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PubMed1994

Patch-clamp analysis of the properties of acetylcholine receptor channels at the normal human endplate.

Normative data were obtained on the kinetic properties of the acetylcholine receptor (AChR) channel at the human motor endplate by patch-clamp analysis. Single channel currents were recorded from 34 endplates of 8 nonweak subjects in the presence of 1 micron acetylcholine (ACh) at 22 +/- 0.5 degrees C. The vast majority of channels opened to a conductance of about 60 pS. The dwell-time distributions of these channels were well described as the sum of two exponential functions. The mean duration of the dominant longer component was 1.9 ms for the open intervals and 3.04 ms for the bursts. At three endplates, a small proportion of the channels had lower conductance and longer open time, resembling immature AChR channels. At 28 endplates it was also possible to obtain an estimate of the rate constant for channel closure (alpha) and approximate estimates for the rate constants of channel opening (beta) and ACh dissociation (k-2). Estimates of k-2 varied by 15% with methods of estimation. This is attributed to errors inherent in estimating the duration of the briefest channel events. The normative data will be useful for evaluating pathologic alterations in the kinetic properties of the AChR channel found in some congenital myasthenic syndromes.

Humans↗

PubMed1988

Patch-clamp study of gamma-aminobutyric acid receptor Cl- channels in cultured astrocytes.

The membrane channels operated by gamma-aminobutyric acid (GABA) were studied in cultured astrocytes from rat cerebral hemispheres by using patch-clamp techniques. The channel properties appeared to be very similar, in many respects, to those present in neuronal cell membranes. The Cl- -selective channels were activated after the sequential binding of two GABA molecules to the receptor, as deduced from the slope of the dose-response curve. Single-channel currents displayed multiple conductance states of 12 pS, 21 pS, 29 pS, and 43 pS, with the main-state conductance being 29 pS. The gating properties could be described by a sequential reaction scheme for agonist-activated channels. GABA-induced whole-cell currents were potentiated by the benzodiazepine receptor agonist diazepam and also, to a lesser extent, by methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate an inverse agonist. In neurons and chromaffin cells, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate reduces the sensitivity of the GABA receptor, indicating that neuronal and glial GABA/benzodiazepine receptor--Cl- channel complexes are different. Glial GABA receptor channels could be of functional importance in buffering extracellular Cl- in the cleft of the GABAergic synapse.

Animals↗

PubMed1988

Patch-clamp study of the ionic currents underlying action potentials in cultured frog pituitary melanotrophs.

The ionic conductance mechanisms underlying the action potential behaviour of frog melanotrophs in primary culture were studied by using the patch-clamp technique in whole-cell configuration. The action potentials spontaneously generated by these cells were predominantly sodium spikes with a calcium component. Voltage-dependent sodium, calcium, potassium and calcium-activated potassium currents were identified and analysed separately. The voltage-dependent sodium current was characterized by its fast kinetic, its low-threshold activation, its voltage-dependent inactivation and a tetrodotoxin sensitivity. Calcium currents were identified on the basis of their ionic selectivity to divalent cations (Ba2+, Ca2+, Co2+) and their time course. Only two of the three well-documented calcium currents could be detected in frog melanotrophs. A sustained calcium current (ICaS) and an inactivating calcium current (ICaN) were elicited by step depolarizations up to -20 mV. ICaN inactivated for membrane potentials more positive than -50 mV; its inactivation appeared to be both voltage- and calcium-dependent. Transient calcium current (ICaT) has never been observed. Two types of potassium currents were identified: voltage-dependent potassium (IKV) and calcium-activated potassium currents, (IK[Ca]). They were both suppressed by tetraethylammonium chloride, whereas only IK(Ca) was blocked by cobalt. These major ionic currents underlying spontaneous electrical activity are assumed to be involved in the process of alpha-melanocyte-stimulating hormone release. The present study provides the ground for future investigations regarding the relationships between the electrical and secretory activities in amphibian pars intermedia cells.

Action Potentials↗

PubMed1991

Patch clamping protoplasts from vascular plants : method for the quick isolation of protoplasts having a high success rate of gigaseal formation.

A method is described for the isolation of protoplasts (Pisum sativum, Phaseolus vulgaris, Avena sativa, Arabidopsis thaliana) in preparation for ion flux studies using patch clamp electrophysiology. Protoplasts that have been exposed to hydrolytic, cell wall degrading, enzymes for as little as 5 minutes form gigaseals (seal resistance higher than 10 giga Ohm) with the patch pipette with success rates greater than 40%. Sealing of these protoplasts is fast, averaging less than 2 minutes. This method yields high rates of gigaseal formation in a variety of tissues from both monocots and dicots and will enhance data collection in ion flux studies of plasma membranes of vascular plants.

Journal Article↗

PubMed1986

A patch-clamp analysis of membrane currents in salamander olfactory receptor cells.

Isolated olfactory receptor cells were obtained from salamander olfactory epithelium and kept in short term culture conditions. They were studied by means of the whole cell patch-clamp technique associated with ionic substitutions and channel blockers. Under physiological ionic gradients, these cells had a resting potential of -39 +/- 10 mV and an input resistance above 2 G omega. Using different channel blockers and ion substitutions, we could separate several distinct components in the overall whole cell current. In most cells, inward current reflected the activation of a TTX resistant conductance which was blocked by cobalt ions. This inward current lasted only for about 5 min of whole cell recording. In a minority of cells, a TTX sensitive sodium current was also observed. The outward K+ current was blocked when the cells were loaded with cesium and tetraethylammonium. It inactivated slowly and incompletely and could act as a depolarization limited in case of intense odour stimulations. Single channel analysis from outside out patches suggested that it corresponded to the activity of 34 pS channels. In some cells a rapidly inactivating K+ current was also present. Single channel activities (27 +/- 6 pS) were commonly recorded with KCl-filled pipettes, at resting or hyperpolarized membrane potentials but not at depolarized potentials. Membrane hyperpolarization increased the open-state probability. A preliminary study with odorant stimulations indicated the existence of a stimulus-induced current probably corresponding to the activation of the chemoreceptive membrane.

Animals↗

PubMed1989

Patch clamp analysis of recovery of sodium channels from inactivation in mammalian myocardium in terms of a Markovian state model.

The kinetics of the recovery of Na channels from inactivation were investigated in ventricular cardiocytes of adult mice by a patch clamp technique. Single channel analysis showed an unaltered slope conductance of unitary currents during the recovery process to be 18.5 pS. The mean open time tau 0 and its monoexponential distribution remained also unchanged. The recovery time course of the apparent probability for a channel not to open coincided perfectly with the recovery time course determined from peak values of macroscopic currents which were recorded from cell-attached patches with up to 80 channels by averaging between 19 and 38 sweeps. Conclusively, the recovery was quantified by the time course of re-accumulation of the channels in a resting state. The initial recovery time course was sigmoidal. A serial Markov state model with three inactivated states in addition to the resting state was most appropriate for its description. The rate constants were assumed to be equal direction. A highly voltage-sensitive reaction towards the last inactivated state and a nearly voltage-independent reaction towards the resting state were obtained. Slow inactivation components were analyzed both during the recovery process (-100 mV ... -130 mV) after long preceding inactivation and at maintained depolarization (-50 mV ... -20 mV). For description, the model was extended by two further, serially coupled inactivated states. All four rate constants describing slow inactivation in the range of hundreds of milliseconds to seconds proved to be voltage-independent. Taken together, the results suggested the absorbing inactivated state I to be composed of five serially coupled inactivated states with only two transitions being steeply voltage-dependent.

Animals↗

PubMed1994

Dependence of LTP induction on postsynaptic depolarization: a perforated patch-clamp study in visual cortical slices of young rats.

1. To see whether there is a threshold of postsynaptic depolarization for induction of long-term potentiation (LTP) or depression (LTD) of synaptic transmission, perforated patch-clamp recordings were carried out under microscopic observation from 61 layer II/III neurons in visual cortical slices of young rats. Electrical stimulation given to nearby neurons was paired with stepwise shifts (30- or 300-ms duration) of clamped membrane potential of the recorded neurons to various levels. 2. Excitatory postsynaptic currents (EPSCs) were elicited by focal stimulation of a nearby pyramidal cell-like neuron. As the intensity of stimulation was increased, EPSCs emerged abruptly with 100% probability, and their peak latencies and amplitudes remained almost constant up to more than twice the threshold, indicating that the EPSCs were elicited monosynaptically. 3. LTP of EPSCs was induced in 11 of the 15 cells after pairing with a step to -20 mV and in 5 of the 14 cells after pairing with a step to -40 mV. No LTP was observed when the postsynaptic cells were clamped at -60, -70, or -90 mV. Significant LTD was not seen at any membrane potential level tested. There was no significant difference between the duration of potential shift of 30 and 300 ms during the pairing procedure in induction probability of LTP and magnitude of LTP, if it was induced. 4. These results suggest that LTP is induced by synaptic inputs associated with postsynaptic depolarization above the threshold around -40 mV at synapses linking layer II/III neurons in the developing visual cortex.

Age Factors↗

PubMed1988

Patch-clamp studies in human macrophages: single-channel and whole-cell characterization of two K+ conductances.

Human peripheral blood monocytes cultured for varying periods of time were studied using whole-cell and single-channel patch-clamp recording techniques. Whole-cell recordings revealed both an outward K current activating at potentials greater than 20 mV and an inwardly rectifying K current present at potentials negative to -60 mV. Tail currents elicited by voltage steps that activated outward current reversed near EK, indicating that the outward current was due to a K conductance. The I-V curve for the macroscopic outward current was similar to the mean single-channel I-V curve for the large conductance (240 pS in symmetrical K) calcium-activated K channel present in these cells. TEA and charybdotoxin blocked the whole-cell outward current and the single-channel current. Excised and cell-attached single-channel data showed that calcium-activated K channels were absent in freshly isolated monocytes but were present in greater than 85% of patches from macrophages cultured for greater than 7 days. Only 35% of the human macrophages cultured for greater than 7 days exhibited whole-cell inward currents. The inward current was blocked by external barium and increased when [K]o increased. Inward-rectifying single-channel currents with a conductance of 28 pS were present in cells exhibiting inward whole-cell currents. These single-channel currents are similar to those described in detail in J774.1 cells (L.C. McKinney & E.K. Gallin, J. Membrane Biol. 103:41-53, 1988).

Barium↗

PubMed1987

A patch clamp study of gamma-aminobutyric acid (GABA)-induced macroscopic currents in rat melanotrophs in cell culture.

1. The macroscopic currents induced in cultured rat melanotrophs by exogenous gamma-aminobutyric acid (GABA) were analysed using the patch clamp recording technique. 2. Using various concentrations of intra- and extracellular chloride it was demonstrated that the conductance activated by GABA was chloride selective. Since these currents were blocked with bicuculline and enhanced with chlordiazepoxide the involvement of GABAA receptors similar to those in the CNS is indicated. 3. When chloride was symmetrically distributed across the membrane the voltage/current relationship was linear; pronounced rectification of GABA mediated currents was evident when there was an asymmetrical distribution of chloride. 4. With concentrations of GABA greater than 10 microM a fading of the current was seen during prolonged (5-10 s) applications. This effect appeared to be due to a decline of conductance rather than a shift of the chloride equilibrium potential. 5. Values for the Hill coefficient derived from dose-response curves suggested that the binding of 2 molecules of GABA to the receptor is required for the activation of the chloride channel. 6. There was no indication of a direct, GABAB receptor-mediated change of conductance.

Action Potentials↗

PubMed1991

Identification of anion and cation pathways in the inner mitochondrial membrane by patch clamping of mouse liver mitoplasts.

Alkalinization of the matrix side of the mitochondrial inner membrane by pH shifts from 6.8 to 8.3 caused a reversible increase in current of 3.2 +/- 0.2 pA (mean +/- SE, n = 21) at +/- 40 mV measured using patch-clamp techniques. The current increase was reversed in a graded fashion by the addition of Mg2+ as well as a reduction in pH. Detection of single-channel events was done at 0.5, 1 and 2 M KCl. The single-channel amplitude in 0.15 M KCl corresponds to approximately 15 pS. Reversal potentials derived from whole patch currents indicated that the inner mitochondrial membrane was primarily cation selective at pH 6.8 with a PK/PCl = 32 (n = 6). Treatment with alkaline pH (8.3) increased the current and anion permeability (PK/PCl = 16, n = 6). The membrane becomes completely cation selective when low concentrations (12 microM) of the drug propranolol are added. The amphiphilic drugs amiodarone (4 microM), propranolol (70 microM) and quinine (0.6 mM) blocked almost all of the current. The pH-dependent current was also inhibited by tributyltin. These results are consistent with the presence of two pathways in the inner mitochondrial membrane. One is cation selective and generally open and the other is anion selective and induced by alkaline pH. The alkaline pH-activated channel likely corresponds to the inner membrane anion channel postulated by others from suspension studies.

Amiodarone↗

PubMed1993

Patch-clamp recording from Müller (glial) cell endfeet in the intact isolated retina and acutely isolated Müller cells of mouse and guinea-pig.

Müller cells span through the entire retina and terminate with the formation of endfeet at the vitreous body. These endfeet are thought to be specialized for maintaining the K+ homeostasis in the retina based on the assumption that voltage signals can passively spread from the cell body to the endfeet. We employed the patch-clamp technique to study the physiological properties of these endfeet in a retinal wholemount preparation from guinea-pig or mouse. After assessing one endfoot with the patch pipette and establishing the whole cell recording configuration, a membrane area which approximately matched the size of one endfoot and proximal process could be voltage-clamped. This morphological correlation could be established by filling the cytoplasm with the fluorescent dye Lucifer Yellow via the patch-pipette. The morphological, immunocytochemical and ultrastructural inspection of the recorded cells revealed that mouse Müller cell endfeet were connected by only a thin stalk to the proximal process. In contrast, guinea-pig endfeet were connected by thick stalks. The endfoot current in the mouse was dominated by a voltage and time-independent K+ conductance. In contrast, in some of the recordings from guinea-pig, delayed and inwardly rectifying K+ currents were observed. These voltage-gated currents were more frequently observed or were facilitated when the membrane area under voltage clamp was increased, blocking the passive K+ currents by Ba2+ in both, mouse and guinea-pig. We thus assume that the voltage-gated currents were not in the endfeet membrane, but rather in the proximal process and could thus be better activated in the guinea-pig with its thicker stalk or after increasing the membrane area under voltage clamp control. Similar results were obtained in freshly isolated Müller cells; in contrast to the cells from the wholemount the voltage-gated currents were more frequently observed. These studies demonstrate that the Müller cell endfoot of the mouse with its vascularized retina is an electrically isolated unit and that voltage signals do not spread to the proximal process. Such a property would, however, be required for the redistribution of K+ via spatial buffer currents. In contrast, guinea-pig Müller glial cells with their stout morphological connection between endfoot and proximal process are better suited to fulfil this task.

Animals↗

PubMed1989

Patch clamp experiments on nicotinic acetylcholine receptor-ion channels in bullfrog sympathetic ganglion cells.

Nicotinic acetylcholine-receptor ion channels (AChR channels) were studied in bullfrog sympathetic ganglion cells cultured for 1 day to 3 weeks, using a patch clamp technique. Microsuperfusion of ACh (2-10 microM) to the ganglion cell under the whole cell clamp produced an inward current at membrane potentials negative to -60 mV, which had a fast onset and decay. This rapid ACh-induced current was accompanied by a large current fluctuation, decreased and increased in amplitude by membrane depolarization and hyperpolarization, respectively, and blocked by d-tubocurarine. Thus, this current must be induced by the nicotinic action of ACh, but not by a muscarinic effect to activate a slow cation-selective current. At depolarized levels more than -50 mV, ACh induced an additional inward current which was slow in time course, accompanied by no or decreased current fluctuation and increased in amplitude by membrane depolarization. Accordingly, this slow ACh-induced current could result from the suppression of a voltage-dependent K+ current (M-current: Brown and Adams 1980) by the muscarinic action of ACh. Fluctuation analysis of the rapid ACh-induced current at potentials negative to -50 mV revealed the elementary conductance of 14 pS and a power spectral density distribution of the double Lorentzian function which yielded the time constants of 5.4 and 62.5 ms at -60 to -80 mV. The variance of either component was independent of the mean current.

Acetylcholine↗

PubMed1990

Limitations of the whole cell patch clamp technique in the control of intracellular concentrations.

Recent experimental studies (Pusch and Neher, 1988) and theoretical studies (Oliva et al., 1988) have found that the pipette tip is a significant barrier to diffusion in the whole cell patch clamp configuration. In this paper, we extend the theoretical analysis of fluxes between the pipette and cell to include transmembrane fluxes. The general conclusions are: (a) within the pipette, ion fluxes are driven primarily by diffusion rather than voltage gradients. (b) At steady state there is a concentration difference between the bulk pipette and intracellular solution that is described by delta c = jRp/Dp, where delta c = 1 mM for a flux, j = 1 fmol/s, through a pipette of resistance, Rp = 1 M omega, filled with a solution of resistivity, p = 100 omega --cm, given a solute diffusion coefficient, D = 10(-5) cm2/s. (c) The time to steady state is always accelerated by membrane transport, regardless of the direction of transport. We apply our analysis to the measurement of transport by the Na/K pump and Na/Ca exchanger in cells from the ventricles of mammalian heart. We find that the binding curve for intracellular Na+ to the Na/K pump will appear significantly less steep and more linear if one does not correct for the concentration difference between intracellular and pipette Na+. Similar shifts in the binding curve for extracellular Na+ to the Na/Ca exchanger can occur due to depletion of intracellular Ca(+)+ when the exchanger is stimulated. Lastly, in Appendix we analyze the effects of mobile and fixed intracellular buffers on the movement of Ca(+)+ between the pipette and cell. Fixed buffers greatly slow the time for equilibration of pipette and intracellular Ca(+)+. Mobile buffers act like a shuttle system, as they carry Ca(+)+ from pipette to cell then diffuse back when they are empty. Vigorous transport by the Na/Ca exchanger depletes mobile buffered calcium, thus stimulating diffusion from the pipette to match the rate of Ca(+)+ transport. Moreover, we find that binding of Ca(+)+ to the exchanger can be affected by the mobile buffer.

Animals↗

PubMed1989

Whole-cell patch-clamp analysis of voltage-dependent calcium conductances in cultured embryonic rat hippocampal neurons.

1. Voltage-dependent calcium currents in embryonic (E18) hippocampal neurons cultured for 1-14 days were investigated using the whole-cell patch-clamp technique. 2. Calcium currents were isolated by removing K+ from both the internal and external solutions. In most recordings the external solution contained tetrodotoxin, tetraethylammonium ions, and low concentrations of Na+, whereas the internal solution contained the large cations and anions, N-methyl-D-glucamine and methanesulphonate, and an adenosine 5'-triphosphate (ATP) regenerating system (Forscher and Oxford, 1985) to retard "run-down" of Ca currents. 3. Under these conditions, the sustained inward current triggered during depolarizing steps was enhanced when extracellular [Ca2+] ([Ca2+]0) was raised from 2 to 10 mM and abolished when [Ca2+]0 was lowered to 0.1 mM or by addition of Co2+ ions. These results indicate that the inward current was carried primarily by Ca2+ ions and was designated ICa. This current may be comparable to the "high-voltage-activated" Ca current described in other preparations. 4. In cells cultured for 1-3 days, ICa was small or absent (less than 20 pA for cells 1 day in culture and less than 80 pA for cells 3 days in culture). Although ICa decayed considerably during depolarizing steps, there was little evidence of the transient calcium current (T current) that was recorded in approximately 40% of cells cultured longer than 6 days. Maximal (i.e., the largest) ICa increased from 20 to 80 pA in 1- to 3-day cells to 150-450 pA in cells cultured for longer than 6 days. 5. The decay of ICa elicited by depolarizations from holding potentials of -60 mV or more negative was usually greatest for the maximal ICa. Replacement of extracellular Ca2+ (4 mM) with Ba2+ (2 mM) resulted in a substantial decrease in the extent of decay of ICa and a shift of the I-V relation in the hyperpolarizing direction. 6. Qualitative data obtained from experiments in which different levels of internal Ca2+ buffering were employed demonstrated that, on average, the decay of ICa was reduced as the capacity and/or rate of buffering was increased. The mean decay of ICa in cells buffered with 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was 9 +/- 7 (SD) %, (n = 12) and 25 +/- 12%, (n = 12) for cells buffered with the same concentration of ethyleneglycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Whole-cell patch clamp measurements and berberine inhibition of hyperpolarization-activated inward current in rabbit sinoatrial node cells.

Single rabbit sinoatrial node (SAN) cells were isolated by means of an enzymatic dispersion procedure and used for the whole-cell patch clamp experiment. At a holding potential of -40 mV, a time- and voltage-dependent inward current, I(f), was activated at different hyperpolarization potentials from -60 mV to -110 mV. The current-voltage relation curve showed that I(f) was activated at potential more negative than -40 mV. Five min after treatment by CsCl 2 mmol.L-1-containing Tyrode solution, I(f) was almost completely blocked. At a hyperpolarization potential of -110 mV, I(f) was reduced from 1.7 +/- 0.2 nA of the control to 1.2 +/- 0.4 nA after superfusing with Tyrode solution containing berberine (Ber) 1 mumol.L-1 for 5 min. And it was difficult to wash out this action. Ber also had inhibitory effects on other currents to a certain extent. The results indicate that I(f) is a Cs(+)-sensitive current and that the negative chronotropic effect of Ber may be due to the inhibition of I(f) that functions as an important pacemaking modulator for the spontaneous depolarization of SAN tissue.

Action Potentials↗

PubMed1992

Patch-clamp recordings of spiking and nonspiking interneurons from rabbit olfactory bulb slices: GABA- and other transmitter receptors.

Transmitter receptor ion channels from previously identified rabbit olfactory bulb neurons were studied by using a thin slice preparation in combination with patch-clamp measurements. PG cells, which closely resembled previously described periglomerular interneurons in their morphology, responded to microapplication of GABA, acetylcholine, norepinephrine and glycine with the activation of distinct ionic currents. JG cells, which belong either to the class of short axon cells or external tufted cells, never showed GABA responses. In mitral cells ionic currents activated by GABA, acetylcholine, norepinephrine and glutamate could be elicited. Further measurements of GABA-activated currents of PG cells were made and indicated that these cells expressed two different types of GABA receptors: one which showed fast desensitization with a decay time constant of about 5 s, and one which slowly desensitized with a decay time constant of about 20-30 s. Both types were completely inhibited by bicuculline methiodide (50 microM). GABA receptors were not blocked by Zn2+ (0.1 mM). From the dose-response relationship of the peak GABA-activated currents, an apparent dissociation constant of 50 microM was derived. From single channel measurements in excised outside-out patches, a single channel conductance of GABA-activated Cl- currents of 24 pS was obtained during continuous application of the agonist. Single channel events had a mean open time of 1.9 ms.

Animals↗

PubMed1991

Patch-clamp studies on the anomalous mole fraction effect of the K+ channel in cytoplasmic droplets of Nitella: an attempt to distinguish between a multi-ion single-file pore and an enzyme kinetic model with lazy state.

Patch-clamp studies have been employed in order to check whether the assumption of a multi-ion single-file pore is necessary for the explanation of the anomalous mole fraction effect or whether this effect can also be explained by a single-barrier enzyme kinetic model. Experiments in the cell-attached configuration were done on the tonoplast membrane of cytoplasmic droplets of Nitella in solutions containing 150 mol m-3 of K+ plus Tl+ with seven different K+/Tl+ ratios. At first sight, the results seem to support the multi-ion single-file pore, because apparent open channel conductivity displays the anomalous mole fraction effect, whereas open-probability has not been found to be dependent on the K+/Tl+ ratio. Changes in open probability would be expected for a single-barrier enzyme kinetic model with a lazy state. On the other hand, the lazy-state model is more successful in explaining the measured I-V curves. The entire slope of the apparent open channel current-voltage curves rotates with changing K+/Tl+ ratios in the whole voltage range between -100 and +80 mV. Numerical calculations on the basis of multi-ion single-file pores could create the anomalous mole fraction effect only in a limited voltage range with intersecting I-V curves. The apparent absence of an effect on open probability which is postulated by the lazy-state model can be explained if switching into and out of the lazy state is faster than can be resolved by the temporal resolution of 1 msec.

Biological Transport↗

PubMed1992

A patch-clamp study on the muscarine-sensitive potassium channel in bullfrog sympathetic ganglion cells.

1. A voltage-independent K+ channel was characterized and effects of muscarine were studied in cultured bullfrog sympathetic ganglion cells using the cell-attached patch-clamp configuration. 2. Three types of single-channel current were recorded from 2- to 10-day-old cultured cells in the presence of tetraethylammonium (2-20 mM), tetrodotoxin (1-2 microM), Cd2+ (0.1 mM) and apamin (20 nM). 3. The most frequently observed channel was a voltage-independent K+ channel which was open at the resting membrane potential and had a conductance of 52.6, 78.9 and 114.9 pS at a [K+]o of 2, 40 and 100 mM, respectively. This channel was designated background K+ channel. 4. Two other channel types were observed less frequently. One had a conductance of 26 pS (external K+, 118 mM) and a long open time of several seconds at the resting membrane potential. The second channel had a smaller conductance (20 pS) and displayed a voltage-dependent activation. 5. The open probability of the background K+ channel varied between patches, ranging from 0.0005 to 0.486. The open time distribution was fitted by a single exponential with a time constant of 0.51 ms. Both of these parameters were independent of the membrane potential. The closed time distribution consisted of at least four exponentials having time constants of 0.17, 3.7, 120 ms and several seconds. 6. Muscarine (10-20 microM) applied to the membrane outside the patch pipette reversibly enhanced the activity of the background K+ channel. This effect was associated with an increase in the open probability, which resulted from an increase in the mean open time concomitant with a decrease in the mean closed time. Muscarine did not change the single-channel conductance of this channel. 7. The effects of muscarine were blocked by atropine (1 microM). 8. It is concluded that there exists a muscarine-sensitive, voltage-independent K+ channel in cultured bullfrog ganglion cells. This K+ channel appears to contribute to the generation of the resting membrane potential and underlie the slow inhibitory postsynaptic potential of these neurones in situ.

Animals↗