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At least 721 records · Page 40Linked to original sources
PubMed1989

Patch clamp studies of single cell-fusion events mediated by a viral fusion protein.

To enter cells, viruses must fuse their envelope with a host cell membrane. Fusion is mediated by specific, membrane-spanning fusion proteins, of which the influenza virus haemagglutinins (HA) are the best characterized. Several HAs have been sequenced, and the crystal structure of the major part of one HA is known. The conditions for fusion and some of the rearrangements in the HA that accompany fusion are well understood, but it remains unclear how HA causes bilayers to fuse. We have observed, in real time, unitary cell-fusion events caused by HA. Fibroblasts expressing HA were induced to fuse with red blood cells by a rapid drop in pH. Fusion was monitored by fluorescence microscopy, and by measuring the membrane conductance and capacitance of the fibroblast. The earliest event observed was the sudden opening of an aqueous pore connecting the cytoplasms of the fusing cells. Initially, the pore conductance often fluctuated between zero and approximately 600 pS, as if the pore were opening and closing repeatedly. Later, it increased over tens of seconds, as if the pore dilated. We suggest that, as in exocytosis, HA-mediated membrane fusion begins with the formation of a narrow pore. Based on the conductance, we estimate the initial diameter of the pore to be no more than twice that of a gap junction channel.

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PubMed1987

Voltage-Dependent K-Channel in Protoplasmic Droplets of Chara corallina: A Single Channel Patch Clamp Study.

Passive transport of potassium through the plasma membrane of a protoplasmic droplet isolated from large internodal cells of Chara corallina Klein ex Willd., em, R.D.W. has been investigated using the patchclamp technique. When the membrane is hyperpolarized the conductance of a single K(+)-channel is of the order of magnitude of 100 picoSiemens and is reduced by tetraethylammonium chloride. Its open time is voltage dependent. This voltage-dependent K(+)-channel displays rectifying properties. The channel density is about 0.1 channel per square micrometer of membrane. When the membrane is depolarized the conductance of a single channel is of the order of magnitude of 30 picoSiemens and is insensitive to tetraethylammonium chloride. These results suggest that K(+)-channels are incorporated in the plasma membrane during membranogenesis of a protoplasmic droplet. They constitute further evidence for the existence of voltage-dependent K(+)-channels in plant cells.

Journal Article↗
PubMed1990

Interaction of the Depolarization-Activated K Channel of Samanea saman with Inorganic Ions: A Patch-Clamp Study.

A depolarization-activated K(+) channel capable of carrying the large K(+) currents that flow from shrinking cells during movements of Samanea saman leaflets has been described in the plasmalemma of Samanea motor cell protoplasts (N Moran et al [1988] Plant Physiol 88:643-648). We now characterize this channel in greater detail. It is selective for K(+) over other monovalent ions, with the following order of relative permeability: K(+) > Rb(+) > Na(+) approximately Cs(+) approximately Li(+). It is blocked by Cs(+) and by Ba(2+) in a voltage dependent manner, exhibiting a ;long-pore' behavior, similarly to various types of K(+) channels in animal systems. Cadmium, known for its blockage of Ca(2+) channels in animal systems, and Gd(3+), closely related to La(3+), which also blocks Ca(2+) channels in animal cells, both block K(+) currents in Samanea in a voltage-independent manner, and without interfering with the kinetics of the currents. The suggested mechanism of block is either (a) by a direct interaction with the K(+) channel, but external to its lumen, or, alternatively, (b) by blocking putative Ca(2+) channels, and preventing the influx of Ca(2+), on which the activation of the K(+) channels may be dependent.

Journal Article↗
PubMed1993

Characterization of synaptically elicited GABAB responses using patch-clamp recordings in rat hippocampal slices.

1. Tight-seal, whole-cell voltage clamp recording techniques were used to characterize monosynaptically evoked GABAB currents in adult rat brain slices maintained at 34-35 degrees C. Responses were recorded from granule cells of the dentate gyrus following the blockade of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-, D-2-amino-5-phosphonovaleric acid (D-AP5)- and picrotoxin-sensitive fast synaptic transmission, so that the remaining synaptic currents could be studied in isolation. 2. Under these conditions, stimulation in the molecular layer elicited a slow outward current which was blocked by the selective GABAB antagonist CGP 35348 in a concentration-dependent manner (200-800 microM). This current was absent in recordings made with pipettes containing 10-15 mM of the lidocaine derivative QX-314 or when caesium was substituted for K+. 3. Increasing the [K+]o e-fold (from 2.5 to 6.8 mM) shifted the reversal potential of the GABAB current from -97.9 to -73.2 mV, as predicted by the Nernst equation. Peak conductance was constant, but in 6.8 mM [K+]o at voltages hyperpolarized to EK (equilibrium potential for potassium), a small outward rectification was evident. 4. The time course of the current could be described by fourth-power exponential activation kinetics with double exponential inactivation. At 34-35 degrees C, the average activation time constant (tau m) was 45.2 ms, while the two inactivation time constants (tau h1 and tau h2) were 110.2 and 516.2 ms, with corresponding weighting factors (wh1 and wh2) of 0.84 and 0.16, respectively. The Q10 (temperature coefficient) values for these time constants were between 1.82 and 2.31. Neither tau m, nor tau h1 and tau h2 were voltage dependent in the range from -45 to -95 mV. 5. Paired-pulse depression of the GABAB current was studied by giving identical conditioning and test stimuli over a wide range (50-5000 ms) of interstimulus intervals (ISIs). The maximal depression (48%) occurred at 200 ms ISI, and the depression lasted for over 5 s. The magnitude of paired-pulse depression was not dependent on the postsynaptic membrane potential. 6. Application of the competitive antagonist CGP 35348 such that the peak current was diminished by approximately 50% had no effect on the activation or inactivation kinetics of the current. Similarly, during paired-pulse depression the kinetics of test currents were identical to those of conditioning currents. These findings support the hypothesis that the mechanism responsible for paired-pulse depression involves a reduction in neurotransmitter release without postsynaptic alterations in K+ channel activation/inactivation kinetics.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed2004

Patch Clamp Study of Serotonin-Gated Currents via 5-HT Type 3 Receptors by Using a Novel Approach SHAM for Receptor Channel Scanning.

We studied 5-hydroxy tryptamine type 3 (5-HT(3)) receptors transfected in tsA-201 cell line to examine serotonin-induced whole cell currents. Using the site-directed mutagenesis technique, we individually mutated each residue in the membrane-spanning M2 segment to histidine. A high proportion of tsA-201 cells cotransfected with the cDNAs of 5-HT(3)R and CD8 produced large amplitude responses (0.5–7.0 nA) to serotonin. The dose-response curve of wild-type (WT) receptor ranging from 0.5 to 500 $\mu$ mole increases its K(d) values, and I_\mathrm{max} of 5-HT(3)R falls at low external pH as if protonation of an acid group is enough to block the channel. Lysine at position 281, a basic residue, is more susceptible to acidification-induced blockade of the 5-HT(3)R channel. Dose-response curves of K281S (replacing lysine at the 281 position with serine) at different pH are not significantly modulated, and histidine substitutions at the three consecutive positions 293, 294, and 296 eliminate the pH block of the channel.

Journal Article↗
PubMed1987

Mouse spinal cord neurons in serum-free culture media: suitability for patch clamp studies on chemical and electrical excitability.

Methods were devised for the serum-free culture of spinal cord neurons derived from 12- to 13-day mouse embryos. Neurons exhibited good attachment if plated for 24 h on poly-d-lysine-coated dishes in the presence of serum. Cultures were subsequently fed with a serum-free medium consisting of minimum essential medium, Earle's salts and the N1 supplement, i.e. insulin, putrescine, transferrin, progesterone and selenium. After 3 weeks in vitro, growth and survival of neurons in this medium were comparable to results obtained using serum-supplemented medium. The presence of putrescine was not essential for the beneficial effects of N1, while insulin was required for long-term survival in serum-free media. Neurons maintained in serum-free media for 3 weeks retained aspects of electrical and chemical excitability characteristic of serum-grown cells.

Animals↗
PubMed1991

[Primary culture of mammalian brain neurons and its application to patch-clamp recording].

A method is described for the primary culture of neurons from adult mammalian brain. The primary culture consists of five processes: 1) preparation of brain slices, 2) microdissection of the discrete area, 3) enzymatic treatment of the tissue, 4) dissociation of cells by mechanical agitation of the tissue fragments, and 5) plating and feeding of dissociated cells. The cells could be maintained in culture for more than several weeks. Whereas neurons freshly dissociated from the adult brain do not respond to exogenously applied neurotransmitter substances, probably due to destruction of receptors by the enzymatic treatment, the neurons regained the ability to respond to a variety of neurotransmitters when they were cultured. Cultured neurons from adult mammalian brain are proving to be an excellent model for physiological as well as pharmacological investigations on the central nervous system.

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PubMed1990

An improved method for isolating cardiac myocytes useful for patch-clamp studies.

An improved method for isolating cardiac myocytes is described. This method consists of the initial dispersion of the myocytes with a new type of collagenase and the following treatment of the cells with a non-specific protease. This method gives more than 80% rod-shaped, viable, and quiescent cells, which are suitable for biochemical as well as electrophysiological experiments. Formation of a G omega seal and subsequent whole-cell recording is easily performed using these cells.

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