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W Almers

Publications and source records attributed to W Almers.

At least 19 recordsLinked to original sources
PubMed1992

Repulsion between tetraethylammonium ions in cloned voltage-gated potassium channels.

Tetraethylammonium ion (TEA+) blocks voltage-gated K+ channels by acting at two sites located at opposite ends of the aqueous pore. This allowed us to test two predictions made by models of ion permeation, namely that K+ channels can be simultaneously occupied by multiple ions and that the ions repel each other. We show that externally applied TEA+ antagonize block by internal TEA+ and vice versa. The antagonism is less than predicted for competitive binding, hence TEA+ may occupy both sites simultaneously. External TEA+ and internal TEA+ reduce each others affinity 4- to 5-fold. In addition, K+ antagonizes block by TEA+ at the opposite side of the membrane, and external TEA+ antagonizes is block by internal Ba2+. The antagonism between ions applied at opposite sides of the membrane may be common to all cations binding to K+ channels.

Animals↗
PubMed1992

Exocytosis and its control at the synapse.

Several new approaches have given fresh insight into the mechanism and control of exocytosis. Electrophysiological and morphological studies show that many or all of the intramembrane particles at presynaptic active zones are voltage-gated Ca2+ channels. The sensitivity and time resolution of voltammetry allow the time course with which a single vesicle releases transmitter to be studied. Membrane proteins of the cell surface and synaptic vesicles have been shown to interact, and may join to form the fusion-pore complex.

Animals↗
PubMed1991

Cloning, expression, and gene structure of a G protein-coupled glutamate receptor from rat brain.

A complementary DNA encoding a G protein-coupled glutamate receptor from rat brain, GluGR, was cloned by functional expression in Xenopus oocytes. The complementary DNA encodes a protein of 1199 amino acids containing a seven-transmembrane motif, flanked by large amino- and carboxyl-terminal domains. This receptor lacks any amino acid sequence similarity with other G protein-coupled receptors, suggesting that it may be a member of a new subfamily. The presence of two introns flanking the central core suggests that GluGR may have evolved by exon shuffling. Expressed in oocytes, GluGR is activated by quisqualate greater than glutamate greater than ibotenate greater than trans-1-aminocyclopentyl-1,3-dicarboxylate, and it is inhibited by 2-amino-3-phosphonopropionate. Activation is blocked by Bordella pertussis toxin. These properties are typical of some metabotropic glutamate receptors.

Amino Acid Sequence↗
PubMed1991

The first milliseconds of the pore formed by a fusogenic viral envelope protein during membrane fusion.

Fibroblasts expressing the influenza virus hemagglutinin on their plasma membrane were patch clamped while they fused to erythrocytes. An increase in the fibroblast's membrane capacitance indicated the opening of the "fusion pore," the first aqueous connection between the fusing cells. We show here that the capacitance increase is preceded by a brief current transient, generated as the erythrocyte discharges its membrane potential through the nascent fusion pore. This signal allows one to calculate the pore conductance during the first milliseconds of its existence. The pore conductance jumps from 0 to approximately 150 pS and then grows more gradually over the subsequent tens of milliseconds until growth is arrested. The initial conductance is similar to that of a large ion channel and suggests that the pore is initially only 1-2 nm wide. Hence, we are probably observing events caused by only a small number of hemagglutinin molecules.

Animals↗
PubMed1990

Properties of the fusion pore that forms during exocytosis of a mast cell secretory vesicle.

During exocytosis, secretory vesicles of mast cells generate a current transient that marks the opening of the fusion pore, the first aqueous connection that forms between the vesicle lumen and the cell exterior. By recording and analyzing such current transients, we have tracked the conductance of the fusion pore over the first millisecond of its existence. The first opening of the pore occurs rapidly, generally within 100 microseconds at 23 degrees C. The electric conductance of the pore is a few hundred picosiemens at first, but gradually increases over the subsequent milliseconds. Evidently the pore opens abruptly and then dilates. The initial conductance of the pore suggests a diameter comparable to that of a large ion channel. From an analysis of "capacitance flicker" we infer that a pore can increase its diameter severalfold and still close again completely. This suggests that several early events in membrane fusion are reversible.

Animals↗
PubMed1990

Cytosolic Ca2+, exocytosis, and endocytosis in single melanotrophs of the rat pituitary.

We have monitored cytosolic [Ca2+] with fura-2 and exocytosis by measuring the membrane capacitance, and we have studied the influence of cytosolic [Ca2+] on secretion in single endocrine cells. As in neurons, cytosolic Ca2+ is sufficient to trigger exocytosis. The rate of secretion grows with the fourth or fifth power of cytosolic [Ca2+], and paired stimuli reveal facilitation. Ca2+ influx through voltage-sensitive Ca2+ channels can stimulate secretion 1000-fold over the basal levels measured biochemically. Unlike neurons, however, melanotrophs continue to secrete for seconds afer a depolarizing pulse, while they extrude or sequester the Ca2+ that has entered through Ca2+ channels. Following episodes of secretion, pituitary cells can retrieve membrane with half-times around 30 s at 32 degrees C, even in the absence of cytosolic K+.

Animals↗
PubMed1990

Membrane channel formation by the lymphocyte pore-forming protein: comparison between susceptible and resistant target cells.

The assembly of pores by the pore-forming protein (perforin) of cytolytic T lymphocytes (CTLs) and natural killer cells on the membranes of different cell lines was studied. Using the patch clamp technique in the whole cell configuration, we measured the conductance increase induced by perforin in susceptible cell lines as well as in resistant CTL lines (CTLLs). The results showed that although the amplitudes of the first observed conductance steps produced in both cell types were comparable, CTLLs required at least 10-fold higher doses of perforin to form membrane pores. Outside-out patches excised from CTLL-R8, on the other hand, appeared to be more susceptible to channel formation by perforin than intact cells, as lower doses were able to induce conductance increases. Once channels were induced in CTL membranes, however, their conductances (greater than 1 nS) were indistinguishable from the ones obtained in susceptible cell lines. Fluorescence measurements with quin-2 showed that perforin induced rapid increases in the intracellular Ca2+ concentration in susceptible EL4 cells. In marked contrast, a perforin dose 60-120-fold higher than the minimal dose required to elicit Ca2+ changes in EL4 cells was not able to induce any measurable Ca2+ increase in CTLL-R8. The data suggest that the resistance of CTLs to lysis mediated by their own mediator perforin is at least in part due to their ability to avoid pore formation by this protein. The mechanism underlying this phenomenon is not yet understood, but the observation that outside-out patches excised from CTLL-R8 are more susceptible to channel formation by perforin than intact cells raises the possibility that an intracellular mechanism may be involved.

Aminoquinolines↗
PubMed1990

Exocytosis.

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Animals↗
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.

Animals↗
PubMed1988

Agonists that suppress M-current elicit phosphoinositide turnover and Ca2+ transients, but these events do not explain M-current suppression.

The hypothesis that acetylcholine, substance P, and LHRH suppress M-current by activating phospholipase C was tested. Each agonist caused turnover of phosphoinositide, as measured by release of inositol phosphates, and a modest transient rise in intracellular free Ca2+ ([ Ca2+]i), as determined with fura-2. Active phorbol esters depressed M-current only 50% and did not prevent further suppression by LHRH. M-current, its control by agonists, and its depression by phorbol esters were not affected by adding inositol trisphosphate or Ca2+ buffers with high or low Ca2+ to the whole-cell, voltage-clamp pipette. We conclude that phospholipase C activation does occur but does not mediate the suppression of M-current by agonists. Caffeine produced large [Ca2+]i transients and acted as an agonist to suppress M-current.

Acetylcholine↗
PubMed1987

Final steps in exocytosis observed in a cell with giant secretory granules.

Secretion by single mast cells was studied in normal and beige mice, a mutant with grossly enlarged secretory vesicles or granules. During degranulation, the membrane capacitance increased in steps, as single secretory vesicles fused with the cell membrane. The average step size was 10 times larger in beige than in normal mice, in agreement with the different granule sizes measured microscopically in the two preparations. Following individual capacitance steps in beige mice, individual granules of the appropriate size were observed to swell rapidly. Capacitance steps are frequently followed by the stepwise loss of a fluorescent dye loaded into the vesicles. Stepwise capacitance increases were occasionally intermittent before they became permanent, indicating the existence of an early, reversible, and incomplete state of vesicle fusion. During such "capacitance flicker," loss of fluorescent dye from vesicles did not occur, suggesting that the earliest aqueous connection between vesicle interior and cell exterior is a narrow channel. Our results support the view that the reversible formation of such a channel, which we term the fusion pore, is an early step in exocytosis.

Animals↗
PubMed1987

Gradual and stepwise changes in the membrane capacitance of rat peritoneal mast cells.

1. The membrane capacitance of mast cells was monitored under voltage clamp, using sinusoidal excitation and a lock-in amplifier. 2. Degranulation was accompanied by stepwise capacitance increases that presumably represent the fusion of single secretory granules with the cell membrane. Besides capacitance steps, we also observed gradual changes in capacitance that occurred even in the absence of degranulation, were independent of the presence of nucleotides in the pipette, and were steeply dependent on cytoplasmic [Ca2+]. 3. Cytoplasmic Ca2+ at concentrations of 0.3-3 microM stimulated a decline in capacitance, with a dose-response curve suggesting control by the binding of Ca2+ to high-affinity intracellular sites. When maximally activated, this mechanism could lead to a loss of about 6% of the cell membrane capacitance, at an average rate of 0.1-0.2% s-1. 4. At even higher cytoplasmic [Ca2+] (greater than 3 microM), the reverse effect was observed. The capacitance increased gradually by up to 40%, at an average rate of 0.4% s-1. Evidently gradual changes in membrane capacitance can occur by two mechanisms, and both are influenced by cytoplasmic [Ca2+]. 5. Ca2+ frequently stimulated an inward current accompanied by an increase in membrane conductance. 6. The effects described above were observed also when only trace amounts of Ca2+ and chelator were added to the cytosol, and when increases in cytosolic [Ca2+] could have occurred only by endogenous mechanisms. It is suggested that these effects occur also in intact cells during the large [Ca2+] increases known to occur before and during degranulation.

Animals↗
PubMed1986

Fast calcium transients in rat peritoneal mast cells are not sufficient to trigger exocytosis.

The calcium concentration, [Ca]i, in single rat peritoneal mast cells was measured by means of the new Ca indicator dye fura-2. Upon stimulation with antigen or compound 48/80, [Ca]i rose for seconds to values greater than 5 microM. These Ca transients did not depend on the presence of extracellular Ca, and they sometimes occurred spontaneously, especially in the presence of exogenous phosphatidylserine. Calcium transients did not necessarily lead to degranulation. Degranulation usually occurred during periods of somewhat elevated [Ca] (0.5-1 microM) following transients but was sometimes observed at [Ca]i less than or equal to 250 nM. We found no evidence that an antigen-induced Ca influx is required for degranulation.

Animals↗