellar information transfer and computation. [source] The voltage-dependent Cl, channel ClC-5 and plasma membrane Cl, conductances of mouse renal collecting duct cells (mIMCD-3)THE JOURNAL OF PHYSIOLOGY, Issue 3 2001J. A. Sayer 1We 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. 2Full-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. 3Slow 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 Ca2+ -activated Cl, conductance was also evident, which appeared to be activated by green fluorescent protein (GFP) transfection. 4A 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. 5Endogenous expression of mClC-5 was demonstrated in mouse renal collecting duct cells (mIMCD-3) by RT-PCR and by immunocytochemistry. 6Using 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. 7Transient 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 Ca2+ -activated Cl, conductance 24 h post-transfection. 8Confocal 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. 9These 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. [source] Phosphatidylinositol 4,5-bisphosphate is important for stomatal openingTHE PLANT JOURNAL, Issue 5 2007Yuree Lee Summary Previously, we demonstrated that a protein that binds phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] inhibits both light-induced stomatal opening and ABA-induced stomatal closing. The latter effect is due to a reduction in free PtdIns(4,5)P2, decreasing production of inositol 1,4,5-trisphosphate and phosphatidic acid by phospholipases C and D. However, it is less clear how PtdIns(4,5)P2 modulates stomatal opening. We found that in response to white light irradiation, the PtdIns(4,5)P2 -binding domain GFP:PLC,1PH translocated from the cytosol into the plasma membrane. This suggests that the level of PtdIns(4,5)P2 increases at the plasma membrane upon illumination. Exogenously administered PtdIns(4,5)P2 substituted for light stimuli, inducing stomatal opening and swelling of guard cell protoplasts. To identify PtdIns(4,5)P2 targets we performed patch-clamp experiments, and found that anion channel activity was inhibited by PtdIns(4,5)P2. Genetic analyses using an Arabidopsis PIP5K4 mutant further supported the role of PtdIns(4,5)P2 in stomatal opening. The reduced stomatal opening movements exhibited by a mutant of Arabidopsis PIP5K4 (At3g56960) was countered by exogenous application of PtdIns(4,5)P2. The phenotype of reduced stomatal opening in the pip5k4 mutant was recovered in lines complemented with the full-length PIP5K4. Together, these data suggest that PIP5K4 produces PtdIns(4,5)P2 in irradiated guard cells, inhibiting anion channels to allow full stomatal opening. [source] The effects of a progesterone metabolite, 5,-dihydroprogesterone, on oxytocin receptor binding in human myometrial membranesBJOG : AN INTERNATIONAL JOURNAL OF OBSTETRICS & GYNAECOLOGY, Issue 6 2003Shirley Astle Objective To determine the effect of the progesterone metabolite 5,-dihydroprogesterone on human oxytocin receptor binding in myometrial membranes and on whole-cell calcium current in single myometrial cells. Design Receptor binding studies in human myometrial membranes prepared from biopsies taken before or after the onset of labour and in Chinese hamster ovary cells expressing the human oxytocin receptor. Whole cell patch-clamp experiments were undertaken on isolated myometrial cells. Setting University research laboratories and University hospital. Population Patients undergoing caesarean section at term either prior to or following onset of labour. Methods Myometrial biopsies were taken from women undergoing caesarean section. The binding affinities of oxytocin, 5,-dihydroprogesterone and atosiban were determined in myometrial membranes and Chinese hamster ovary cells expressing the human oxytocin receptor. The effect of 5,-dihydroprogesterone on inward current was also determined in isolated myometrial cells. Main outcome measures Receptor binding affinity and electrophysiological inward current. Results 5,-Dihydroprogesterone did not reduce oxytocin receptor binding in myometrial membranes or Chinese hamster ovary cells expressing the human oxytocin receptor. Nor did it influence calcium current under whole-cell patch conditions in single myometrial cells. In contrast, atosiban inhibited binding in myometrial membranes prepared from samples taken either prior to or following labour (Ki= 112 and 108 nM, respectively). The affinity of atosiban for the oxytocin receptor was much lower than oxytocin (Ki= 5 and 6 nM in samples taken before or after labour, respectively) in myometrial membranes and in Chinese hamster ovary cells expressing the human oxytocin receptor (Ki= 63 M and 1 nM for atosiban and oxytocin, respectively). Conclusions We conclude that 5,-dihydroprogesterone is unlikely to regulate myometrial activity as a result of a direct effect on oxytocin receptor binding or inward calcium current. [source] Electrophysiological characterization of the SK channel blockers methyl-laudanosine and methyl-noscapine in cell lines and rat brain slicesBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2004Jacqueline Scuvée-Moreau We have recently shown that the alkaloid methyl-laudanosine blocks SK channel-mediated afterhyperpolarizations (AHPs) in midbrain dopaminergic neurones. However, the relative potency of the compound on the SK channel subtypes and its ability to block AHPs of other neurones were unknown. Using whole-cell patch-clamp experiments in transfected cell lines, we found that the compound blocks SK1, SK2 and SK3 currents with equal potency: its mean IC50s were 1.2, 0.8 and 1.8 ,M, respectively. IK currents were unaffected. In rat brain slices, methyl-laudanosine blocked apamin-sensitive AHPs in serotonergic neurones of the dorsal raphe and noradrenergic neurones of the locus coeruleus with IC50s of 21 and 19 ,M, as compared to 15 ,M in dopaminergic neurones. However, at 100 ,M, methyl-laudanosine elicited a constant hyperpolarization of serotonergic neurones of about 9 mV, which was inconsistently (i.e. not in a reproducible manner) antagonized by atropine and hence partly due to the activation of muscarinic receptors. While exploring the pharmacology of related compounds, we found that methyl-noscapine also blocked SK channels. In cell lines, methyl-noscapine blocked SK1, SK2 and SK3 currents with mean IC50s of 5.9, 5.6 and 3.9 ,M, respectively. It also did not block IK currents. Methyl-noscapine was slightly less potent than methyl-laudanosine in blocking AHPs in brain slices, its IC50s being 42, 37 and 29 ,M in dopaminergic, serotonergic and noradrenergic neurones, respectively. Interestingly, no significant non-SK effects were observed with methyl-noscapine in slices. At a concentration of 300 ,M, methyl-noscapine elicited the same changes in excitability in the three neuronal types than did a supramaximal concentration of apamin (300 nM). Methyl-laudanosine and methyl-noscapine produced a rapidly reversible blockade of SK channels as compared with apamin. The difference between the IC50s of apamin (0.45 nM) and methyl-laudanosine (1.8 ,M) in SK3 cells was essentially due to a major difference in their k,1 (0.028 s,1 for apamin and 20 s,1 for methyl-laudanosine). These experiments demonstrate that both methyl-laudanosine and methyl-noscapine are medium potency, quickly dissociating, SK channel blockers with a similar potency on the three SK subtypes. Methyl-noscapine may be superior in terms of specificity for the SK channels. British Journal of Pharmacology (2004) 143, 753,764. doi:10.1038/sj.bjp.0705979 [source] | ||||||||||||||||||||||