Biomedical subjects

P Brehm

Publications and source records attributed to P Brehm.

At least 19 recordsLinked to original sources

PubMed1995

A single site on the epsilon subunit is responsible for the change in ACh receptor channel conductance during skeletal muscle development.

Four critically positioned amino acids on each of the alpha, beta, delta, and gamma subunits of the Torpedo nicotinic acetylcholine receptor are determinants of channel conductance. Our results show that the gamma and epsilon subunits of Xenopus muscle receptors are identical at all four positions, despite the fact that alpha 2 beta delta epsilon receptors have a 50% greater conductance than alpha 2 beta delta gamma receptors. Instead, the functional difference is conferred by a single charged residue that lies extracellular to all four positions, corresponding to a location in the Torpedo receptor previously shown to have no influence on conductance. Substitution of a positively charged lysine residue in gamma by the neutral methionine in epsilon at this extra-cellular position is responsible for the increased conductance during maturation of the amphibian neuromuscular junction.

Amino Acid Sequence↗

PubMed1995

A single pulse of nerve growth factor triggers long-term neuronal excitability through sodium channel gene induction.

The continuous presence of nerve growth factor (NGF) is thought to be required for the elaboration of neuronal-like traits in PC12 cells. Surprisingly, we find that a 1 min exposure to NGF is sufficient to engage a longer-term genetic program leading to the acquisition of membrane excitability. Whereas continuous exposure to NGF causes the induction of a family of sodium channels, the effect of a brief exposure is to induce selectively expression of the peripheral nerve-type sodium channel gene PN1, through a distinct signaling pathway requiring immediate-early genes. A 1 min exposure of PC12 cells to interferon-gamma also causes PN1 gene induction, suggesting that the "triggered" NGF and interferon-gamma signaling pathways share common molecular intermediates.

Animals↗

PubMed1993

Expression of subunit-omitted mouse nicotinic acetylcholine receptors in Xenopus laevis oocytes.

1. Nicotinic acetylcholine (ACh) receptors in developing vertebrate skeletal muscle exhibit functional heterogeneity in both conductance and kinetics. To assess the contributions of receptors differing in subunit composition to the heterogeneity, various combinations of mouse alpha beta delta gamma epsilon subunit RNAs were tested for the ability to express functional receptors in Xenopus oocytes. 2. Two combinations of dual-subunit RNAs (alpha delta and alpha gamma) resulted in detectable ACh-activated currents and six different combinations of three or more subunit RNAs produced significant numbers of functional channels. The order of combinations yielding the greatest amount of current was alpha beta gamma > alpha beta delta = alpha delta epsilon > alpha delta gamma > alpha delta > alpha gamma. 3. The extent to which a channel type with three different subunits was expressed was highly dependent upon the ratios of RNAs coding for the different subunits. For alpha beta delta receptors the efficiency of expression was alpha: beta: delta (1/5:1/5:1) >> (1:1:1) >> (1/5:1:1/5) > (1:1/5:1/5). 4. The level of expression of three-subunit combinations was also critically dependent upon the order of RNAs injected. When alpha delta or alpha gamma RNA combinations were co-injected 2 days prior to the injection of beta RNA, the expression was 2-5 times greater than when alpha beta injection was followed by injection of delta or gamma RNA. 5. Single-channel measurements revealed that alpha beta delta channels were not expressed in the presence of alpha beta delta epsilon RNAs, even under conditions when the amount of delta RNA injected was 5-fold higher than the amount of epsilon RNA. 6. These data indicate that the functional expression of subunit-omitted receptors depends critically upon the relative amounts of the five different subunit RNAs. Receptors composed of three different subunits express in the presence of the subunit RNAs characteristic of embryonic muscle (alpha beta delta gamma), but are not observed with the combination of RNAs characteristic of adult muscle (alpha beta delta epsilon).

Animals↗

PubMed1993

The epsilon subunit confers fast channel gating on multiple classes of acetylcholine receptors.

During vertebrate skeletal muscle development, multiple forms of long-open-time (slow-type) ACh receptor channels are replaced by at least two different types of short-open-time (fast-type) ACh receptors. Expression of ACh receptors in Xenopus oocytes indicates that the substitution of an epsilon subunit for a gamma subunit may account for both types of fast-gated channel types in adult muscle. Unlike the various forms of the embryonic receptor, in which functional diversity is achieved through alterations in subunit composition, the two major fast-gated forms expressed in oocytes have identical subunit composition. These findings provide a structural basis for both types of short-open-time ACh receptor types found in adult muscle.

Animals↗

PubMed1990

Multiple conductance classes of mouse nicotinic acetylcholine receptors expressed in Xenopus oocytes.

Acetylcholine receptor (AcChoR) subunit mRNAs transcribed from mouse BC3H-1 cDNAs were injected into Xenopus oocytes and the expressed AcChoR channels were examined by single channel recording. Injection of alpha-, beta-, gamma-, and delta-subunit mRNAs produced two predominant channel classes with conductances of approximately 50 and approximately 12 pS, while infrequent openings of approximately 25-pS channels were also observed. Injection of alpha-, beta-, and gamma-subunit mRNAs produced a single class of approximately 12-pS AcChoR channels, which resembled the smallest conductance channels present in alpha beta gamma omega-injected oocytes. Assembly of delta-less channels may thus explain the lowest conductance AcChoR channels in alpha beta gamma delta-injected oocytes and might also account for similar channels that have been observed in vertebrate skeletal muscle.

Amino Acid Sequence↗

PubMed1990

Cloning and functional characterization of a complementary DNA encoding the murine fibroblast bombesin/gastrin-releasing peptide receptor.

The amphibian tetradecapeptide bombesin and its mammalian homolog gastrin-releasing peptide are neurotransmitters and paracrine hormones, and are mitogenic for fibroblast and small cell lung carcinoma cell lines. cDNAs encoding the bombesin/gastrin-releasing peptide receptor (BR) expressed by murine Swiss 3T3 fibroblasts were isolated using electrophysiological and luminometric Xenopus oocyte expression assays. Oocytes microinjected with BR transcripts responded to concentrations of bombesin from 1 x 10(-10) to 1 x 10(-6) M. These responses showed homologous desensitization and could be specifically blocked by bombesin antagonists. Sequence analysis showed that the BR has seven membrane-spanning domains and five potential N-linked glycosylation sites. Data base analysis showed that the BR is most homologous to the tachykinin receptors. Although tyrosine kinase activity has been associated with BR function, no tyrosine kinase homologies occur within the BR sequence.

Amino Acid Sequence↗

PubMed1989

Resolving the structural basis for developmental changes in muscle ACh receptor function: it takes nerve.

The nicotinic acetylcholine (ACh) receptor undergoes extensive alterations in functional properties during muscle development. One such alteration, the developmental acquisition of the 'junctional' form of the channel, has been attributed to post-translational modification of pre-existing 'non-junctional' receptor channels. However, the discovery that a switch between the epsilon- and gamma-subunits of the muscle ACh receptor results in the 'junctional' form of the channel suggests a transcriptional mechanism of control. Although this issue is by no means settled, recent molecular biological and electrophysiological studies offer new ideas as to how innervation regulates the expression of functionally distinct forms of this receptor/channel.

Animals↗

PubMed1989

Intercellular signaling as visualized by endogenous calcium-dependent bioluminescence.

Bioluminescence in the hydrozoan coelenterate Obelia results from calcium activation of a photoprotein contained in light-emitting cells (photocytes) scattered in the animal's endoderm. The influx of calcium into nonluminescent endodermal cells through conventional voltage-dependent calcium channels is required for the excitation-luminescence coupling. Our results suggest that the subsequent diffusion of this calcium, via gap junctions, into the neighboring photocytes triggers a localized luminescence response. Following intense stimulation, the local rise in calcium elicits a secondary wave of luminescence that is supported by a voltage-independent calcium permeability mechanism in the photocyte plasma membrane. These two mechanisms for elevating internal calcium in light-emitting cells can account for the spatial and temporal features of intracellular luminescence in Obelia.

Animals↗

PubMed1989

The single-channel basis for the slow kinetics of synaptic currents in vertebrate slow muscle fibers.

The time course of synaptic currents is significantly longer in slow than in fast twitch muscle fibers. To examine the underlying basis for these slow synaptic currents, single-channel recordings were made from the synapses of slow muscle fibers. Our analysis indicates that low conductance acetylcholine receptor (AChR) channels predominate in innervated slow fibers. The high level of expression of low conductance channels is in contrast to fast twitch fibers, in which these channels are expressed in significant numbers only in embryonic or denervated muscle. Analysis of the distribution of open durations for the low conductance channel class suggests that the open time of this AChR class is the major determinant in shaping the slow time course of synaptic current decay. The predominant contribution of low conductance channel openings to synaptic currents of slow muscle fibers indicates a well-defined physiological role for this class of AChRs.

Action Potentials↗

PubMed1988

Regulation of acetylcholine receptor channel function during development of skeletal muscle.

The nicotinic acetylcholine (ACh) receptor channel mediates synaptic transmission at the neuromuscular junction. During the development of skeletal muscle, ACh receptors undergo changes in distribution, antigenic determinants, degradation rate, and function. Now that these developmental hallmarks have been identified, attention has turned toward understanding both the structural bases for such changes and the role of nerve in triggering these changes. Recently, a much clearer understanding of one of these developmental processes, namely, the alterations in channel function, has emerged through both sensitive patch-clamp measurements and the application of recombinant DNA technology. In light of these new advances, we now reevaluate the processes governing the developmental changes in the functional properties of the ACh receptor.

Electric Conductivity↗

PubMed1988

Vasoactive intestinal peptide activates Ca2(+)-dependent K+ channels through a cAMP pathway in mouse lacrimal cells.

The action of vasoactive intestinal peptide (VIP) on Ca2(+)-dependent K+ currents, in dissociated mouse lacrimal cells, was investigated using patch clamp techniques. In whole cell recordings, VIP (10-100 pM) increased the magnitude of the Ca2(+)-dependent K+ current. In single channel recordings, VIP increased the fraction of time the large charybdotoxin-sensitive Ca2(+)-activated K+ channel spent in the open state. The activity of this channel was also increased by adding forskolin or 8-bromo cAMP to the bath. Additionally, application of either cAMP or catalytic subunit of cAMP-dependent protein kinase directly to the cytoplasmic surface of excised inside out patches reversibly lengthened the time Ca2(+)-activated K+ channels spent in the open state. These data suggest that VIP stimulates Ca2(+)-activated K+ channels by a cAMP-dependent pathway in mouse lacrimal acinar cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1988

Selective induction of brain type II Na+ channels by nerve growth factor.

Cells derived from a rat pheochromocytoma (PC12 cells) can generate an action potential only upon treatment with nerve growth factor. Using electrophysiological methods, we found that the appearance of action potentials in nerve growth factor-treated PC12 cells can be explained by an increase in the density of Na+ channels. The functional properties of Na+ channels in PC12 cells are similar to those described for peripheral nerves but appear to be different from Na+ channels synthesized in Xenopus oocytes injected with brain type II Na+ -channel mRNA. To determine if PC12 cells express the brain type II Na+ -channel gene, we performed RNase-protection analyses using probes that can distinguish between the brain type I and type II Na+ -channel mRNAs. The results from these studies indicate that undifferentiated PC12 cells express the type II but not the type I Na+ -channel gene. Treatment with nerve growth factor increases expression of the type II Na+ -channel gene but has no effect on type I gene expression. Our findings suggest that Na+ -channel excitability in PC12 cells is due to the specific induction of the brain type II gene by nerve growth factor.

Action Potentials↗

PubMed1987

Transcriptional and translational requirements for developmental alterations in acetylcholine receptor channel function in Xenopus myotomal muscle.

Two functionally distinct types of acetylcholine (ACh) receptor channels are present on embryonic Xenopus myotomal muscle. During differentiation of this muscle, both in vivo and in dissociated cell culture, the occurrence of the high conductance "fast" channel type increases relative to the low conductance "slow" channel type. In order to ascertain whether new receptor synthesis is required for this rapid switch in channel types we examined the effects of inhibitors of transcription (alpha-amanitin and actinomycin D) and translation (cycloheximide) on developing muscle in culture. Inhibition of protein synthesis resulted in greater than 95% reduction in ACh receptor incorporation and also reversibly blocked the developmental appearance of the high conductance channel type. Inhibition of mRNA synthesis only slightly reduced the rate of receptor incorporation into muscle membrane over a 24-hr period but reversibly blocked appearance of the high conductance channel. These findings suggest that the high conductance ACh receptor channel type does not result from post-translational modifications of the low conductance type, but rather from transcription of a different mRNA encoding one or more of the ACh receptor subunits.

Amanitins↗

PubMed1987

Acetylcholine receptor channels on adult mouse skeletal muscle are functionally identical in synaptic and nonsynaptic membrane.

It has been proposed that acetylcholine receptor channels exhibit a functionally distinct "junctional" form at the region of synaptic contact between nerve and muscle. As a direct test of this idea, we compared acetylcholine-activated single-channel currents from the synaptic membrane to those obtained from nonsynaptic sites on freshly dissociated adult mouse toe muscle. We observed, at locations along the entire length of the cell, openings by a channel with a high conductance (70 pS) and brief open time (approximately 2 msec), characteristic of the classical "junctional type" of acetylcholine receptor. In 8 out of 10 synaptic and in 9 out of 19 nonsynaptic recordings, we also observed infrequent openings by a low-conductance (45-pS) channel traditionally associated only with nonsynaptic regions. In these recordings the low-conductance acetylcholine receptor channel averaged only 3% of the total channel openings. Comparisons of synaptic and nonsynaptic patches indicated no trend toward an increased proportion of low-conductance channel openings with increased distance from the synapse. These findings support the view that the functional properties of the acetylcholine receptor channel do not depend on proximity to the synapse in innervated mouse skeletal muscle.

Acetylcholine↗

PubMed1987

Single channel properties of newly synthesized acetylcholine receptors following denervation of mammalian skeletal muscle.

We have examined the single channel properties of newly synthesized acetylcholine (ACh) receptors in denervated adult mouse muscle. Patch-clamp recordings were made on freshly isolated fibers from flexor digitorum brevis (fdb) muscles that had been denervated in vivo for periods up to 3 wk. Muscles were treated with alpha-bungarotoxin (alpha-BTX), immediately before denervation, in order to block pre-existing receptors. Denervated fibers exhibited two types of ACh receptor channels, which differed in terms of single channel conductance (45 and 70 pS) and mean channel open time (approximately 7 and 2.5 ms, respectively). In contrast to innervated muscle, where only 3% of the total openings were contributed by the low-conductance channel type, greater than 80% of the openings in the nonsynaptic membrane of denervated muscle were of this type. Importantly, a similar increase in the proportion of low-conductance channels was observed for recordings from synaptic membrane after denervation. These data argue against the proposal that, in denervated muscle, the low-conductance channels undergo continued conversion to the high-conductance type focally at the site of former synaptic contact. Rather, our findings provide additional support for the idea that the functional properties of ACh receptors are governed uniformly by the state of innervation of the fiber and not by proximity to the site of synaptic contact.

Animals↗

PubMed1987

Acetylcholine reduces inward rectification on thymus-derived macrophage cells in culture.

Cultures prepared from dissociated rat thymus were examined 1-2 weeks after plating. Macrophage cells were identified by their adherence, morphological appearance, and ability to phagocytize carbon particles or heat-inactivated Staphylococcus aureus. Whole cell current recordings from macrophage cells revealed an inward current at potentials more negative than the equilibrium potential for potassium and an outward current at potentials more positive than -40 mV in normal recording solution. Acetylcholine or muscarine caused a reduction in inward current but did not alter the outward current. The inward current and acetylcholine effect were seen at less negative potentials by decreasing the potassium equilibrium potential and both were blocked by the addition of cesium to the external recording solution. These results indicated that the inward current was mediated by potassium through the inward or anomalous rectifier. Physiologically, the action of acetylcholine on the inward rectifier of these macrophage cells may be mediated by cholinergic innervation of the thymus.

Acetylcholine↗

PubMed1984

Properties of non-junctional acetylcholine receptor channels on innervated muscle of Xenopus laevis.

Patch-clamp recordings of current through acetylcholine-activated channels were made from non-junctional membrane of innervated myotomal muscle from Xenopus laevis. Two classes of acetylcholine (ACh) receptor channels were identified on the basis of current amplitudes. Both amplitude classes exhibited current-voltage relations which deviated from linearity as the extrapolated reversal potential was approached (-5 to -12 mV). Over the range of greatest linearity the conductances of the two classes were 64 and 44 pS. Both event classes were blocked by alpha-bungarotoxin. At the normal resting membrane potential (approximately -95 mV) the larger conductance channel (gamma) exhibited an apparent mean channel open time of less than 1 ms, compared to approximately 2 ms for the smaller gamma class. The apparent open time was voltage-dependent, changing e-fold with a 63 mV hyperpolarization for the high gamma channel and 93 mV hyperpolarization for the low gamma channel. At low ACh concentrations (0.1-0.3 microM) both amplitude classes exhibited bursts of successive openings separated by brief closures of less than 0.5 ms. Bursts were separated by longer closed intervals of 1 to greater than 100 ms. Closed interval histograms revealed corresponding populations of brief and long closures, indicating that at least two kinetic processes are required to describe the distribution of closed intervals. In the absence of exogenous ACh, channels were observed in an occasional patch which showed a conductance and extrapolated reversal potential similar to ACh-activated channels. In such patches the event frequency could occasionally be altered by adjusting the negative pressure applied to the patch. The two main conductance classes of ACh activated channels were observed to coexist in most patches. However, the most frequent event observed in non-junctional membrane of innervated muscle corresponded to the high gamma class. In this respect, the non-junctional ACh receptors bore a greater similarity to junctional ACh receptors than to non-junctional receptors reported for denervated muscle.

Acetylcholine↗

PubMed1984

Acetylcholine receptor channel properties during development of Xenopus muscle cells in culture.

Developmental changes in acetylcholine (ACh) receptor channel function on aneural cultures of embryonic myotomal muscle cells were examined using the patch-clamp technique. At all stages of differentiation two different unitary-event amplitudes were observed, corresponding to high-gamma (single-channel conductance) (64 pS) and low-gamma (46 pS) channel types. No change in conductance occurred for either channel type during the 6-day in vitro period examined. At resting membrane potential (-85 mV) the low-gamma channel exhibited a mean open time of approximately 2 ms which, on the average, was 2-3-fold longer than that measured for the high-gamma channel. Neither the estimated mean channel open time nor the voltage dependence of the open state measured for either channel type changed during development. In recordings with low ACh concentration (0.1-0.25 microM) both high-gamma and low-gamma channel types exhibited non-stationary opening probabilities over the recording period. Usually the opening rate of both channel types decreased with time following seal formation, however, the 'drop-out' rate was faster for the low-gamma channel. A developmental increase in the proportion of high-gamma events occurred between day 1 (16%) and day 5 (56%) in culture, paralleling the time-dependent changes in the channel kinetics based on ACh-activated membrane noise. We conclude that the development of non-junctional muscle membrane is associated with increased expression of high-gamma channels and that this process is primarily responsible for the previously reported developmental alterations in macroscopic ACh receptor channel currents.

Animals↗