Molecular and antigenic structure of nicotinic acetylcholine receptors.
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Animals↗
Biomedical subjects
V Gerzanich
Publications and source records attributed to V Gerzanich.
alpha 5 Subunit alters desensitization, pharmacology, Ca++ permeability and Ca++ modulation of human neuronal alpha 3 nicotinic receptors.
Functional effects of human alpha 5 nicotinic ACh receptor (AChR) subunits coassembled with alpha 3 and beta 2 or with alpha 3 and beta 4 subunits, were investigated in Xenopus oocytes. The presence of alpha 5 subunits altered some properties of both alpha 3 AChRs and differentially altered other properties of alpha 3 beta 2 AChRs vs. alpha 3 beta 4 AChRs. alpha 5 subunits increased desensitization and Ca++ permeability of all alpha 3 AChRs. The Ca++ permeabilities of both alpha 3 beta 2 alpha 5 and alpha 3 beta 4 alpha 5 AChRs were comparable to that of alpha 7 AChRs. As we have shown previously, alpha 5 subunits increased the ACh sensitivity of alpha 3 beta 2 AChRs 50-fold but had little effect on alpha 3 beta 4 AChRs. alpha 5 caused only subtle changes in the activation potencies of alpha 3 AChRs for nicotine, cytisine and 1,1-dimethyl-4-plenylpiperazinium (DMPP). However, alpha 5 increased the efficacies of nicotine and DMPP on alpha 3 beta 2 AChRs but decreased them on alpha 3 beta 4 AChRs. Immunoisolation of cloned human AChRs expressed in oocytes showed that alpha 5 efficiently coassembled with alpha 3 plus beta 2 and/or beta 4 subunits. As expected, human AChRs immunoisolated from SH-SY5Y neuroblastoma cells showed that AChRs containing alpha 3 and probably alpha 5 subunits were present, but alpha 4 AChRs were not. In brain, by contrast, alpha 4 beta 2 AChRs were shown to predominate over alpha 3 AChRs. Some of the brain alpha 4 beta 2 AChRs were found to contain alpha 5 subunits.
Animals↗
Mutation causing autosomal dominant nocturnal frontal lobe epilepsy alters Ca2+ permeability, conductance, and gating of human alpha4beta2 nicotinic acetylcholine receptors.
A mutation (S247F) in the channel-lining domain (M2) of the alpha4 nicotinic acetylcholine receptor (AChR) subunit has previously been linked genetically to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). To better understand the functional significance of this mutation, we characterized the properties of mutant and wild-type human alpha4beta2 AChRs expressed in Xenopus oocytes. Both had similar expression levels and EC50 values for ACh and nicotine. Substantial use-dependent functional upregulation was found for mutant alpha4beta2 AChRs, but not for wild type. Mutant AChR responses showed faster desensitization, slower recovery from desensitization, less inward rectification, and virtually no Ca2+ permeability as compared with wild-type alpha4beta2 AChRs. Addition of the alpha5 subunit restored Ca2+ permeability to the mutant alpha4beta2alpha5 AChRs. At -80 mV, wild-type alpha4beta2 AChR single channel currents exhibited two conductances, each with two mean open times (gamma1 = 17 pS, tau1 = 3.7 msec, and tau2 = 23.4 msec; gamma2 = 28 pS, tau1 = 1.9 msec, and tau2 = 8.1 msec). In contrast, mutant AChRs exhibited only one conductance of 11 pS, with tau1 = 1.9 msec and tau2 = 4.1 msec. The net effect of the mutation is to reduce AChR function. This could result in the hyperexcitability characteristic of epilepsy if the mutant AChRs were part of an inhibitory circuit, e.g., presynaptically regulating the release of GABA. In the minority of AChRs containing the alpha5 subunit, the overall functionality of these AChRs could be maintained despite the mutation in the alpha4 subunit.
Acetylcholine↗
Chronic nicotine treatment up-regulates alpha3 and alpha7 acetylcholine receptor subtypes expressed by the human neuroblastoma cell line SH-SY5Y.
Chronic exposure to nicotine has been reported to increase the number of nicotinic acetylcholine receptors (AChRs) in brain. The mechanism of up-regulation for the alpha4beta2 AChR subtype, which accounts for the majority of high affinity nicotine binding in mammalian brain, has previously been shown to involve a decrease in the rate of alpha4beta2 AChR turnover. Here, we report an investigation of the extent and mechanism of nicotine-induced up-regulation of alpha3 AChRs and alpha7 AChR subtypes expressed in the human neuroblastoma cell line SH-SY5Y. Up-regulation of human alpha3 AChRs and alpha7 AChRs, unlike alpha4beta2 AChRs, requires much higher nicotine concentrations than are encountered in smokers; the extent of increase of surface AChRs is much less; and the mechanisms of up-regulation are different than with alpha4beta2 AChRs. The mechanisms of up-regulation may be different for alpha3 AChRs or alpha7 AChRs. Chronic treatment with nicotine or carbamylcholine, but not d-tubocurarine, mecamylamine, or dihydro-beta-erythroidine, induced a 500-600% increase in the number of alpha3 AChRs but only a 30% increase in alpha7 AChRs. Chronic nicotine treatment did not increase affinity for nicotine or increase the amount of RNA for alpha3 or alpha7 subunits. The effect of nicotine on up-regulation of alpha7 AChRs was partially blocked by either d-tubocurarine or mecamylamine. The effect of nicotine treatment on the number of alpha3 AChRs was only slightly blocked by the antagonists d-tubocurarine, mecamylamine, or dihydro-beta-erythroidine at concentrations that efficiently block alpha3 AChR function. Most of the nicotine-induced increase in alpha3 AChRs was found to be intracellular. The alpha3 AChRs, which accumulate intracellularly, were shown to have been previously exposed on the cell surface by their susceptibility to antigenic modulation. The data suggest that chronic exposure to nicotine may induce a conformation of cell surface alpha3 AChRs that at least in this cell line are consequently internalized but not immediately destroyed.
Endocytosis↗
"Orphan" alpha6 nicotinic AChR subunit can form a functional heteromeric acetylcholine receptor.
Previously, a rat brain cDNA was reported that was designated alpha6 because of its homology with nicotinic acetylcholine receptor (AChR) alpha subunits, being especially similar to alpha3, but no acetylcholine-gated cation channels were detected when it was expressed in Xenopus laevis oocytes alone or in combination with other known rat AChR subunits. We cloned chicken alpha6 and human beta4 AChR subunits and tested for acetylcholine-gated cation channels with alpha6 by expression in X. laevis oocytes alone or in pairwise combination with chicken alpha3, beta2, or beta4 or with human alpha3, beta2, or beta4 AChR subunits. Chicken alpha6 formed detectable functional AChRs only when expressed together with the human beta4 subunit. The alpha6beta4 AChR-mediated currents show strong inward rectification and dependence on extracellular Ca2+. It exhibited a distinct pharmacological profile with an EC50 value of 28 microM for acetylcholine, 24 nM for (+)-epibatidine, 6.6 microM cytisine, and 15 microM 1,1-dimethyl-4-phenylpiperazinium. Both cytisine and 1,1-dimethyl-4-phenylpiperazinium behaved as partial (approximately 30%) agonists. Remarkably, nicotine (EC50 = 22 microM) was an even weaker partial agonist (approximately 18%) and had a relatively long-lasting inhibitory effect. Coexpression of the previously cloned rat alpha6 subunit with the human the beta4 subunit also resulted in functional alpha6beta4 AChRs with properties resembling those of the chicken/human alpha6beta4 AChRs. Therefore, alpha6 can function as part of AChRs with unusual pharmacological properties.
Acetylcholine↗
Chronic nicotine exposure differentially affects the function of human alpha3, alpha4, and alpha7 neuronal nicotinic receptor subtypes.
Because chronic exposure to nicotine and nicotinic drugs might both activate and desensitize nicotinic acetylcholine receptors (AChRs), we sought to determine whether prolonged exposure to nicotine concentrations encountered in tobacco users differentially affects electrophysiological properties of major subtypes of human neuronal nicotinic AChRs. Xenopus laevis oocytes were injected with subunit cRNAs encoding (1) homomeric alpha7 AChRs, (2) heteromeric alpha4beta2 AChRs and (3) heteromeric alpha3 AChRs formed from combinations of alpha3, beta2, beta4 and alpha5 cRNAs. Acute activation required micromolar concentrations of nicotine. Chronic exposure to submicromolar concentrations of nicotine irreversibly inactivated many alpha4beta2 AChRs and alpha7 AChRs but inhibited alpha3 AChRs much less. Thus, although alpha3 AChRs are present in the brain in much smaller amounts than are alpha4beta2 AChRs or alpha7 AChRs, alpha3 AChRs in brain and autonomic ganglia may be able to play a relatively large role in acute responses to endogenous ACh or subsequent doses of nicotine after chronic exposure to nicotine. The behavioral effects of nicotine may typically reflect the sustained inhibition of alpha4beta2 AChRs and alpha7 AChRs in combination with the residual susceptibility of alpha3 AChRs and perhaps some other AChR subtypes for acute activation. Tolerance for nicotine exhibited by tobacco users may reflect the long-term irreversible functional inactivation of alpha4beta2 AChRs and alpha7 AChRs produced by chronic exposure to nicotine.
Animals↗
Assembly of human neuronal nicotinic receptor alpha5 subunits with alpha3, beta2, and beta4 subunits.
Nicotinic acetylcholine receptors formed from combinations of alpha3, beta2, beta4, and alpha5 subunits are found in chicken ciliary ganglion neurons and some human neuroblastoma cell lines. We studied the co-expression of various combinations of cloned human alpha3, beta2, beta4, and alpha5 subunits in Xenopus oocytes. Expression on the surface membrane was found only for combinations of alpha3beta2, alpha3beta4, alpha3beta2alpha5, and alpha3beta4alpha5 subunits but not for other combinations of one, two, or three of these subunits. alpha5 subunits assembled inside the oocyte with beta2 but not with alpha3 subunits or other alpha5 subunits. alpha5 subunits coassembled very efficiently with alpha3beta2 or alpha3beta4 combinations. The presence of alpha5 subunits had very little effect on the binding affinities for epibatidine of receptors containing also alpha3 and beta2 or alpha3 and beta4 subunits. The presence of alpha5 subunits increased the rate of desensitization of both receptors containing also alpha3 and beta2 or alpha3 and beta4 subunits. In the case of receptors containing alpha3 and beta4 subunits, the addition of alpha5 subunits had little effect on the responses to acetylcholine or nicotine. However, in the case of receptors containing alpha3 and beta2 subunits, the addition of alpha5 subunits reduced the EC50 for acetylcholine from 28 to 0.5 microM and the EC50 for nicotine from 6.8 to 1.9 microM, while increasing the efficacy of nicotine from 50% on alpha3beta2 receptors to 100% on alpha3beta2alpha5 receptors. Both alpha3beta2 and alpha3beta2alpha5 receptors expressed in oocytes sedimented at the same 11 S value as native alpha3-containing receptors from the human neuroblastoma cell line SH-SY5Y. In the receptors from the neuroblastoma alpha3, beta2, and alpha5 subunits were co-assembled, and 56% of the receptor subtypes containing alpha3 subunits also contained beta2 subunits. The beta2 subunit-containing receptors from SH-SY5Y cells exhibited the high affinity for epibatidine characteristic of receptors formed from alpha3 and beta2 or alpha3, beta2, and alpha5 subunits rather than the low affinity exhibited by receptors formed from alpha3 and beta4 or alpha3, beta4, and alpha5 subunits. Nicotine, like the structurally similar toxin epibatidine, also distinguishes by binding affinity two subtypes of receptors containing alpha3 subunits in SH-SY5Y cells. The affinities of alpha3beta2 receptors expressed in oocytes were similar to the affinities of native alpha3 containing receptors from SH-SY5Y cells for acetylcholine, cytisine, and 1,1-dimethyl-4-phenylpiperazinium.
Acetylcholine↗
Structure and function of neuronal nicotinic acetylcholine receptors.
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Animals↗
P2x-purinoceptors of myenteric neurones from the guinea-pig ileum and their unusual pharmacological properties.
1. Whole-cell and outside-out patch clamp recordings were used to characterize the physiological and pharmacological properties of the P2x-purinoceptors of myenteric neurones from the guinea-pig ileum. 2. Adenosine 5'-triphosphate (ATP) and analogues (1-3000 microM) evoked a rapid inward current in > 90% of all recorded neurones. The reversal potential of this current was dependent on the extracellular sodium concentration, at +14 +/- 1.9, 0 +/- 1.6 and -12 +/- 1 mV for 166, 83 and 42 mM of sodium, respectively. The fast activation and inactivation of this current occurred even when guanosine 5'-triphosphate (GTP) was omitted from the pipette solution or substituted with an equimolar concentration of guanosine 5'-o-[2-thiotriphosphate] (GTP-gamma-S). Single channel currents were observed when these outside-out membrane patches were exposed to ATP (10-30 microM). These channels have a unitary conductance of about 17 picosiemens. 3. The rank-order of potency of the agonists used to induce the whole-cell currents was: ATP-gamma-S = ATP = 2-methylthio-ATP (2-Me-S-ATP) > > alpha, beta-methylene ATP = beta, gamma-methylene ATP; adenosine and uridine 5'-triphosphate (UTP) (up to 1 mM) were inactive. 4. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (1-30 microM) antagonized the effects of ATP (1 mM) with an IC50 of 4 microM. alpha, beta-Methylene ATP (100 microM) did not affect the ATP (30 microM)-induced current. Cibacron Blue 3GA increased the ATP activated cationic current whereas Basilen Blue E-3G had a very weak antagonistic effect (IC50 > or = 3 mM). Suramin potentiated the currents induced by ATP through a mechanism that was independent of its inhibitory effect on ectonucleotidase activity, as suramin also potentiated the effect of alpha, beta-methylene ATP (an ATP analogue that is resistant to nucleotidases). 5. In conclusion, the myenteric P2x-purinoceptor shares some properties with other purinoceptors in particular with the P2x4- and P2x6-purinoceptors. This receptor has also some unusual pharmacological properties suggesting that myenteric neurones express a novel subtype of P2x-purinoceptors. The properties of this receptor, however, might be a result of the combination of two or more of the homomeric purinoceptors so far characterized.
Adenosine Triphosphate↗
Neuronal nicotinic receptor subtypes.
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Animals↗
Comparative pharmacology of epibatidine: a potent agonist for neuronal nicotinic acetylcholine receptors.
Pharmacological properties of the (+)- and (-)-isomers of synthetic epibatidine, exo-2-(6-chloro-3-pyridyl)-7-azabicyclo-[2.2.1]heptane, were compared with nicotine and acetylcholine on several subtypes of chicken and human nicotinic acetylcholine receptors (AChRs). Both isomers of epibatidine behaved as extremely potent full agonists on chicken (alpha 3 beta 2, alpha 3 beta 4, alpha 4 beta 2, alpha 7, and alpha 8) and human (alpha 3 beta 2, alpha 3 beta 4, and alpha 7) neuronal AChRs expressed in Xenopus oocytes. Currents induced by epibatidine were effectively blocked by the nicotinic antagonists hexamethonium and mecamylamine. Apparent affinity was 100 to 1000-fold higher for epibatidine than for nicotine or acetylcholine. EC50 values ranged from 1 nM (for homomeric chicken alpha 8) to 2 microM (for homomeric chicken alpha 7). Epibatidine showed comparatively lower affinity for muscle-type AChRs from Torpedo and humans (EC50 values, 1.6 and 16 microM respectively). In binding assays, epibatidine was used on AChR subtypes immunoisolated from chicken brain and retina (alpha 4 beta 2, alpha 7, and alpha 8), the human neuronal cell line SH-SY5Y (alpha 3 and alpha 7), Torpedo electric organ (alpha 1 beta 1 gamma delta), or the human rhabdomyosarcoma cell line TE671 (alpha 1 beta 1 gamma delta). Both isomers of epibatidine exhibited extremely high affinity for all neuronal AChRs tested, with KI values ranging from 0.6 pM (human alpha 3 AChRs) to 0.6 microM (chicken alpha 7 AChRs). In contrast, epibatidine had lower affinity for Torpedo muscle-type AChRs (KI approximately 5 microM). Racemic [3H]epibatidine was an effective labeling reagent for human alpha 3 beta 2 AChRs, exhibiting a KD (0.14 nM) similar to the KI values observed for unlabeled (+)-epibatidine (0.23 nM) or (-)-epibatidine (0.16 nM).
Animals↗
Homomers of alpha 8 and alpha 7 subunits of nicotinic receptors exhibit similar channel but contrasting binding site properties.
alpha 8 subunits of alpha-bungarotoxin-sensitive chick neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes from cRNA are shown to form homomeric, acetylcholine-gated, rapidly desensitizing, inwardly rectifying, Ca(2+)-permeable cation channels similar to those of alpha 7 homomers. alpha 8 forms oligomers of several sizes, of which < 14% are expressed on the oocyte surface, which is less efficient than for alpha 7 homomers. alpha 8 homomers are more sensitive to agonists but less sensitive to antagonists than are alpha 7 homomers, and some agonists for alpha 8 homomers are partial agonists or antagonists for alpha 7 homomers. The pharmacological properties of homomers of alpha 8 and alpha 7 subunits generally reflect those of native alpha 8 and alpha 7 receptors.
Animals↗
ATP closes a potassium and opens a cationic conductance through different receptors in neurons of guinea pig submucous plexus.
Intracellular recordings were made to study the actions of ATP and related nucleotides on neurons from the guinea pig submucous plexus. Local application of ATP, by pressure, induced a depolarization in most AH-type neurons, which had a latency of several milliseconds, lasted for about 5 sec, appeared to reverse at about +4 mV and occurred concomitantly with a reduction in input resistance. Pressure application of ATP also depolarized the S-type neurons. In most of these cells the depolarization had two phases: the first component resembled the depolarization observed in AH cells and the second component was much slower in onset and was longer lasting (30-90 sec). The slower component was associated with an increase in input resistance, reversed polarity near the potassium equilibrium potential and was observed in isolation in 30% of S neurons. Superfusion of ATP or other analogs (0.03-10 microns) induced a slow depolarization in most of S neurons with the following rank order of potency: 2-methylthio-ATP > ATP > adenosine-5'-o-3-thiotriphosphate = ADP; alpha, beta-methylene ATP and beta, gamma-methylene ATP were inactive (10-100 microM). When whole-cell recordings were used, fast superfusion with ATP or other analogs (3-1000 microM) evoked, at negative membrane potentials, a rapidly desensitizing inward current. This current reversed polarity at about 0 mV and was much reduced in low extracellular sodium concentration. The rank order of potency of the used agonists was: ATP = adenosine-5'-o-3-thiotriphosphate = 2-methylthio-ATP > > alpha,beta-methylene ATP = beta,gamma-methylene ATP; adenosine, AMP or ADP (1 mM) were inactive.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Triphosphate↗
Nicotine-induced increase in neuronal nicotinic receptors results from a decrease in the rate of receptor turnover.
Chronic nicotine exposure in tobacco smokers or experimental animals is known to cause an increase in brain binding sites for nicotine. It has been proposed that this is an adaptive response of neurons to accumulation of chronically desensitized receptors. Acetylcholine receptors of the same (alpha 4)2(beta 2)3 subunit composition as the predominant subtype of brain nicotinic receptors with high affinity for nicotine have been expressed in Xenopus oocytes and in a permanently transfected fibroblast cell line. Chronic exposure of these cells to nicotine or another agonist is shown to result in an increase in receptor amount, indicating that nicotine-induced up-regulation reflects properties of the alpha 4 beta 2 receptor protein, rather than being an adaptive response unique to the neurons in which these receptors are normally expressed. The nicotine concentration dependence, time course, and extent of receptor up-regulation are similar to those reported for receptors in brain. Up-regulation does not appear to require ion flow through the ion channel, because it is also caused by mecamylamine, which blocks the ion channel, and because after prolonged exposure to nicotine most receptors become permanently unable to open their channels in response to nicotine binding. The noncompetitive antagonist mecamylamine blocks open channels more effectively, and so it is more effective at blocking channels in the presence of nicotine. Mecamylamine and nicotine are also synergistic in causing receptor up-regulation. Ligands that cause up-regulation appear to induce a conformation of the receptor that is removed from the surface and degraded more slowly.
Animals↗
Human alpha 7 acetylcholine receptor: cloning of the alpha 7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional alpha 7 homomers expressed in Xenopus oocytes.
The alpha-bungarotoxin-binding acetylcholine receptors from the human neuroblastoma cell line SH-SY5Y were found to cross-react with some monoclonal antibodies to alpha 7 subunits of nicotinic acetylcholine receptors from chicken brain. The human alpha 7 subunit cDNA from SH-SY5Y was cloned, revealing 94% amino acid sequence identity to rat alpha 7 subunits and 92% identity to chicken alpha 7 subunits. Native human alpha 7 receptors showed affinities for some ligands similar to those previously observed with native chicken alpha 7 receptors, but for other ligands there were large species-specific differences in binding affinity. These results paralleled properties of alpha 7 homomers expressed in Xenopus oocytes. Human alpha 7 homomers exhibited rapidly desensitizing, inwardly rectifying, agonist-induced, cation currents that triggered Ca(2+)-sensitive Cl- channels in the oocytes. A change in efficacy from partial agonist for chicken alpha 7 homomers to full agonist for human alpha 7 homomers was exhibited by 1,1-dimethyl-4-phenylpiperazinium. This result reveals a large species-specific pharmacological difference, despite small differences in alpha 7 sequences. This is important for understanding the effects of these drugs in humans and for identifying amino acids that may contribute to the acetylcholine binding site, for analysis by in vitro mutagenesis. These results also characterize properties of native alpha 7 receptors and alpha 7 homomers that will provide criteria for functional properties expected of structural subunits, when these can be identified, cloned, and coexpressed with alpha 7 subunits.
Amino Acid Sequence↗
Reporter epitopes: a novel approach to examine transmembrane topology of integral membrane proteins applied to the alpha 1 subunit of the nicotinic acetylcholine receptor.
The development of a novel immunological method called the "reporter epitope" technique to probe the transmembrane topology of integral membrane proteins is described. Using this method, synthetic oligonucleotides encoding epitopes (reporter epitopes) for well characterized monoclonal antibodies (reporter mAbs) were inserted at various locations within the human acetylcholine receptor (AChR) alpha 1 subunit cDNA. The engineered subunits were then expressed along with Torpedo beta 1, gamma, and delta subunits in Xenopus oocytes, and the transmembrane location of the site of insertion was determined by the binding of the 125I-labeled reporter mAbs to whole oocytes. Control reporter epitope insertions at alpha 347 exhibited the expected cytoplasmic location. Reporter epitopes inserted at alpha 429 are located on the extracellular surface. Reporter epitopes that are 16-48 amino acids long do not disrupt assembly or function of hybrid AChRs when inserted near the carboxy terminus (at alpha 429) or in the large cytoplasmic domain (at alpha 347). However, because two reporter epitopes inserted at alpha 157 obliterated subunit assembly and a third reporter epitope when tolerated at this position was inaccessible from the extracellular surface and only marginally accessible after detergent solubilization of the AChRs, a definitive transmembrane location for this region was not possible. Nonetheless, the use of this approach has been successfully demonstrated, and it may be generally applicable to the study of other integral membrane proteins.
Amino Acid Sequence↗
On the excitatory effects of ATP and its role as a neurotransmitter in coeliac neurons of the guinea-pig.
1. The effects of ATP on neurons from guinea-pig coeliac ganglia were studied to evaluate the possibility that this nucleotide acts as an excitatory neurotransmitter substance. 2. In experiments with intracellular microelectrodes, ATP (> or = 10 nM) depolarized coeliac neurons from the resting potential and produced an increase in the membrane conductance. These excitatory effects of ATP were observed in isolated coeliac ganglia, in acutely dissociated neurons or in cultured neurons. ATP also produced membrane conductance increases in neurons clamped at the resting potential using a single electrode voltage clamp. 3. When studied in the whole-cell configuration of the patch clamp (intracellular Cs+ to block K+ currents; -50 mV holding potential), ATP evoked inward currents in a manner more potent and efficacious than acetylcholine (ACh). 4. Whole-cell currents induced by ATP were inwardly rectifying and reversed at -13 mV in normal Na+ solutions. Changes in extracellular Na+ concentration altered the reversal potential in a manner predicted by the Goldman-Hodgkin-Katz bi-ionic equation with a ratio of Na+ to Cs+ permeability (PNa/PCs) = 0.6. 5. Single channel currents were evoked by ATP in excised (outside-out) patches. Current-voltage relationships for single channel currents exhibited inward rectification. The mean single channel conductance was 22 pS at -50 mV. 6. Antagonists of ATP-gated channels (suramin, Reactive Blue 2) reduced the effects of ATP but not ACh. 7. Antagonists at nicotinic receptors/ion channels (hexamethonium or tubocurarine) reduced the effects of ACh but not ATP. 8. Excitatory synaptic currents were observed in cultures of coeliac neurons. Synaptic currents possessed similar current-voltage relationships to currents produced by ATP, were increased in frequency by K+ depolarization in a Ca(2+)-dependent manner, and were selectively antagonized by ATP antagonists. 9. Local K+ depolarization of the ends of neurites evoked single channel currents characteristic of ATP in outside-out patches when patches were positioned near the region of apparent synaptic contact but not when patches were positioned at remote regions. 10. The results suggest that ATP receptors are linked to ion channels and mediate excitatory synaptic transmission between coeliac neurons.
Acetylcholine↗
ATP mediates excitatory synaptic transmission in mammalian neurones.
Adenosine 5'-triphosphate (ATP, 0.1-100 microM), produced inward currents in patch-clamped coeliac neurones from guinea-pig when studied in either the whole cell configuration or in excised (outside-out) patches. The P2-purinoceptor antagonists suramin (80-230 microM) or reactive blue 2 (2-20 microM) depressed the ATP-induced currents but not those produced by acetylcholine. Excitatory post-synaptic currents (e.p.s.cs) were observed in cultured neurones. E.p.s.cs had similar current-voltage relationships to currents evoked by ATP in excised patches and were reduced by suramin or reactive blue 2 to a similar extent as ATP currents. The results suggest that ATP is the excitatory neurotransmitter in cultures of these neurones.
Acetylcholine↗