Acetycholine receptor production and incorporation into membranes of developing muscle fibers

Using electrophysiological and quantitative autoradiographic techniques, we studied the kinetics of acetylcholine (ACh) receptor production and incorporation into membranes of muscle fibers developing in culture. These studies were performed by utilizing ¹²⁵I-labeled α-Bungarotoxin (α-BGT) which binds irreversibly to ACh receptors. α-BGT binding to ACh-sensitive muscle cells in culture correlates well with the level of ACh sensitivity. α-BGT binds to myotubes with two different apparent rates. The slow component of binding is due to the incorporation of new receptors into the membrane at a rate of 90 receptors/μm² per hour. However, the ACh receptor density increases at a rate of only 35 receptors/μm² per hour as the result of a concurrent increase in cell surface area. The α-BGT-receptor complexes turn over slowly and the rate of receptor incorporation is not affected by the presence of α-BGT. Inhibition of protein synthesis with cycloheximide depresses receptor incorporation, the percent inhibition increasing with time in cycloheximide. Overnight treatment in actinomycin D has no effect, but inhibition of ATP synthesis with dinitrophenol and iodoacetate or incubation in the cold inhibits the appearance of new ACh receptors.     

Potassium inward rectifier and acetylcholine receptor channels in embryonic Xenopus muscle cells in culture

Embryonic muscle cells of the frog Xenopus laevis were isolated and grown in culture and single-channel recordings of potassium inward rectifier and acetylcholine (ACh) receptor currents were obtained from cell-attached membrane patches. Two classes of inward rectifier channels, which differed in conductance, were apparent. With 140 mM potassium chloride in the electrode, one channel class had a conductance of 28.8 ± 3.4 pS (n = 21), and, much more infrequently, a smaller channel class with a conductance of 8.6 ± 3.6 pS (n = 7) was recorded. Both channel classes had relatively long mean channel open times, which decreased with membrane hyperpolarization. The probability of finding a patch of membrane with an inward rectifier channel was high (66%) and many membrane patches contained more than one inward rectifier channel. The open state probability (with no applied potential) was high for both inward rectifier channel classes so that 70% of the time there was a channel open. Seventy-three percent of the membrane patches with ACh receptor channels (n = 11) also had at least one inward rectifier channel present when the patch electrode contained 0.1 μM ACh. Inward rectifier channels were also found at 71% of the sites of high ACh receptor density (n = 14), which were identified with rhodamine-conjugated α-bungarotoxin. The results indicate that the density of inward rectifier channels in this embryonic skeletal muscle membrane was relatively high and includes sites of membrane that have synaptic specializations. © 1996 John Wiley & Sons, Inc.     

A physiological study on acetylcholine receptor expressed in Xenopus oocytes from cloned cDNAs

Nicotinic ACh receptor was expressed in Xenopus oocytes by injecting mRNAs produced from cloned cDNAs encoding the four subunits of ACh receptor of Torpedo californica. ACh responses recorded from oocytes 3 days after injection of the mRNAs were reversibly blocked by d-tubocurarine (1-2 microM), indicating that the newly synthesized receptor is of nicotinic type. The reversal potential of ACh response was found at around -1 - -5 mV. The reversal potential was not changed by removal of extracellular C1-, suggesting that the ionic channel of the newly expressed ACh receptor is permeable only to cations. Repetitive applications of ACh caused desensitization of the receptor. The rate of the desensitization was greater when the membrane potential was more negative. Subunit deletion studies showed that all four subunits are required for the formation of ACh receptors with normal ACh sensitivity. However, ACh receptors without delta subunit responded to ACh with low sensitivity. Studies on ACh receptor mutants with -subunits altered by site directed mutagenesis of the cDNA suggest that the anphipathic segment is involved in the channel function of the receptor as well as the four hydrophobic segments since partial deletion of amino acids in these segments essentially abolished ACh sensitivity with relatively little change in 125I-alpha-bungarotoxin binding activity.     

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.     

Single acetylcholine-activated channel currents in developing muscle cells

The properties of single acetylcholine-activated ion channels in developing rat myoblasts and myotubes in tissue culture have been investigated using the gigaohm seal patch clamp technique. Two classes of ACh-activated channels were identified. The major class of channels (accounting for >95% of all channel openings) has a conductance of 35 pS and a mean open time of 15 msec (at room temperature and ?80 mV). The minor class of channels has a larger conductance (55 pS) and a briefer mean open time (2–3 msec). Functional ACh-activated channels are present in undifferentiated mononucleated myoblasts 1–2 days in culture, although the channel density on such cells is low. Over the next week in culture, as the myoblasts fuse to form multinucleate myotubes, there is a marked increase in channel density and an increase in the proportion of large conductance channels. No significant change, however, occurs in channel conductance or open time (within a given class of channels) during this period. At high concentrations of ACh, channels desensitize and channel openings occur in groups, similar to what has been previously described in adult muscle. The rate of channel opening within a group of openings increases with increasing agonist concentration while mean open time is independent of agonist concentration, as expected from simple models of drug action. During a group of openings, the channel is open for half the time (i.e., channel opening rate is equal to channel closing rate) at a concentration of approximately 6 μm ACh.     

Channel open time of acetylcholine receptors on Xenopus muscle cells in dissociated cell culture

The kinetics of acetylcholine (ACh) receptor channels on cultured myotomal muscle cells from Xenopus embryos were studied by analyzing focally recorded membrane currents. The mean open time for receptor channels on embryonic muscle cells grown in dissociated cell cultures showed a time-dependent decrease similar to that seen in vivo. The changes in power density spectra are consistent with the hypothesis that the decrease results from the appearance of a class of ACh receptor with a short mean channel open time (0.7 msec) and a decrease in the proportion of receptors with a long mean channel open time (3 msec). The addition of dissociated neural tube cells to muscle cell cultures resulted in an unexpected increase in mean channel open time for ACh receptors in both synaptic and nonsynaptic regions. These studies demonstrate that ACh receptor function may be altered in cultured muscle cells.     

Functional properties of acetylcholine receptors coexpressed with the 43K protein in heterologous cell systems.

The nicotinic acetylcholine (ACh) receptor is an integral membrane protein which mediates synaptic transmission at the skeletal neuromuscular junction. A key event in the development of the neuromuscular junction is the formation of high density aggregates of ACh receptors in the postsynaptic membrane. Receptor clustering has been attributed, in part, to their association with a peripheral membrane protein of Mr 43,000 (43K protein). We have addressed whether the association of the 43K protein can alter the single channel properties of the ACh receptor, and thus influence neuromuscular transmission at developing synapses, by expressing ACh receptors with and without the 43K protein in heterologous expression systems. We found that coexpression of the 43K protein with the receptor did not significantly alter either its single channel conductance or its mean channel open time. This was true in oocytes and also in COS cells where it was possible to localize 43K-induced clusters by fluorescence microscopy and to record from those clustered receptors. These data are in agreement with previous single channel studies which have shown that the properties of diffusely distributed and clustered receptors in native muscle cells from both mice and Xenopus do not differ.     

Functional properties and developmental regulation of nicotinic acetylcholine receptors on embryonic chicken sympathetic neurons

Measurement of acetylcholine (ACh)-induced currents indicates that the sensitivity of embryonic sympathetic neurons increases following innervation in vivo and in vitro. We have used single-channel recording to assess the contribution of changes in ACh receptor properties to this increase. Early in development (before synaptogenesis), we detect three classes of ACh-activated channels that differ in their conductance and kinetics. Molecular studies indicating a variety of neuronal receptor subunit clones suggest a similar diversity. Later in development (after innervation), changes in functional properties include increases in conductance and apparent mean open time, the addition of a new conductance class, as well as apparent clustering and segregation of channel types. These changes in channel function are compatible with the developmental increase in ACh sensitivity.     

Development of acetylcholine sensitivity during myogenesis

The development of acetylcholine (ACh) sensitivity during myogenesis has been studied by iontophoretic application of ACh and intracellular recording from myogenic cells from rat forelimbs cultured in vitro. The fine structure of the cells was then examined by electron microscopy. The development of ACh sensitivity is correlated with the appearance of thick and thin filaments and precedes myofibril formation. All myotubes are sensitive to ACh. Myogenic cells arising by cell division in vitro can become sensitive to ACh and construct myofibrils without cell fusion. When cell fusion is inhibited by calcium ion deficiency or when cell division is blocked by FUdR, many mononucleate, striated, ACh-sensitive cells appear in culture. While ACh sensitivity appears at the onset of muscle differentiation, ACh receptors seem to play no role in the early events of myogenesis, as evidenced by the failure of receptor block or of desensitization to interfere with myogenesis. The concurrent appearance of myofilaments and ACh sensitivity is discussed in relation to the early events and control mechanisms of myogenesis.     

血管紧张素Ⅱ对培养心肌细胞c-fos表达和蛋白质合成的作用

本实验在培养新生大鼠心肌细胞上,探讨血管紧张素Ⅱ对心肌细胞ｃ－ｆｏｓ ｍＲＮＡ表达和蛋白质合成的影响。结果显示,血管紧张素Ⅱ能诱导ｃ－ｆｏｓ ｍＲＮＡ的表达,增加蛋白质含量,并呈量－效关系,还能加速＾３Ｈ－亮氨酸的掺入速度。上述这些作用可血管紧张素Ⅱ受体拮抗ｓａｒａｌａｓｉｎ所阻断,提示这些作用是受体介导的。     

Evidence for active chloride accumulation in normal and denervated rat lumbrical muscle

Intracellular Cl- activity (aiCl) was measured with Cl(-)-sensitive microelectrodes in normal and denervated rat lumbrical muscle. In normal muscle bathed in normal Krebs solution, aiCl lay close to that predicted by the Nernst equation. The addition of 9-anthracene carboxylic acid, which blocks Cl- conductance, caused aiCl to increase far above that predicted by a passive distribution. Furosemide (10 microM) reversibly blocked this accumulation. After muscle denervation, aiCl progressively increased for 1-2 wk. The rise occurred in two stages. The initial stage (1-3 d after denervation) reflected passive Cl- accumulation owing to membrane depolarization. At later times, aiCl continued to increase, with no further change in membrane potential, which suggests an active uptake mechanism. This rise approximately coincided with the natural reduction in membrane conductance to Cl- that occurs several days after denervation. Na+ replacement, K+ replacement, and furosemide each reversibly blocked the active Cl- accumulation in denervated muscle. Quantitative estimates suggested that there was little difference between Cl- flux rates in normal and denervated muscles. The results can be explained by assuming that, in normal muscle, an active accumulation mechanism operates, but that Cl- lies close to equilibrium owing to the high membrane conductance to Cl-. The rise in aiCl after denervation can be accounted for by the membrane depolarization, the reduction in membrane Cl- conductance, and the nearly unaltered action of an inwardly directed Cl- "pump."     

A novel cholinergic receptor mediates inhibition of chick cochlear hair cells.

The central nervous system provides feedback regulation at several points within the peripheral auditory apparatus. One component of that feedback is inhibition of cochlear hair cells by release of acetylcholine (ACh) from efferent brainstem neurons. The mechanism of hair cell inhibition, and the character of the presumed cholinergic receptor, however, have eluded understanding. Both nicotinic and muscarinic, as well as some non-cholinergic ligands can affect the efferent action. We have made whole-cell, tight-seal recordings from short (outer) hair cells isolated from the chick's cochlea. These are the principal targets of cochlear efferents in birds. ACh hyperpolarizes short hair cells by opening a cation channel through which Ca2+ enters the cell and subsequently activates Ca(2+)-dependent K+ current (Fuchs & Murrow 1991, 1992). Both curare and atropine are effective-antagonists of cholinergic inhibition at 3 microM, whereas trimethaphan camsylate and strychnine block at 1 microM. The normally irreversible nicotinic antagonist, alpha-bungarotoxin, reversibly blocked the hair cell response, as did kappa-bungarotoxin. The half-blocking concentration for alpha-bungarotoxin was 26 nM. It is proposed that the hair cell AChR is a ligand-gated cation channel related to the nicotinic receptor of nerve and muscle.     

Voltage dependent calcium channels in cerebellar granule cell primary cultures

Voltage activated calcium channels were studied in rat cerebellar granule cells in primary culture. Macroscopic currents, carried by 20mM Ba2+, were measured in the whole-cell configuration. Slowly inactivating macroscopic currents, with a maximum value at a membrane potential around 5 mV, were recorded between the 1st and the 4th day in culture. These currents were completely blocked by 5mM Co2+, partially blocked by 10 microM nifedipine, and increased by 2 to 5 microM BAY K-8644. Two types of channels, in the presence of 80 mM Ba2+, were identified by single channel recording in cell-attached patches. The first type, which was dihydropyridine agonist sensitive, had a conductance of 18 pS, a half activation potential of more than 10 mV and did not inactivate. This type of channel was the only type found during the first four days in culture, although it was also present up to the 11th day. The second type of channel was dihydropyridine insensitive, had a conductance of 10 pS, a half activation potential less than -15 mV, and displayed voltage dependent inactivation. This second type of channel was found in cells for more than four days in culture.     

The Ach-evoked Ca2+-activated K+ Current in Mouse Mandibular Secretory Cells. Single Channel Studies

Although acetylcholine (ACh) is able to activate voltage- and Ca²⁺-sensitive K⁺ (BK) channels in mouse mandibular secretory cells, our recent whole cell studies have suggested that these channels, like those in sheep parotid secretory cells, do not contribute appreciably to the conductance that carries the ACh-evoked whole cell K⁺ current. In the present study, we have used cell-attached patch clamp methods to identify and characterize the K⁺ channel type responsible for carrying the bulk of this current. When the cells were bathed in a NaCl-rich solution the predominant channel type activated by ACh (1 μmol/l or 50 nmol/l) had a conductance only of 40 pS; it was not blocked by TEA but it was sensitive to quinine and it conducted Rb⁺ to an appreciable extent. BK channels, which could be seen in some but not all patches from resting cells, also showed increased activity when ACh was added to the bath, but they were much less conspicuous during ACh stimulation than the 40-pS channels. When the cells were bathed in a KCl-rich rather than a NaCl-rich solution, a small-conductance K⁺ channel, sensitive to quinine but not to TEA, was still the most conspicuous channel to be activated by ACh although its conductance was reduced to 25 pS. Our studies confirm that the ACh-evoked whole-cell K⁺ current is not carried substantially by BK channels and show that it is carried by a small-conductance K⁺ channel with quite different properties. Received: 28 September 1995/Revised: 26 December 1995