Formation of functional alpha3beta4alpha5 human neuronal nicotinic receptors in Xenopus oocytes: a reporter mutation approach.

1. The alpha5 subunit participates to the formation of native neuronal nicotinic receptors, particularly in autonomic ganglia. Like the related beta3 subunit, alpha5 forms functional recombinant receptors if expressed together with a pair of typical alpha and beta subunits, but its effect on the properties of the resulting alphabetaalpha5 receptor depends on the alpha and beta subunits chosen and on the expression system. We used a reporter mutation approach to test whether alpha5, like beta3, is incorporated as a single copy in human alpha3beta4alpha5 receptors expressed in oocytes. 2. As previously reported, the main indication of the presence of alpha5 in alpha3beta4alpha5(wt) was an increase in apparent receptor desensitization (compared with alpha3beta4 receptors). If the alpha3beta4alpha5 receptor bore a 9'T mutation in the second transmembrane domain of either alpha3 or beta4, alpha5 incorporation produced a decrease in ACh sensitivity (by 4 fold for alpha3(LT)beta4alpha5 vs. alpha3(LT)beta4 and by 40 fold for alpha3beta4(LT)alpha5 vs. alpha3beta4(LT)). The much greater effect observed in alpha3beta4(LT)alpha5 receptors accords with the hypothesis that alpha5 takes the place of a beta subunit in the receptor. 3. Introducing a 9'T mutation in alpha5 had no effect on the agonist sensitivity of alpha3beta4alpha5 receptors, but reduced apparent desensitisation, as judged by the sag in the current response to high agonist concentrations. 4. Introducing the 9'T mutation in alpha3 or beta4 in the triplet receptor reduced the EC(50) for ACh by a similar extent (7 and 9 fold, respectively), suggesting that alpha3beta4alpha5 receptors contain two copies each of alpha and beta and therefore only one copy of alpha5.

Stoichiometry of human recombinant neuronal nicotinic receptors containing the b3 subunit expressed in Xenopus oocytes.

The neuronal nicotinic subunit beta3 forms functional receptors when co-expressed with both an alpha and a beta subunit, such as alpha3 and beta4. We examined the subunit stoichiometry of these 'triplet' alpha3beta4beta3 receptors by expression in Xenopus oocytes of the alpha3, beta4 and beta3 subunits, either in wild-type form or after insertion of a reporter mutation. The mutation chosen was the substitution of a conserved hydrophobic residue in the second transmembrane domain of the subunits (leucine or valine 9THORN ) with a hydrophilic threonine. In other ion channels within the nicotinic superfamily, this mutation type consistently increases the potency of agonists. In muscle-type nicotinic receptors, the magnitude of this effect is approximately constant for each mutant subunit incorporated. In alpha3beta4beta3 receptors, the ACh EC50 was decreased by approximately 17-fold when this mutation was in alpha3 alone and only by fourfold when beta3 alone was mutated. Mutating beta4 was equivalent to mutating alpha3, suggesting that the 'triplet' receptor contains one copy of beta3 and two copies each of alpha3 and beta4. Mutating beta3 and alpha3 or beta3 and beta4 reduced the ACh EC50 further, to values two- to threefold lower than those seen when only alpha3 or beta4 carried the mutation. In 'pair' alpha3beta4 receptors (known to contain two alpha and three beta subunits), mutating beta4 had a greater effect on the ACh EC50 than mutating alpha3, in agreement with an alpha:beta ratio of 2:3 and a constant and independent effect of each copy of the mutation. Our results suggest that alpha3beta4beta3 neuronal nicotinic receptors contain one copy of beta3 and two copies each of alpha3 and beta4 and confirm that in pair alpha3beta4 receptors the alpha/beta subunits are present in a 2:3 ratio.

A reporter mutation approach shows incorporation of the "orphan" subunit beta3 into a functional nicotinic receptor.

We have investigated whether the neuronal nicotinic subunit beta3 can participate in the assembly of functional recombinant receptors. Although beta3 is expressed in several areas of the central nervous system, it does not form functional receptors when expressed heterologously together with an alpha or another beta nicotinic subunit. We inserted into the human beta3 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 beta3(V273T) in Xenopus oocytes together with both the alpha3 and the beta4 subunits resulted in the predicted changes in the properties of the resulting nicotinic receptor when compared with those of alpha3 beta4 receptors. This indicated that some of the receptors incorporated the mutant beta3 subunit, as part of a "triplet" alpha3 beta4 beta3 receptor. The proportion of triplet receptors was dependent on the ratios of the alpha3:beta4:beta3 cRNA injected. We conclude that, like the related alpha5 subunit, the beta3 subunit can form functional receptors only if expressed together with both alpha and beta subunits.

Properties of human glycine receptors containing the hyperekplexia mutation alpha1(K276E), expressed in Xenopus oocytes.

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.

Recombinant nicotinic receptors, expressed in Xenopus oocytes, do not resemble native rat sympathetic ganglion receptors in single-channel behaviour.

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.

The ion channel properties of a rat recombinant neuronal nicotinic receptor are dependent on the host cell type.

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.

Morphine selectively depresses the slowest, NMDA-independent component of C-fibre-evoked synaptic activity in the rat spinal cord in vitro.

The effects of morphine on the depolarizing synaptic responses produced in motoneurons by electrical stimulation of primary sensory neurones have been recorded in hemisected spinal cord preparations (8- to 12-day-old rat pups). Morphine at concentrations of 0.1-20 microM reduced a slow, long-lasting (latency greater than 1 s, duration up to 10 s) component of the ventral root potential (VRP) evoked by C-fibre strength stimulation of the dorsal root. At 2 microM the reduction in area of this slow synaptic potential was 71.7 +/- 0.9% of control values (n = 15). The earliest components of the C-fibre strength VRP (the first 100 ms) and the responses to A beta strength stimuli were unaffected by the opioid even at 10-20 microM. The intermediate, NMDA receptor antagonist (D-AP5, 40 microM)-sensitive component (which lasts 100-1000 ms) was reduced by 34 +/- 2.2% of control (n = 15), which was significantly less than the reduction of the later NMDA-independent component (P < 0.001). Morphine (0.1-20 microM) also depressed the cumulative depolarization generated by the temporal summation of synaptic responses evoked by brief trains of C-fibre strength stimuli (1 or 10 Hz). A significantly greater reduction at the lower frequency of stimulation (56.3 +/- 2.0%) than at the higher (20.3 +/- 1.69%, n = 10, measured at 2 microM morphine) was found (P < 0.005). The effects of morphine were reversible upon wash-out or superfusion with the opioid receptor antagonist naloxone (2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of neuronal nicotinic receptors in rat sympathetic neurones with subunit pairs expressed in Xenopus oocytes.

1. The agonist sensitivity of nicotinic acetylcholine receptors in rat superior cervical ganglion (SCG) neurones was compared with that of cloned receptors expressed in Xenopus oocytes by pairwise injections of alpha 3-beta 2 or alpha 3-beta 4 neuronal nicotinic subunit combinations. 2. Agonist responses in rat SCG neurones indicated that cytisine was the most potent agonist and lobeline the least potent (rank order of potency: cytisine > dimethylphenylpiperazinium iodide (DMPP) > nicotine > ACh > carbachol > lobeline). 3. Receptors expressed in oocytes by injection of alpha 3 and beta 2 subunits had a relatively high sensitivity to DMPP and low sensitivity to cytisine (rank order of potency: DMPP > ACh > lobeline > carbachol > nicotine > cytisine), whereas receptors composed of alpha 3 and beta 4 subunits had a high sensitivity to cytisine and low sensitivity to DMPP (rank-order of potency: cytisine > nicotine approximately ACh > DMPP > carbachol > lobeline). 4. With the exception of responses to DMPP, agonist sensitivity measurements suggest that nicotinic receptors in the rat SCG are composed of alpha 3 and beta 4 subunits. The results are discussed in terms of the receptor subunit mRNAs known to be expressed in the rat SCG and previous evidence of functional heterogeneity of rat SCG nicotinic acetylcholine receptors.

Small-caliber afferent inputs produce a heterosynaptic facilitation of the synaptic responses evoked by primary afferent A-fibers in the neonatal rat spinal cord in vitro.

1. The effect of brief primary afferent inputs on the amplitude and duration of the synaptic potentials evoked in ventral horn (VH) neurons by the activation of other unconditioned primary afferents was studied by current-clamp intracellular recording in the neonatal rat hemisected spinal cord in vitro. Low-frequency (1 Hz) trains of stimulation were applied to a lumbar dorsal root (Conditioning root) for 20-30 s. Test excitatory synaptic potentials (EPSPs) were evoked by single electrical shocks applied to an adjacent Test dorsal root. 2. Test and Conditioning inputs were generated at stimulation strengths sufficient to activate A beta-, A delta- and C-afferent fibers successively. At A delta- and C-fiber strength the EPSPs lasted for 4-6 s, and, during the repetitive Conditioning inputs, these summated to produce a progressively incrementing cumulative depolarization that slowly decayed back to the control Vm over tens of seconds. 3. Dorsal root conditioning produced heterosynaptic facilitation, defined as an enhancement of Test EPSPs above their DC matched controls, in 7 out of 20 neurons. To facilitate the unconditioned afferent input, the intensity of conditioning stimulation had to exceed the threshold for the activation of thin myelinated (A delta) afferents: conditioning at A beta-fiber strength had no effect, whereas A delta- and C-fiber strength conditioning were equally effective. 4. Heterosynaptic facilitation of only A beta- or A delta-fiber-evoked Test EPSPs was observed, no enhancement of C-fiber strength Test EPSPs could be demonstrated. The facilitation manifested as increases in the EPSP peak amplitude, area or the number of action potentials evoked. 5. Conditioning trials that produced heterosynaptic facilitation generated cumulative depolarizations larger than those produced by ineffective conditioning trials (9.1 +/- 3.1 vs. 3.3 +/- 0.5 mV after 20 s conditioning at resting Vm, mean +/- SE, n = 6 and 13, respectively; P < 0.05). The slope of the Vm trajectory during the summation of the conditioning EPSPs was higher in trials resulting in heterosynaptic facilitation, at 0.31 +/- 0.10 mV/s in neurons with heterosynaptic facilitation and 0.06 +/- 0.02 mV/s in cells without heterosynaptic facilitation (P < 0.05). 5. Four of the 20 VH neurons in our sample responded to A delta/C-fiber conditioning with action-potential windup: all 4 also displayed heterosynaptic facilitation. 6. Heterosynaptic facilitation decayed after the completion of the conditioning stimulus with a time course that was parallel to but not superimposable on that of the slow Vm depolarization evoked by the conditioning.(ABSTRACT TRUNCATED AT 400 WORDS)

Rate of rise of the cumulative depolarization evoked by repetitive stimulation of small-caliber afferents is a predictor of action potential windup in rat spinal neurons in vitro.

1. The summation of depolarizing postsynaptic potentials (PSPs) evoked by stimulation of primary afferent fibers in lumbar dorsal roots was studied in dorsal and ventral horn neurons in the rat hemisected spinal cord in vitro with current-clamp intracellular recording techniques. PSPs evoked by activation of A delta and/or C fibers could summate after repetitive stimulation at low frequencies: this resulted in a progressive, long-lasting change in the neuronal membrane potential (Vm) (cumulative depolarization). Cumulative depolarization was not observed after stimulation at A beta-fiber intensity or at frequencies < 0.2 Hz. 2. Effective PSP summation resulting in a significant cumulative depolarization (> 5 mV) was observed only if the PSPs evoked were longer than 4 s. On the other hand, there was no correlation between the amplitude of cumulative depolarization achieved in a neuron and the absolute duration of the PSP or the neuronal resting membrane properties (Vm and input resistance). 3. With relation to the changes in Vm during the stimulation train, three patterns of response to repetitive stimuli could be detected in both dorsal horn (DH) and ventral horn (VH) neurons: 1) little or no depolarization throughout the train; 2) an initial depolarization, which either remained stationary or decayed to the resting Vm after the first 3-4 s of stimulation; or 3) a progressive cumulative depolarization increasing throughout the train. 4. Depolarizing the cell by DC current injection resulted in a steeper rise in Vm in response to repetitive stimuli in one half of VH neurons and in one out of nine DH neurons tested. 5. Action potential windup, an increase in the number of action potentials elicited by each stimulus as the train progresses, was observed both in DH and in VH cells. C-fiber strength stimulation was the most effective in eliciting windup. Windup was associated with Vm responses to repetitive stimulation belonging to the progressive cumulative depolarization pattern. Indeed, cumulative depolarizations that resulted in windup had significantly steeper slopes for the Vm trajectory than those that did not. In those neurons in which DC depolarization by intracellular current injection increased the slope of the Vm trajectory, DC depolarization could also bring about action potential windup. 6. These results indicate that, although the generation of long-lasting "slow" PSPs by high-threshold primary afferents is necessary for the summation of synaptic activity in the spinal cord at low frequencies, the rate and pattern of the summation does not depend on the absolute duration of the PSPs.(ABSTRACT TRUNCATED AT 400 WORDS)

Long duration ventral root potentials in the neonatal rat spinal cord in vitro; the effects of ionotropic and metabotropic excitatory amino acid receptor antagonists.

Long duration, primary afferent evoked ventral root potentials (VRP's) have been recorded in vitro from hemisected spinal cords prepared from 8-12-day-old rat pups. Single shock stimulation of a dorsal root at stimulus strengths sufficient to recruit C/group IV afferent fibres evoked a long duration (11.9 +/- 1.2 s) ipsilateral VRP in all preparations. This long duration VRP consisted of two components, (i) a slow wave, time to peak 137.0 +/- 5.1 ms, the amplitude of which was reduced to 8.7% of mean control value in the presence of the N-methyl-D-aspartate (NMDA) antagonist D-AP5 (40 microM), (ii) a prolonged wave with a time to peak of 2.0 +/- 0.2 s which was partially resistant to D-AP5 (40 microM). Both the slow and the prolonged waves were unaffected following superfusion with the metabotropic excitatory amino acid (EAA) receptor antagonist L-AP3 (100-200 microM). Low frequency (1-10 Hz) repetitive stimulation (20 s duration) of high threshold dorsal root afferents evoked a temporal summation of synaptic activity which generated a progressively depolarizing VRP. This cumulative VRP was graded with frequency of stimulation (0.89 +/- 0.13 to 1.25 +/- 0.19 mV). The cumulative VRP was followed by a post-stimulus depolarization which outlasted the period of repetitive stimulation by tens of seconds (47.6 +/- 8.4 to 91.2 +/- 19.9 s). In the presence of AP5 the amplitude of the cumulative VRP was depressed to 54.5 +/- 11.5% of control values when low frequency (1.0 Hz) stimulation was used. The proportion of the cumulative VRP resistant to D-AP5 increased as the frequency of stimulation was increased to 10 Hz. The decay time of the post-stimulus depolarization was unaffected by AP5. Neither the amplitude nor the post-stimulus depolarization of the cumulative VRP was affected by 200 microM L-AP3. It is suggested that both an AP5 sensitive and AP5 insensitive potential contribute to the long duration VRP evoked in the neonatal rat spinal cord following single shock high threshold afferent stimulation. Moreover, the AP5 insensitive prolonged depolarization is manifest following sustained low frequency stimuli and higher frequency inputs.