Single-channel conductances of NMDA receptors expressed from cloned cDNAs: comparison with native receptors.

To cast light on the subunit composition of native NMDA-type glutamate receptors, four cloned subunits of the NMDA receptor have been expressed, in pairs, in Xenopus oocytes, and their single-channel properties have been measured. The conductances of the channels, and their characteristic patterns of sublevel transitions, turn out to be useful diagnostic criteria for subunit composition. The NR1-NR2A and NR1-NR2B combinations (which have identical TM2 sequences) are very similar to each other. Both have 50 pS openings and brief 40 pS sublevels (in 1 mM external Ca2+), with similar mean lifetimes and frequencies. They also show close quantitative resemblance to the channels of hippocampal CA1 and dentate gyrus cells and of cerebellar granule cells, except that the NR1-NR2A combination has a lower glycine sensitivity than the native channels. In contrast, the NR1-NR2C combination produces a channel with 36 pS and 19 pS conductances of similar (brief) duration; these closely resemble the 38-18 pS channels that have previously been observed in large cerebellar neurons in culture (together with 50 pS channels).     

Molecular regulation of postsynaptic differentiation at the neuromuscular junction

The neuromuscular junction (NMJ) is a synapse that develops between a motor neuron and a muscle fiber. A defining feature of NMJ development in vertebrates is the re-distribution of muscle acetylcholine (ACh) receptors (AChRs) following innervation, which generates high-density AChR clusters at the postsynaptic membrane and disperses aneural AChR clusters formed in muscle before innervation. This process in vivo requires MuSK, a muscle-specific receptor tyrosine kinase that triggers AChR re-distribution when activated; rapsyn, a muscle protein that binds and clusters AChRs; agrin, a nerve-secreted heparan-sulfate proteoglycan that activates MuSK; and ACh, a neurotransmitter that stimulates muscle and also disperses aneural AChR clusters. Moreover, in cultured muscle cells, several additional muscle- and nerve-derived molecules induce, mediate or participate in AChR clustering and dispersal. In this review we discuss how regulation of AChR re-distribution by multiple factors ensures aggregation of AChRs exclusively at NMJs.     

Substance P modulates single-channel properties of neuronal nicotinic acetylcholine receptors.

Substance P (SP) is present in avian sympathetic ganglia and accelerates the decay rate of acetylcholine (ACh)-evoked macroscopic currents in sympathetic neurons. We demonstrate here that SP modulates ACh-elicited single channels in a manner consistent with an enhancement of ACh receptor (AChR) desensitization. Furthermore, since AChR channel function was monitored in cell-attached patches with SP applied to the extra-patch membrane, the peptide must act via a second messenger mechanism. SP specifically decreases the net ACh-activated single-channel current across the patch membrane by decreasing both channel opening frequency and mean open time kinetics. These experiments demonstrate that a peptide can modulate neuronal AChR function by a second messenger mechanism.     

Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating

The nicotinic receptor (AChR) is a pentamer of homologous subunits with an alpha(2)betaepsilondelta composition in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 15' of the M1 domain is phenylalanine in alpha subunits while it is isoleucine in non-alpha subunits. Given this peculiar conservation pattern, we studied its contribution to muscle AChR activation by combining mutagenesis with single-channel kinetic analysis. AChRs containing the mutant alpha subunit (alphaF15'I) as well as those containing the reverse mutations in the non-alpha subunits (betaI15'F, deltaI15'F, and epsilonI15'F) show prolonged lifetimes of the diliganded open channel resulting from a slower closing rate with respect to wild-type AChRs. The kinetic changes are not equivalent among subunits, the beta subunit, being the one that produces the most significant stabilization of the open state. Kinetic analysis of betaI15'F of AChR channels activated by the low-efficacious agonist choline revealed a 10-fold decrease in the closing rate, a 2.5-fold increase in the opening rate, a 28-fold increase in the gating equilibrium constant in the diliganded receptor, and a significant increase opening in the absence of agonist. Mutations at betaI15' showed that the structural bases of its contribution to gating is complex. Rate-equilibrium linear free-energy relationships suggest an approximately 70% closed-state-like environment for the beta15' position at the transition state of gating. The overall results identify position 15' as a subunit-selective determinant of channel gating and add new experimental evidence that gives support to the involvement of the M1 domain in the operation of the channel gating apparatus.     

Cholinergic receptor pathways involved in apoptosis, cell proliferation and neuronal differentiation

Acetylcholine (ACh) has been shown to modulate neuronal differentiation during early development. Both muscarinic and nicotinic acetylcholine receptors (AChRs) regulate a wide variety of physiological responses, including apoptosis, cellular proliferation and neuronal differentiation. However, the intracellular mechanisms underlying these effects of AChR signaling are not fully understood. It is known that activation of AChRs increase cellular proliferation and neurogenesis and that regulation of intracellular calcium through AChRs may underlie the many functions of ACh. Intriguingly, activation of diverse signaling molecules such as Ras-mitogen-activated protein kinase, phosphatidylinositol 3-kinase-Akt, protein kinase C and c-Src is modulated by AChRs. Here we discuss the roles of ACh in neuronal differentiation, cell proliferation and apoptosis. We also discuss the pathways involved in these processes, as well as the effects of novel endogenous AChRs agonists and strategies to enhance neuronal-differentiation of stem and neural progenitor cells. Further understanding of the intracellular mechanisms underlying AChR signaling may provide insights for novel therapeutic strategies, as abnormal AChR activity is present in many diseases.     

Potassium channels in human and avian fibroblasts

The cell-attached and excised inside-out patch-clamp techniques were used to study single-channel characteristics of potassium channels in cultured human and avian fibroblasts. Six different potassium channels were distinguished with conductances of 235 +/- 25, 190 +/- 57, 114 +/- 27, 77 +/- 14, 40 +/- 6 and 21 +/- 4 pS in symmetric 140 mM potassium solutions. The channels were separable by their conductances, ion-selectivities, voltage-sensitivities and kinetic properties. All six channels were found in both fully differentiated human skin fibroblasts and primary cultures of 72 h chick sclerotome. The largest channel (235 pS) had a steep bimodal voltage dependence, being open only around the resting membrane potential. It was imperfectly selective for potassium, having a relative sodium:potassium permeability of 0.3. The 190 pS channel was very potassium-selective, had an S-shaped voltage sensitivity and was calcium-dependent. The two intermediate-size channels (114 and 77 pS) had open probabilities of less than 0.5 under all of the conditions we used. They were not completely selective for potassium and were not voltage-sensitive. The two smallest channels (40 and 21 pS) were not well characterized. They both had open probabilities of less than 0.2 and showed no evidence of voltage-sensitivity. The 40 pS channel seemed highly potassium-selective. A suction stimulus was used to test all observed channels for mechanosensitivity but none of the six potassium channels was mechanosensitive. Another small channel, with very clear mechanical sensitivity, was seen on a few occasions; this channel has not yet been characterized.     

Reconstitution of vacuolar ion channels into planar lipid bilayers.

Vacuolar ion channels were characterized after reconstitution into planar lipid bilayers. (1) Channel activity was observed after incorporation of tonoplast-enriched microsomal membranes, purified tonoplast membranes or of solubilized tonoplast proteins. (2) Channels of varying single-channel conductances were detected after reconstitution. In symmetrical 100 mmol l-1 KCl, conductances between 1 and 110 pS were frequently measured; the largest number of independent reconstitution events was seen for single-channel conductances of 16-25 pS (28 experiments), 30-42 pS (26), 49-56 pS (15) and 64-81 pS (15). Channel current usually increased linearly with voltage. (3) In asymmetrical solutions, cation-, non-selective and, for the first time for the tonoplast, anion-selective channels were detected. Ca(2+)-dependent regulation of channel opening was not observed in our reconstitution system. (4) Permeability was also observed for Cl-, NO3-, SO4(2-) and phosphate. (5) After fractionation of tonoplast proteins by size exclusion chromatography, ion channel activity was recovered in specific fractions. (6) Some of these fractions catalyzed sulfate transport after reconstitution into liposomes. The results suggest that different channels are active at the tonoplast membrane at a larger number than has been concluded from previous work.     

Stretch-activated current through single ion channels in the abdominal stretch receptor organ of the crayfish

Single stretch-activated ion channels were studied on the soma and primary dendrites of stretch receptor neurons of the crayfish Orconectes limosus. When the membrane of the patch was deformed by applying suction to the pipette, a marked nonlinear increase in single-channel activity could be observed in two types of channels. These were indistinguishable on the basis of their single-channel conductances but differed in their voltage range of activation. One type showed strong inward rectification (RSA channel) and the second type was largely voltage independent (SA channel). A linear relationship was found between negative pressure and the natural logarithm of the channels' open probability. For an e-fold change in pressure, the average sensitivity was 8.7 +/- 0.4 (SD, n = 5) mmHg for the RSA channel and 5.6 +/- 2.2 (n = 5) mmHg for the SA channel. Both channels were found to be permeable to mono- and divalent cations. Current-voltage relationships were linear with slope conductances for the SA channel of: 71 +/- 11 (SD, n = 3) pS for K+, 50 +/- 7.4 (n = 5) pS for Na+, and 23 pS for Ca++. Similar values were found for the RSA channel. The data suggest that the SA channel is responsible for the mechanotransduction process in the stretch receptor neuron.     

Functional properties of human nicotinic AChRs expressed by IMR-32 neuroblastoma cells resemble those of alpha3beta4 AChRs expressed in permanently transfected HEK cells.

We characterized the functional and molecular properties of nicotinic acetylcholine receptors (AChRs) expressed by IMR-32, a human neuroblastoma cell line, and compared them to human alpha3 AChRs expressed in stably transfected human embryonic kidney (HEK) cells. IMR-32 cells, like neurons of autonomic ganglia, have been shown to express alpha3, alpha5, alpha7, beta2, and beta4 AChR subunits. From these subunits, several types of alpha3 AChRs as well as homomeric alpha7 AChRs could be formed. However, as we show, the properties of functional AChRs in these cells overwhelmingly reflect alpha3beta4 AChRs. alpha7 AChR function was not detected, yet we estimate that there are 70% as many surface alpha7 AChRs in IMR-32 when compared with alpha3 AChRs. Agonist potencies (EC(50) values) followed the rank order of 1,1-dimethyl-4-phenylpiperazinium (DMPP; 16+/-1 microM) > nicotine (Nic; 48 +/- 7 microM) > or = cytisine (Cyt; 57 +/- 3 microM) = acetylcholine (ACh; 59 +/- 6 microM). All agonists exhibited efficacies of at least 80% relative to ACh. The currents showed strong inward rectification and desensitized at a rate of 3 s(-1) (300 microM ACh; -60 mV). Assays that used mAbs confirmed the predominance of alpha3- and beta4-containing AChRs in IMR-32 cells. Although 18% of total alpha3 AChRs contained beta2 subunits, no beta2 subunit was detected on the cell surface. Chronic Nic incubation increased the amount of total, but not surface alpha3beta2 AChRs in IMR-32 cells. Nic incubation and reduced culture temperature increased total and surface AChRs in alpha3beta2 transfected HEK cells. Characterization of various alpha3 AChRs expressed in HEK cell lines revealed that the functional properties of the alpha3beta4 cell line best matched those found for IMR-32 cells. The rank order of agonist potencies (EC(50) values) for this line was DMPP (14 +/- 1 microM) = Cyt (18 +/- 1 microM) > Nic (56 +/- 15 microM > ACh (79 +/- 8 microM). The efficacies of both Cyt and DMPP were approximately 80% when compared with ACh and the desensitization rate was 2 s(-1). These data show that even with the potential to express several human nicotinic AChR subtypes, the functional properties of AChRs expressed by IMR-32 are completely attributable to alpha3beta4 AChRs.     

Molecular basis of the differential sensitivity of nematode and mammalian muscle to the anthelmintic agent levamisole

Levamisole is an anthelmintic agent that exerts its therapeutic effect by acting as a full agonist of the nicotinic receptor (AChR) of nematode muscle. Its action at the mammalian muscle AChR has not been elucidated to date despite its wide use as an anthelmintic in humans and cattle. By single channel and macroscopic current recordings, we investigated the interaction of levamisole with the mammalian muscle AChR. Levamisole activates mammalian AChRs. However, single channel openings are briefer than those activated by acetylcholine (ACh) and do not appear in clusters at high concentrations. The peak current induced by levamisole is about 3% that activated by ACh. Thus, the anthelmintic acts as a weak agonist of the mammalian AChR. Levamisole also produces open channel blockade of the AChR. The apparent affinity for block (190 microm at -70 mV) is similar to that of the nematode AChR, suggesting that differences in channel activation kinetics govern the different sensitivity of nematode and mammalian muscle to anthelmintics. To identify the structural basis of this different sensitivity, we performed mutagenesis targeting residues in the alpha subunit that differ between vertebrates and nematodes. The replacement of the conserved alphaGly-153 with the homologous glutamic acid of nematode AChR significantly increases the efficacy of levamisole to activate channels. Channel activity takes place in clusters having two different kinetic modes. The kinetics of the high open probability mode are almost identical when the agonist is ACh or levamisole. It is concluded that alphaGly-153 is involved in the low efficacy of levamisole to activate mammalian muscle AChRs.     

Characterization of inositol-1,4,5-trisphosphate-gated channels in the plasma membrane of rat olfactory neurons

It is generally accepted that inositol-1,4,5-trisphosphate (InsP3) plays a role in olfactory transduction. However, the precise mode of action of InsP3 remains controversial. We have characterized the conductances activated by the addition of 10 microM InsP3 to excised patches of soma plasma membrane from rat olfactory neurons. InsP3 induced current fluctuations in 25 of 121 inside-out patches. These conductances could be classified into two groups according to the polarity of the current at a holding potential of +40 to +60 mV (with Ringer's in the pipette and pseudointracellular solution in the bath). Conductances mediating outward currents could be further divided into large- (64 +/- 4 pS, n = 4) and small- (16 +/- 1.7 pS, n = 11) conductance channels. Both small- and large-conductance channels were nonspecific cation channels. The large-conductance channel displayed bursting behavior at +40 mV, with flickering increasing at negative holding potentials to the point where single-channel currents were no longer discernible. The small-conductance channel did not display flickering behavior. The conductance mediating inward currents at +40 to +60 mV reversed at +73 +/- 4 mV (n = 4). The current traces displayed considerable fluctuations, and single-channel currents could not be discerned. The current fluctuations returned to baseline after removal of InsP3. The power density spectrum for the excess noise generated by InsP3 followed a 1/f dependence consistent with conductance fluctuations in the channel mediating this current, although other mechanisms are not excluded. These experiments demonstrate the presence of plasma membrane InsP3-gated channels of different ionic specificity in olfactory receptor cells.     

A carboxy-terminal fragment of colicin Ia forms ion channels

A carboxy-terminal, 18 kD fragment of colicin Ia, a bacterial toxin, forms ion channels in artificial phospholipid bilayers. This fragment, which comprises a quarter of the intact 70 kD molecule, is resistant to extensive protease digestion and probably constitutes a structural domain of the protein. The ion channels formed by the 18 kD fragment are functionally heterogeneous, having conductances that range from 15 to 30 pS at positive voltages and from 70 to 250 pS at negative voltages, and open lifetimes that range from at least 25 msec to 5 sec. In contrast, ion channels formed by whole colicin Ia open only at negative voltages, at which their conductances range from 6 to 30 pS, and their open lifetimes range from 1 sec to 3 min. Additionally, the open state of the 18 kD fragment channel is characterized by noisy fluctuations in current, while the open state of the whole molecule ion channel is often marked by numerous, stable subconductance states. Since the properties of the fragment channel differ substantially from those of the whole molecule channel, we suggest that portions of the molecule outside of the 18 kD fragment are involved in forming the whole molecule ion channel.     

Properties of Acetylcholine Receptor Ion Channels in the Acutely Dissociated Neurons of the Marginal Division in the Rat Striatum

Cell-attached mode of patch clamp technique was employed to investigate the properties of acetylcholine (ACh)-induced ion channels in acutely dissociated neurons from the marginal division (MrD) of rat striatum. Two types of conductance states (25 pS and 60 pS) were recorded. The 25 pS channel (more than 80%) was the main type in the neurons of MrD and was described here. The amplitudes of inward currents increased with hyperpolorization and the reversing potential was about 0 mV. Both single short opening and long burst openings were observed in MrD neurons. Two time constants of these two kinds of ion channels are 0.29 ms, 1.84 ms and 1.96 ms, 18.24 ms, respectively. Average close time can be fitted with two exponential functions, the two time constants are 1.7 ms and 54 ms. Probability of channel opening is about 0.012 and no voltage-dependence was found. The properties of reversing potential, voltage-independence and the form of agonist to the ion channels indicated that the recorded channel currents flow through AChR channels. The mAChR is involved in slow synaptic transmission and Ach can not induce the opening of mAChR ion channel. The binding site of ACh to AChR and the nAChR ion channel are the same protein, ACh can only activate nAChR ion channel directly. Therefore, the recorded ion channels in the present study are nAChR ion channels. The results suggest that nAChR ion channels exist in the neurons of MrD and the MrD probably is involved in learning and memory mechanism of the brain.     

Decremental Response to High-Frequency Trains of Acetylcholine Pulses but Unaltered Fractional Ca2+ Currents in a Panel of “Slow-Channel Syndrome” Nicotinic Receptor Mutants

The slow-channel congenital myasthenic syndrome (SCCMS) is a disorder of the neuromuscular junction caused by gain-of-function mutations to the muscle nicotinic acetylcholine (ACh) receptor (AChR). Although it is clear that the slower deactivation time course of the ACh-elicited currents plays a central role in the etiology of this disease, it has been suggested that other abnormal properties of these mutant receptors may also be critical in this respect. We characterized the kinetics of a panel of five SCCMS AChRs (αS269I, βV266M, εL221F, εT264P, and εL269F) at the ensemble level in rapidly perfused outside-out patches. We found that, for all of these mutants, the peak-current amplitude decreases along trains of nearly saturating ACh pulses delivered at physiologically relevant frequencies in a manner that is consistent with enhanced entry into desensitization during the prolonged deactivation phase. This suggests that the increasingly reduced availability of activatable AChRs upon repetitive stimulation may well contribute to the fatigability and weakness of skeletal muscle that characterize this disease. Also, these results emphasize the importance of explicitly accounting for entry into desensitization as one of the pathways for burst termination, if meaningful mechanistic insight is to be inferred from the study of the effect of these naturally occurring mutations on channel function. Applying a novel single-channel–based approach to estimate the contribution of Ca²⁺ to the total cation currents, we also found that none of these mutants affects the Ca²⁺-conduction properties of the AChR to an extent that seems to be of physiological importance. Our estimate of the Ca²⁺-carried component of the total (inward) conductance of wild-type and SCCMS AChRs in the presence of 150 mM Na⁺, 1.8 mM Ca²⁺, and 1.7 mM Mg²⁺ on the extracellular side of cell-attached patches turned out be in the 5.0–9.4 pS range, representing a fractional Ca²⁺ current of ∼14%, on average. Remarkably, these values are nearly identical to those we estimated for the NR1-NR2A N-methyl-d-aspartate receptor (NMDAR), which has generally been considered to be the main neurotransmitter-gated pathway of Ca²⁺ entry into the cell. Our estimate of the rat NMDAR Ca²⁺ conductance (using the same single-channel approach as for the AChR but in the nominal absence of extracellular Mg²⁺) was 7.9 pS, corresponding to a fractional Ca²⁺ current of 13%.     

Single-channel and whole-cell currents evoked by acetylcholine in dissociated sympathetic neurons of the rat

Single acetylcholine-activated channels have been recorded from neurons dissociated from the sympathetic chain of 17-21 day old rats. The mean single channel conductance is 35 pS in normal medium containing 1 mM calcium, and 51 pS in the absence of calcium. The measured current amplitudes are about five times more variable than at the frog endplate, at least in part because the current, while the channel is open, is much noisier than when it is shut. Single activations of the receptor by acetylcholine (ACh) produce a burst of openings; the distribution of the burst length has two components, the longer of which is of primary importance in synaptic transmission. Whole-cell currents, in response to ACh (up to 30 microM), show strong inward rectification with no outward current being detectable. This phenomenon is similar whether the intracellular ion is sodium or cesium, whether or not divalent cations are present, and whether or not atropine is present. Nevertheless, outward single-channel currents (of normal conductance) are detectable in isolated outside-out patches.     

The alpha 5 gene product assembles with multiple acetylcholine receptor subunits to form distinctive receptor subtypes in brain.

The acetylcholine receptor (AChR) alpha 5 gene has been classified as a member of the AChR gene family based on sequence homology. Expression studies, however, have yet to identify a function for the alpha 5 gene product or even to demonstrate an interaction with known AChR subunits. We report here that the alpha 5 gene product is identical to the 49 kd protein previously found on immunoblots of AChRs purified from brain and ciliary ganglia. In brain the alpha 5 gene product is present both in alpha 3- and in alpha 4-based receptor subtypes, while in the ganglion it is found in an alpha 3-based receptor subtype concentrated in postsynaptic membrane. Immunoprecipitation experiments with subunit-specific monoclonal antibodies indicate that some native AChRs are likely to have at least three kinds of subunits, with two being of the alpha type. These findings support new views about the construction of AChRs in neurons.     

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.     

Single-channel recordings from purified acetylcholine receptors reconstituted in bilayers formed at the tip of patch pipets

The channel of the purified acetylcholine receptor from Torpedo californica electric organ reconstituted in lipid vesicles was assayed by direct electrical recording using patch-clamp pipets. High-resistance seals were obtained by gentle suction of vesicles into the pipet or after the formation of lipid bilayers from monolayers at the tip of the pipet. Single-channel currents were activated by three cholinergic ligands: acetylcholine, carbamylcholine, and suberyldicholine. The single-channel conductance, gamma, was 40 +/- 5 pS in 0.5 M NaCl, irrespective of the agonist used. The distributions of channel open times were fitted by a sum of two exponentials. The lifetimes of the two exponential components were a factor of 2 longer for suberyldicholine than for acetylcholine or carbamylcholine. At desensitizing concentrations of agonists the single events appeared in paroxysms of channel activity followed by quiescent periods. These results suggest that the full cycle of solubilization, purification, and reconstitution of this membrane receptor can be achieved without impairment of channel function.     

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.