Proc Natl Acad Sci U S A 1995 Oct 24;92(22):10327-10329
The phenomenon of desensitization is universal, but its mechanism is still ill-understood and controversial. A recently published study [Lin, F. & Stevens, C. F. (1994) J. Neurosci, 14, 2153-2160] attempted to cast light on the mechanism of desensitization of N-methyl-D-aspartate (NMDA) receptors, in particular the vexed question of whether the channel must open before it can desensitize. During the desensitizing preexposure to agonist in those experiments, more desensitization was produced when channel openings were observed than when no openings were observed. The conclusion that "desensitization occurs more rapidly from the open state" unfortunately was based on a stochastic fallacy, and we present here a theoretical treatment and illustration showing that the observed behavior is predicted by a simple mechanism in which desensitization can occur only from a shut state.
Proc R Soc Lond B Biol Sci 1995 Nov 22;262(1364):205-213
Co-expression of wild-type and mutated NMDA NR1 (N598R) subunits in Xenopus oocytes has been used to determine the stoichiometry of the NMDA receptor-channel. When expressed together, wild-type NR2A and mutant NR1 (N598R) subunits produced channels with a main conductance of 2.6 pS and a sublevel of 1.2 pS. These conductances were clearly different from those obtained from wild-type NR1 and wild-type NR2A channels which gave characteristic 50 pS events with a 40 pS sublevel. When wild-type and mutant NR1 subunits were co-expressed together with NR2A subunits a different channel type with a main conductance of 15.2 pS and a sublevel of 11.4 pS was obtained, as well as the 'all wild-type' and 'all mutant' channels described above. These results indicate that there are likely to be two copies of the NR1 subunit in each NMDA receptor complex.
Nature 1995 Oct 5;377(6548):428-431
ATP is known to depolarize sensory neurons, and may play a role in nociceptor activation when released from damaged tissue. Here we report the molecular cloning and characterization of a new member of the P2X receptor family, P2X3, expressed by these cells. The channel transcript was present in a subset of rat dorsal-root-ganglion sensory neurons, some of which express nociceptor-associated markers; it was absent in other tissues that were tested, including sympathetic, enteric and central nervous system neurons. Moreover, when expressed in Xenopus oocytes, the channel showed an ATP-dependent cation flux. P2X3 is the only ligand-gated channel known to be expressed exclusively by a subset of sensory neurons. The remarkable selectivity of expression of the channel coupled with its sensory neuron-like pharmacology suggests that this channel may transduce ATP-evoked nociceptor activation.
Comment in: Nature 1995 Oct 5;377(6548):385-6
Comment in: Nature 1995 Dec 14;378(6558):658
J Physiol (Lond) 1996 May 15;493( Pt 1):167-173
1. Spontaneous and evoked non-NMDA receptor-mediated EPSCs
were recorded from cerebellar granule cells in slices at
approximately 24 and approximately 34 degrees C. The EPSC decay
was fitted with the sum of two exponential functions. 2. The time
courses of non-NMDA receptor deactivation and desensitization
were determined with fast concentration jumps of glutamate onto
patches from cultured granule cells. Deactivation (decay time
constant tau = 0.6 ms at 24 degrees C) was substantially faster
than desensitization (tau = 4 ms). Both processes were fitted by
single exponential functions. 3. The decay of the fast component
of the spontaneous EPSC (tau EPSCfast = 0.9 ms at 23 degrees C)
was marginally slower than deactivation but too fast to be
determined by desensitization. Our results suggest that the decay
of this component is set by both the rate of decline of
transmitter concentration and channel deactivation. 4. A simple
diffusion model predicts that the time course of transmitter in
the cleft declines slowly during the later stages of its action.
The slow phase of transmitter removal could account for the time
course of the slow component of the spontaneous EPSC (tau
EPSCslow = 8 ms at 23 degrees C).
Published erratum appears in J. Physiol. (Lond) 1996 Nov 1;496(Pt 3):891
Proc R Soc Lond B Biol Sci 1996 Aug 22;263(1373):1079-1086
We have investigated the single-channel and whole-cell behaviour of recombinant N-methyl-D-aspartate (NMDA) receptors formed from NR1a and NR2D receptor subunits expressed in Xenopus oocytes. The EC50 for apparent steady-state activation of NR1a/NR2D receptors by glutamate was 450 nM, while extracellular MG2+ produced a voltage-dependent block of glutamate responses with an IC50 of 440 microM at -70 mV. At negative holding potentials glutamate-activated NR1a/NR2D single-channel currents, in 0.85 mM external Ca2+, had slope conductances of 35 pS for the main level, and 17 pS for the sublevel; direct transitions occurred between these two conductance levels. On average 35 pS events had mean open times of 1.01 +/- 0.04 ms, whereas the mean open times of 17 pS events were consistently longer (1.28 +/- 0.06 ms). In 5 mM external Ca2+ the larger conductance level was reduced to 20 pS whereas in Ca(2+)-free solutions it was increased to 50 pS. The frequency of transitions between the main and subconductance levels showed temporal asymmetry: 35-17 pS transitions were more frequent (61%) than 17-35 pS transitions. This asymmetry was not affected by alterations in the external Ca2+ concentration (up to 5 mM). In conclusion, the NR1a/NR2D channel is, like NR1a/NR2C, a 'low conductance' NMDA channel, but it can be distinguished from NR1a/NR2C channels on the basis of transition asymmetry and differences in the open times of its main and sub-conductance levels.
Nature 1996 Jan 18;379(6562):257-262
Dorsal root ganglion sensory neurons associated with C-fibres, many of which are activated by tissue-damage, express an unusual voltage-gated sodium channel that is resistant to tetrodotoxin. We report here that we have identified a 1,957 amino-acid sodium channel in these cells that shows 65% identity with the rat cardiac tetrodotoxin-insensitive sodium channel, and is not expressed in other peripheral and central neurons, glia or non-neuronal tissues. In situ hybridization shows that the channel is expressed only by small-diameter sensory neurons in neonatal and adult dorsal root and trigeminal ganglia. The channel is resistant to tetrodotoxin when expressed in Xenopus oocytes. The electrophysiological and pharmacological properties of the expressed channel are similar to those described for the small-diameter sensory neuron tetrodotoxin-resistant sodium channels. As some noxious input into the spinal cord is resistant to tetrodotoxin, block of expression or function of such a C-fibre-restricted sodium channel may have a selective analgesic effect.
J Physiol (Lond) 1997 Apr 1;500( Pt 1):123-138
1. In order to establish the subunit composition of neuronal nicotinic receptors in rat superior cervical ganglia (SCG), their single-channel properties were compared with those of recombinant receptors expressed in Xenopus oocytes, using outside-out excised patch recording. 2. The mean main conductance of SCG channels from adult and 1-day-old rats was 34.8 and 36.6 pS, respectively. Less frequent openings to lower conductances occurred both as isolated bursts and as events connected to the main level by direct transitions. There was considerable interpatch variability in the values of the lower conductances. 3. Nicotinic receptors from oocytes expressing alpha3beta4 and alpha4beta4 subunits had chord conductances lower than that of SCG neurones (22 pS for alpha3beta4 and 29 pS for alpha4beta4). 4. Prolonged recording from both native and recombinant channels was precluded by 'run-down', i.e. channel activity could be elicited for only a few minutes after excision. Nevertheless, SCG channel openings were clearly seen to occur as short bursts (slowest component, 38 ms), whereas recombinant channels opened in very prolonged bursts of activity, the major component being the slowest (480 ms). 5. Addition of the alpha5 subunit to the alpha3beta4 pair produced channels with a higher conductance than those observed after injection of the pair alone (24.9 vs. 22 pS), suggesting incorporation of alpha5 into the channel. Addition of the beta2 subunit did not change alpha3beta4 single-channel properties. In one out of fourteen alpha3alpha5beta4 patches, both ganglion-like, high conductance, short burst openings and recombinant-type, low conductance, slow burst openings were observed. 6. Channels produced by expression in Xenopus oocytes of neuronal nicotinic subunits present in rat SCG as a rule differ from native ganglion receptors in single-channel conductance and gross kinetics. While it is possible that an essential nicotinic subunit remains to be cloned, it is perhaps more likely that oocytes either cannot assemble neuronal nicotinic subunits efficiently into channels with the correct composition and stoichiometry, or that they produce post-translational channel modifications which differ from those of mammalian neurones.
FEBS Lett 1997 Jun 2;409(1):49-52
Sensory neurons express a sodium channel (SNS) that is highly resistant to block by tetrodotoxin (IC50 = 60 microM). SNS is 65% homologous to the cardiac sodium channel, in which a single hydrophilic residue in the SS2 segment is critical for tetrodotoxin resistance. By site-directed mutagenesis, we have substituted phenylalanine for serine at the equivalent position in SNS: this mutated (S356F) SNS channel is functionally similar to wild-type SNS when expressed in Xenopus oocytes, but is potently blocked by tetrodotoxin and saxitoxin with IC50s of 2.8 nM and 8.2 nM, respectively. These data provide clues to the rational design of selective blockers of SNS with potential as analgesic drugs.
J Physiol (Lond) 1997 Dec 1;505( Pt 2):299-306
Wellcome Laboratory for Molecular Pharmacology, Department of Pharmacology, University College London, UK.
1. A stable mammalian cell line (L-alpha 3 beta 4) has been established which expresses the cloned rat neuronal nicotinic acetylcholine receptor (nAChR) subunits alpha 3 and beta 4, which are the most abundant in autonomic ganglia. Ion channel properties of nAChRs expressed in L-alpha 3 beta 4 cells were investigated by single-channel and whole-cell recording techniques, and compared with both rat alpha 3 beta 4 nAChRs expressed in Xenopus oocytes, and endogenous nicotinic receptors in rat superior cervical ganglion (SCG) neurones, using identical solutions for all cell types. 2. Acetylcholine (ACh) caused activation of single ion channel currents with a range of amplitudes. Some channels had high conductances (30-40 pS), and relatively brief lifetimes; these resembled the predominant native channel from SCG. Other channels had low conductances (20-26 pS) and long bursts of openings which were quite unlike native channels, but which were similar to channels formed by alpha 3 beta 4 in oocytes. Both types often occurred in the same patch. 3. Cytisine was about 3 times more potent than ACh (low-concentration potency ratio) in L-alpha 3 beta 4 cells, which is not dissimilar to the 5-fold potency ratio found in both SCG and oocytes, whereas 1,1-dimethyl-4-phenylpiperazinium (DMPP) was less potent than ACh in some cells (as in the oocyte), but more potent in others (as in SCG). 4. While the channels expressed in L-alpha 3 beta 4 cells do not mimic exactly those expressed in rat SCG, they differ considerably from the same subunit combination expressed in oocytes. Larger conductance, SCG-like channels were detected frequently in L-alpha 3 beta 4, but were rarely, if ever, seen in oocytes injected with alpha 3 and beta 4 mRNA. Our results indicate that ion channel properties such as single-channel conductance can be influenced by the choice of heterologous expression system.
J Physiol (Lond) 1998 Feb 15;507( Pt 1):25-40
1. Inherited defects in human glycine receptors give rise to hyperekplexia (startle disease). We expressed human glycine receptors in Xenopus oocytes, in order to examine the pharmacological and single-channel properties of receptors that contain a mutation, alpha1(K276E), associated with an atypical form of hyperekplexia. 2. Equilibrium concentration-response curves showed that recombinant human alpha1(K276E)beta receptors had a 29-fold lower glycine sensitivity than wild-type alpha1beta receptors, and a greatly reduced Hill coefficient. The maximum response to glycine also appeared much reduced, whereas the equilibrium constant for the glycine receptor antagonist strychnine was unchanged. 3. Both wild-type and mutant channels opened to multiple conductance levels with similar main conductance levels (33 pS) and weighted mean conductances (41.5 versus 49.8 pS, respectively). 4. Channel openings were shorter for the alpha1(K276E)beta mutant than for the wild-type alpha1beta, with mean overall apparent open times of 0.82 and 6.85 ms, respectively. 5. The main effect of the alpha1(K276E) mutation is to impair the opening of the channel rather than the binding of glycine. This is shown by the results of fitting glycine dose-response curves with particular postulated mechanisms, the shorter open times of mutant channels, the properties of single-channel bursts, and the lack of an effect of the mutation on the strychnine-binding site.
J. Neurosci. 1998 ;18, 581-589
The NMDA type of ligand-gated glutamate receptor requires the presence of both glutamate and glycine for gating. These receptors are hetero-oligomers of NR1 and NR2 subunits. Previously it was thought that the binding sites for glycine and glutamate were formed by residues on the NR1 subunit. Indeed, it has been shown that the effects of glycine are controlled by residues on the NR1 subunit, and a "Venus flytrap" model for the glycine binding site has been suggested by analogy with bacterial periplasmic amino acid binding proteins. By analysis of 10 mutant NMDA receptors, we now show that residues on the NR2A subunit control glutamate potency in recombinant NR1/NR2A receptors, without affecting glycine potency. Furthermore, we provide evidence that, at least for some mutated residues, the reduced potency of glutamate cannot be explained by alteration of gating but has to be caused primarily by impairing the binding of the agonist to the resting state of the receptor. One NR2A mutant, NR2A(T671A), had an EC50 for glutamate 1000-fold greater than wild type and a 255-fold reduced affinity for APV, yet it had single-channel openings very similar to those of wild type. Therefore we propose that the glutamate binding site is located on NR2 subunits and (taking our data together with previous work) is not on the NR1 subunit. Our data further imply that each NMDA receptor subunit possesses a binding site for an agonist (glutamate or glycine).
Journal of Biological Chemistry, 1998, 273 , 15317-15320.
We have investigated whether the neuronal nicotinic subunit b 3 can participate in the assembly of functional recombinant receptors. While b 3 is expressed in several areas of the central nervous system, it does not form functional receptors when heterologously expressed. together with another a or another b nicotinic subunit. We inserted into the human b 3 subunit a reporter mutation (V273T) which, if incorporated into a functional receptor, would be expected to increase its agonist sensitivity and maximum response to partial agonists. Expressing the mutant b 3V273T in Xenopus oocytes together with both the a 3 and the b 4 subunit resulted in the predicted changes in the properties of the resulting nicotinic receptor, when compared with those of a 3b 4 receptors. This indicated that some of the receptors incorporated the mutant b 3 subunit, as part of a triplet a 3b 4 b 3 receptor. The proportion of triplet receptors was dependent on the ratios of the a 3:b 4:b 3 cRNA injected. We conclude that, like the related a 5 subunit, the b 3 subunit can form functional receptors only if expressed together with both a and b subunits.
Journal of Physiology (London). 1998, 510, 1-18.Erratum (sorry!) Journal of Physiology (London). 1998, 512, 955.
1. We have expressed recombinant NR1a/NR2A and NR1a/NR2D N-methyl-d-aspartate (NMDA) receptor-channels in Xenopus oocytes and made recordings of single-channel and macroscopic currents in outside-out membrane patches. For each receptor type we measured (a) the individual single-channel activations evoked by low glutamate concentrations in steady-state recordings, and (b) the macroscopic responses elicited by brief concentration jumps with high agonist concentrations, and we explore the relationship between these two sorts of observation.
2. Low concentration (5 100 nM) steady-state recordings of NR1a/NR2A and NR1a/NR2D single-channel activity generated shut-time distributions that were best fitted with a mixture of five and six exponential components respectively. Individual activations of either receptor type were resolved as bursts of openings, which we refer to as super-clusters.
3. During a single activation, NR1a/NR2A receptors were open for 36% of the time but NR1a/NR2D receptors were open for only 4% of the time. For both, distributions of super-cluster durations were best fitted with a mixture of six exponential components. Their overall mean durations were 35.8 ms and 1602 ms respectively.
4. Steady-state super-clusters were aligned on their first openings and averaged. The average was well fitted by a sum of exponentials with time-constants taken from fits to super-cluster length distributions. It is shown that this is what would be expected for a channel that shows simple Markovian behaviour.
5. The current through NR1a/NR2A channels following a concentration jump from zero to 1 mM glutamate for 1 ms was well fitted by three exponential components with time constants of 13 ms (rising phase), 70 ms and 350 ms (decaying phase). Similar concentration jumps on NR1a/NR2D channels were well fitted by two exponentials with means of 45 ms (rising phase) and 4408 ms (decaying phase) components. During prolonged exposure to glutamate, NR1a/NR2A channels desensitised with a time-constant of 649 ms while NR1a/NR2D channels exhibited no apparent desensitisation.
6. We show that under certain conditions, the time constants for the macroscopic jump response should be the same as those for the distribution of super-cluster lengths, though the resolution of the latter is so much greater that it cannot be expected that all the components will be resolvable in a macroscopic current. Good agreement was found for jumps on NR1a/NR2D receptors, and for some jump experiments on NR1a/NR2A. However the latter were rather variable and some were slower than predicted. Slow decays were associated with patches that had large currents.