SEARCH · PubMed Health

Results for “patch clamp”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 919 records · Page 51Linked to original sources

PubMed1993

AMPA-type glutamate receptors--nonselective cation channels mediating fast excitatory transmission in the CNS.

In recent years, considerable progress in our understanding of the molecular events underlying excitatory synaptic transmission has been made. This progress was mainly achieved by technical advances, among them the patch-clamp technique in brain slices (Edwards et al., 1989), fast application of agonists (Franke et al., 1987), and cloning and functional expression of GluR channels of the nonNMDA type (e.g., Hollmann et al., 1989). A suitable model for studying excitatory postsynaptic currents (EPSCs) in the brain slice with patch-clamp techniques is the mossy fiber synapse on CA3 pyramidal cells of rat hippocampus (MF-CA3 synapse). This synapse is located close to the cell soma and should provide almost ideal space-clamp conditions. A comparison of MF-CA3 EPSCs with the currents activated by fast application of glutamate on membrane patches isolated from CA3 cell somata suggests that the concentration of glutamate in the synaptic cleft during excitatory synaptic transmission is high (about 1 mM) and that the transmitter remains in the synaptic cleft only briefly (about 1 ms). It seems likely that desensitization influences the peak amplitude of the EPSC in several ways. Brief pulses of glutamate cause desensitization, from which the glutamate receptor channels recover only slowly, and micromolar ambient glutamate concentrations produce desensitization at equilibrium. From the functional properties of recombinant GluR channels, in situ hybridization data, and patch-clamp experiments on different neuronal and nonneuronal cell types, a picture of the molecular identity of native channels emerges. In neurons of the hippocampus the pharmacological features of these channels were similar to recombinant channels assembled from subunits of the AMPA/kainate subtype which are strongly expressed in these cells. The native channels are characterized by outward rectification of the steady-state I-V and low Ca permeability, similar to recombinant channels containing the GluR-B subunit. This is consistent with the ubiquitous expression of this subunit in hippocampal neurones. In contrast, GluR channels from cerebellar glial cells, which uniquely in the central nervous system lack the expression of GluR-B subunits, show double rectification and high Ca permeability. The results suggest that the native functional nonNMDA glutamate receptor channels in the CNS are assembled form subunits of the AMPA/kainate subtype in a cell-specific way, with the functional properties of GluR channels in neurones being dominated by the GluR-B subunit.

Animals↗

PubMed1998

A novel crystallization method for visualizing the membrane localization of potassium channels.

The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5 microS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel "agarose-hemiclamp" technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel.

Animals↗

PubMed1990

Characteristics of single Na+ channels of adult human skeletal muscle.

The patch-clamp technique was used to study Na+ channels of human skeletal muscle. Preparations were from biopsies of quadriceps muscle from adults who were not suffering from neuromuscular diseases. Activity of Na+ channels was recorded from inside-out patches when the membrane potential was stepped from a holding potential of -110 mV to potential above a threshold of about -65 mV. Single channel activity increased within minutes after hyperpolarizing the patch due to recovery from ultra-slow inactivation. Up to ten Na+ channels were active in individual patches. Macroscopic currents were reconstructed by averaging single channel currents. The time-to-peak current declined from 1.6 ms at -60 mV to 0.5 ms at + 10 mV. The currents decayed mono-exponentially with time constants between 12.1 ms at -60 mV and 0.4 ms at + 10 mV (21 C). The conductance of single Na+ channels was 1.65 pS and the mean open time was voltage-dependent. At -50 mV, the mean open time was 0.4 ms, while positive to -10 mV it increased to values above 1 ms. In the threshold potential range, the number of openings per depolarizing pulse was larger than the number of channels under the patch-clamp pipette, indicating reopening of Na+ channels at this potential. Openings could be observed only rarely 10 ms after onset of depolarization and the macroscopic current produced by late openings was less than 0.1% of the peak current. Human skeletal muscle is thus suitable for investigation with the patch-clamp technique and the determination of properties of Na+ channels with this technique could be the basis for an assessment of possible defects of these channels in diseased muscle.

Biopsy↗

PubMed2001

Electrophoretic plugging of nuclear pores by using the nuclear hourglass technique.

The nuclear hourglass technique (NHT) was recently introduced as a novel technique that measures the electrical nuclear envelope (NE) conductance of isolated Xenopus laevis oocyte nuclei. The main conclusion drawn from NHT work so far is that nuclear pore complexes (NPCs) of oocytes are in an electrically open state under physiological conditions, with a mean conductance of 1.7 nS per NPC. Since nuclear patch-clamp data indicate that usually NPCs are electrically closed, our work has been challenged by the notion that NHT cannot assure a high resistance seal ("gigaseal") between glass wall and NE like that required for patch-clamp experiments. Thus, NHT could have dramatically underestimated NE electrical resistance. Here we demonstrate that NHT does not require a gigaseal for accurate NE conductance measurements. In addition, we present experimental conditions where mean single NPC electrical conductance is reduced 26-fold due to electrophoretic plugging by negatively charged nucleoplasmic macromolecules. In addition, data indicate that under physiological conditions (i.e., when macromolecules are offered in the cytosolic solution) the nuclear surface is heavily folded, underestimating "true" NE surface by a factor of 2.6. When "true" NE surface area is taken into consideration, modified values of mean single NPC conductances of 654 pS for electrically open conditions and 25 pS for electrically plugged conditions can be calculated. We conclude that the large overall NE conductance detected with the nuclear hourglass technique in intact Xenopus laevis oocyte nuclei can be explained by the sum of single NPC conductances in the pS range, as long as open probability is high. This confirms previous patch-clamp work concerning single NPC conductance, but disagrees with the view that mean open probability of NPC channels is usually low.

Active Transport, Cell Nucleus↗

PubMed2005

Endogenous Ca2+ buffer concentration and Ca2+ microdomains in hippocampal neurons.

Ca2+-binding proteins are ubiquitously expressed throughout the CNS and serve as valuable immunohistochemical markers for certain types of neurons. However, the functional role of most Ca2+-binding proteins has to date remained obscure because their concentration in central neurons is not known. In this study, we investigate the intracellular concentration of the widely expressed Ca2+-binding protein calbindin-D28k in adult hippocampal slices using patch-clamp recordings and immunohistochemistry. First, we show that calbindin-D28k freely exchanges between patch pipette and cytoplasm during whole cell patch-clamp recordings with a time constant of approximately 10 min. Substituting known concentrations of recombinant calbindin-D28k in patch pipettes enabled us to determine the endogenous calbindin-D28k concentration by postrecording immunohistochemistry. Using this calibration procedure, we find that mature granule cells (doublecortin-) contain approximately 40 microm, and newborn granule cells (doublecortin+) contain 0-20 microm calbindin-D28k. CA3 stratum radiatum interneurons and CA1 pyramidal cells enclose approximately 47 and approximately 45 microm calbindin-D28k, respectively. Numerical simulations showed that 40 microm calbindin-D28k is capable of tuning Ca2+ microdomains associated with action potentials at the mouth of single or clustered Ca2+ channels: calbindin-D28k reduces the increment in free Ca2+ at a distance of 100 and 200 nm by 20 and 35%, respectively, and strongly accelerates the collapse of the Ca2+ gradient after cessation of Ca2+ influx. These data suggest that calbindin-D28k equips hippocampal neurons with approximately 160 microm mobile, high-affinity Ca2+-binding sites (kappa(S) approximately 200) that slow and reduce global Ca2+ signals while they enhance the spatiotemporal fidelity of submicroscopic Ca2+ signals.

Aging↗

PubMed2006

[Pacemaker current gene expression of rat mesenchymal stem cells and identification of mesenchymal stem cells expressing human pacemaker current gene (hHCN2)].

OBJECTIVE: To study pacemaker current gene expression of mesenchymal stem cells (MSCs) and the electrophysiological property of MSCs expressing human pacemaker current gene. METHODS: Pacemaker current gene expression of MSCs were studied by real-time quantitative polymerase chain reaction (real-time PCR) and pcDNA3-hHCN2 was transfected with Lipofectin 2000 into MSCs. hHCN2 expression at mRNA and at protein levels in the transfected cells were identified by real-time PCR and Western blot, respectively. The ionic currents of cloned hHCN2 (IhHCN2) were recorded and the current characteristics were studied through the whole-cell patch clamp technique. RESULTS: mHCN1, mHCN2, mHCN3, mHCN4 represent (0.08+/-0.01)%, (77.16+/-0.03)%, (0.24+/-0.01)%, (22.53+/-0.02)% of total HCN mRNA in MSCs as determined by real-time PCR. Transfected hHCN2 ionic currents were recorded by whole-cell patch clamp and current density-voltage curves were obtained. The threshold for activation of IhHCN2 was approximately -80 mV and this current could be blocked by Cs+ (4 mmol/L). hHCN2 expression in transfected MSCs was detected both at mRNA and protein levels. CONCLUSIONS: 1. mHCN2 and mHCN4 represent the major populations of total HCN mRNA in MSCs. 2. Plasmid pcDNA3-hHCN2 by Lipofectin could be successfully transfected into MSCs with IhHCN2 recorded by whole-cell patch clamp technique, this study provides a basis for future antiarrhythmic gene therapy.

Animals↗

PubMed2003

Microstructured apertures in planar glass substrates for ion channel research.

We have developed planar glass chip devices for patch clamp recording. Glass has several key advantages as a substrate for planar patch clamp devices. It is a good dielectric, is well-known to interact strongly with cell membranes and is also a relatively in-expensive material. In addition, it is optically neutral. However, microstructuring processes for glass are less well established than those for silicon-based substrates. We have used ion-track etching techniques to produce micron-sized apertures into borosilicate and quartz-glass coverslips. These apertures, which can be easily produced in arrays, have been used for high resolution recording of single ion channels as well as for whole-cell current recordings from mammalian cell lines. An additional attractive application that is greatly facilitated by the combination of planar geometry with the optical neutrality of the substrate is single-molecule fluorescence recording with simultaneous single-channel measurements.

Animals↗

PubMed2000

Norepinephrine facilitates inhibitory transmission in substantia gelatinosa of adult rat spinal cord (part 2): effects on somatodendritic sites of GABAergic neurons.

BACKGROUND: It has been reported previously that norepinephrine, when applied to the spinal cord dorsal horn, excites a subpopulation of dorsal horn neurons, presumably inhibitory interneurons. In the current study, the authors tested whether norepinephrine could activate inhibitory interneurons, specifically those that are "GABAergic." METHODS: A transverse slice was obtained from a segment of the lumbar spinal cord isolated from adult male Sprague-Dawley rats. Whole-cell patch-clamp recordings were made from substantia gelatinosa neurons using the blind patch-clamp technique. The effects of norepinephrine on spontaneous GABAergic inhibitory postsynaptic currents were studied. RESULTS: In the majority of substantia gelatinosa neurons tested, norepinephrine (10-60 microM) significantly increased both the frequency and the amplitude of GABAergic inhibitory postsynaptic currents. These increases were blocked by tetrodotoxin (1 microM). The effects of norepinephrine were mimicked by the alpha1-receptor agonist phenylephrine (10-80 microM) and inhibited by the alpha1-receptor-antagonist WB-4101 (0.5 microM). Primary-afferent-evoked polysynaptic excitatory postsynaptic potentials or excitatory postsynaptic currents in wide-dynamic-range neurons of the deep dorsal horn were also attenuated by phenylephrine (40 microM). CONCLUSION: The observations suggest that GABAergic interneurons possess somatodendritic alpha1 receptors, and activation of these receptors excites inhibitory interneurons. The alpha1 actions reported herein may contribute to the analgesic action of intrathecally administered phenylephrine.

Adrenergic alpha-1 Receptor Agonists↗

PubMed1985

Potassium current in the squid giant axon.

The squid giant axon was the first preparation to be investigated with the voltage clamp technique over 30 years ago by Cole (1949) and Hodgkin et al. (1952). During the intervening years it has continued to serve as a useful preparation for the development of other new techniques such as internal perfusion (Baker et al., 1962), gating current measurements (Armstrong and Bezanilla, 1974), and patch clamp measurements (Conti and Neher, 1980). It also has served as a useful comparative preparation for investigations of sodium and potassium currents in other excitable membrane preparations. This article has focused on the activation kinetics and the instantaneous current-voltage relation of the potassium component. The squid axon is well suited for studies of IK, because it appears to have only a single type of potassium channel, and the leakage current is relatively small under ideal conditions. The IK component is activated in a sigmoidal manner following membrane depolarization. It deactivates with a single exponential time constant following return of the membrane potential to the holding level, although the deactivation time constant varies with changes in the external potassium concentration. There has not, as yet, appeared a self-consistent model which describes all of these results. The current-voltage relation is a nonlinear function of driving force, which is approximately described by the Goldman-Hodgkin-Katz equation, although a model of the IV based on single file diffusion of ions through a channel is more in tune with the modern view of the ion permeation process (Hodgkin and Keynes, 1955; Hille and Schwarz, 1978; Clay and Shlesinger, 1977, 1983, 1984). Further progress in this area will probably be achieved both by the traditional techniques and by the patch clamp technique. The traditional method is well suited for studying tail current kinetics and the slow inactivation process. The patch clamp technique is well suited for studying the distribution of channels in the membrane and the kinetics of channel gating in steady state conditions.

Animals↗

PubMed2003

Generation and characterization of a cell line with inducible expression of Ca(v)3.2 (T-type) channels.

Establishment of stable cell lines that constitutively express Ca(2+) channels at high density and that are useful for in vitro studies may be complicated by problems with seal quality and duration during whole-cell patch-clamp electrophysiology. The current studies describe the generation and characterization of cells that express the human alpha1H T-type Ca(2+) channel under the control of a tetracycline-inducible expression system. Western blot and immunostaining studies revealed that expression of the alpha1H protein occurred only in the presence of tetracycline. Using the whole-cell patch-clamp method, the cells displayed peak inward currents of 1.15 +/- 0.14 nA in response to voltage-clamp steps. The T-type Ca(2+) current was inhibited by the T-type Ca(2+) channel antagonist, mibefradil, with an IC(50) of 160 nM. This cell line, with inducible channel expression, sealed with longer duration during whole-cell patch-clamp recording when compared with a cell line that constitutively expresses the alpha1H Ca(2+) channel. Ca(2+) influx through this channel could also be detected after the addition of extracellular Ca(2+). The amount of Ca(2+) influx was dependent on the [Ca](o) with an EC(50) of 4 mM. The Ca(2+) influx was also inhibited by mibefradil with a potency (IC(50) = 183 nM) similar to that observed in the voltage-clamp studies. Overall, this inducible alpha1H Ca(2+) channel-expressing cell line is useful for the study of human T-type Ca(2+) channel function, and offers advantages over a similar cell line that constitutively expresses the channel.

Calcium↗

PubMed1995

Internodal cells of the giant green alga Chara as an expression system for ion channels.

Internodal cells of the giant green alga Chara corallina were utilized as an expression system for two nicotinic acetylcholine receptor subtypes (nAChR) derived from rat muscle. From Chara internodes that were pressure-injected with the respective cRNA, cytoplasmic droplets were formed, and functional expression of channel proteins in the membrane delineating the droplets was confirmed by patch-clamp techniques. The droplet membrane was recently identified as the original tonoplast, patch-clamp recordings on cytoplasmic droplets were easily performed and single channel activity could be measured with high resolution. The properties of the recombinant nAChR observed in this membrane were similar to those reported for the channel expressed in Xenopus oocytes, and for the native channel recorded in situ. The Chara expression system is, therefore, suitable for functional expression of animal messenger RNAs, without the risk of signal contamination by intrinsic ion channels. It offers a low-cost and practical alternative to the use of Xenopus oocytes for the investigation of heterologously expressed ion channels.

Animals↗

PubMedDate not supplied

Hemolysate inhibits L-type Ca2+ channels in rat basilar smooth muscle cells.

Erythrocyte lysate increases intracellular Ca2+ ([Ca2+]i) and contracts cerebral arteries in vitro and has been suggested to be the cause for cerebral vasospasm. We investigated the effect of hemolysate on L-type Ca2+ channels directly by using patch clamp techniques in freshly isolated single smooth muscle cells from rat basilar artery. Patch clamp studies revealed a whole-cell current which resembles the L-type Ca2+ current reported by others. Hemolysate reduced the amplitude of the L-type Ca2+ channel current. The effect of hemolysate was reversible by washout and repeatable. Hemolysate was separated into two fractions by using filter membranes. The fraction > 1 kDa which contains oxyhemoglobin and other proteins mimicked the effect of hemolysate, while the fraction < 1 kDa and ATP were without effect. We conclude that hemolysate does not increase [Ca2+]i by activation of L-type Ca2+ channel.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2005

Neuron-transistor coupling: interpretation of individual extracellular recorded signals.

The electrical coupling of randomly migrating neurons from rat explant brain-stem slice cultures to the gates of non-metallized field-effect transistors (FETs) has been investigated. The objective of our work is the precise interpretation of extracellular recorded signal shapes in comparison to the usual patch-clamp protocols to evaluate the possible use of the extracellular recording technique in electrophysiology. The neurons from our explant cultures exhibited strong voltage-gated potassium currents through the plasma membrane. With an improved noise level of the FET set-up, it was possible to record individual extracellular responses without any signal averaging. Cells were attached by patch-clamp pipettes in voltage-clamp mode and stimulated by voltage step pulses. The point contact model, which is the basic model used to describe electrical contact between cell and transistor, has been implemented in the electrical simulation program PSpice. Voltage and current recordings and compensation values from the patch-clamp measurement have been used as input data for the simulation circuit. Extracellular responses were identified as composed of capacitive current and active potassium current inputs into the adhesion region between the cell and transistor gate. We evaluated the extracellular signal shapes by comparing the capacitive and the slower potassium signal amplitudes. Differences in amplitudes were found, which were interpreted in previous work as enhanced conductance of the attached membrane compared to the average value of the cellular membrane. Our results suggest rather that additional effects like electrodiffusion, ion sensitivity of the sensors or more detailed electronic models for the small cleft between the cell and transistor should be included in the coupling model.

Action Potentials↗

PubMed1999

Volume-regulated anion and organic osmolyte channels in mouse zygotes.

Whole-cell currents in mouse zygotes were measured using the patch-clamp technique in whole-cell mode. Upon exposure to hypotonic medium, patch-clamped zygotes increased in volume and developed a large swelling-activated current. The swelling-activated current was blocked by Cl- channel blockers, and the magnitude of the current and reversal potential were dependent on the Cl- gradient. Thus, the swelling-activated current had the properties of a current mediated by anion channels. However, in addition to being permeable to Cl- and I- (with I- having the greater permeability), there was also a significant swelling-activated conductance to aspartate and taurine, indicating that the swelling-activated channels in zygotes conduct not only inorganic anions but organic osmolytes as well. This swelling-activated anion and organic osmolyte pathway likely underlies the ability of zygotes to recover from an increase in volume, and it may function to regulate intracellular amino acid concentrations.

Animals↗

PubMed1998

Molecular biology and electrophysiology of calcium-activated potassium channels from lens epithelium.

PURPOSE: We structurally and functionally characterized the alpha and beta subunits of the human lens epithelium Ca(++)-activated potassium channel (BK). METHODS: The two subunits were sequenced following RT-PCR with multiple primer pairs. The subunits were cloned using a PCR approach and were expressed in tsA-201 cells for patch clamp recording. Green fluorescence protein-channel subunit fusion proteins were characterized by patch clamping and were imaged by fluorescence microscopy. RESULTS: Alpha subunits alone make a large single-channel conductance, potassium-selective channel with modest Ca++ sensitivity. Beta subunits alone make no channel but, when coexpressed with the alpha subunit, make a channel with increased Ca++ sensitivity, although still less than for natural channels of this type. GFP-BK subunit fusion proteins continue to function and result in a fluorescing channel, which can be localized by fluorescence microscopy. The alpha subunit codes for a "minimal" BK channel in that none of its potential alternative splicing sites contains an "extra" exon. CONCLUSIONS: The Ca(++)-activated potassium channel known as BK has the nucleotide sequences of its alpha and beta subunits represented in messenger RNA of cultured human lens epithelium. It is the first identified channel, to date, which imparts internal Ca++ dependence to lens epithelial potassium conductance.

Amino Acid Sequence↗

PubMed1992

The action of anaesthetics on stimulus-secretion coupling and synaptic activity.

1. Anaesthetics are known to depress excitatory synaptic transmission and the mechanism of this inhibition has been investigated using bovine adrenal chromaffin cells as an experimental model. 2. These cells are homologous with post-ganglionic sympathetic neurons and have well characterized receptor and secretory mechanisms. They are amenable both to the direct measurement of evoked secretion with its associated ion fluxes, and to electrophysiological investigation using the patch clamp technique. 3. These approaches have been used to study the influence of anaesthetics on pre- and post-synaptic mechanisms involved in stimulus-secretion coupling. 4. A variety of agents inhibited secretion evoked by direct depolarization, and this was shown to be due to a reduction in calcium influx. 5. Direct inhibition of voltage-gated calcium currents was confirmed by whole-cell patch clamp measurements. 6. In addition, anaesthetics powerfully modulated nicotinic receptor mediated events: carbachol-evoked secretion was more sensitive to anaesthetics than that stimulated by high potassium. 7. The mechanism of anaesthetic action on the nAChR was examined in more detail with patch-clamp experiments. 8. These showed that anaesthetics reduced the probability of channels being in the open state, largely as a result of reduction in mean channel open time. 9. The data are discussed in relation to excitatory synaptic transmission.

Anesthetics↗

PubMed2000

Low frequency voltage clamp: recording of voltage transients at constant average command voltage.

We implemented a simple feedback system that modifies the conventional current clamp mode of a patch clamp amplifier so that transient potentials, such as action potentials and synaptic potentials, can be measured as in the usual current clamp, while the average membrane potential is kept constant at a value chosen by the user. The circuit thus works like the current clamp for high frequency signals and like a voltage clamp for low frequency signals. We delineate its transfer properties and give application examples.

Animals↗

PubMed1996

Muscarine activates a nonselective cation current through a M3 muscarinic receptor subtype in rat dorsolateral septal nucleus neurons.

1. In the present study, we examined the cellular mechanism and receptor type responsible for a muscarine-induced inward current (Imi) in neurons of rat dorsolateral septal nucleus (DLSN) using single-microelectrode voltage-clamp and "slice" patch-clamp techniques. 2. Imi was associated with an increase of membrane conductance in 75% of DLSN neurons. There was no voltage-dependence of Imi between -60 and -140 mV; it exhibited a reversal potential of -17.0 +/- 5.3 mV (n = 14) determined by extrapolation of Imi and voltage relationship recorded using whole cell patch recording. Lowering extracellular sodium (26 mM) or potassium (1.4 mM) ions depressed Imi. 3. Imi was concentration dependent; 3 and 100 microM muscarine produced the minimum [22 +/- 4.6 pA, (mean +/- SE) n = 8] and maximum (167 +/- 28 pA, n = 7) responses, respectively. An EC50 was determined to be 15 microM (n = 8). Oxotremorine-methiodide (1-100 microM) also produced an inward current with similar potency compared with muscarine. On the other hand, McN-A-343 and pilocarpine (3-100 microM) did not produce any inward current in DLSN neurons. 4. Atropine (1 microM) completely reduced Im produced by 30 microM muscarine, whereas pirenzepine (PZP) shifted the concentration-response curve for muscarine in a parallel manner to the right. The EC50 for muscarine was shifted to 32, 52, and 204 microM by 0.2, 0.5, and 2 microM PZP, respectively. The apparent Kd value for PZP estimated by Schild plot analysis was 190 nM (n = 5). 5. Methoctramine (1 microM) also competitively depressed Imi; the calculated EC50 values were 26, 41, and 107 microM in concentrations of 0.2, 2, and 10 microM methoctramine, respectively. The apparent Kd for methoctramine was 420 nM. In contrast, AF-DX 116 (1 microM) did not significantly inhibit Imi. 6. Intracellular dialysis with guanosine 5'-O-(3-thiotriphosphate), a nonhydrolyzable analogue of GTP, suppressed irreversibly Imi. Pretreatment of DLSN neurons with pertussis toxin (PTX) did not prevent Imi (n = 8). 7. We suggest that muscarine causes this inward current by activating a M3 subtype of muscarinic receptor, which is coupled to a PTX-insensitive GTP-protein in rat DLSN neurons.

Animals↗