Fading and rebound of P2X2 currents at millimolar ATP concentrations caused by low pH

When ATP is applied at high concentrations (above 1 mM) to PC12 cells, it produces a rapidly desensitizing peak current followed by a rebound of the current after termination of the ATP application. We expressed P2X₂ receptors, which are thought to mediate the ATP currents of PC12 cells, in HEK293 cells and studied the effects of acidification on this `fading and rebound'' phenomenon. We found that the desensitization disappeared after adjusting the low pH (<5.0) of the millimolar ATP concentrations to a more physiological value (pH 7.3). Furthermore, the fading and rebound could also be induced at much lower ATP concentrations by decreasing the pH of the ATP containing application solutions. Thus, it appears this phenomenon is not caused directly by high concentrations of ATP, but is due to a concomitant acidification that occurs when high concentrations of ATP are dissolved in only moderately buffered application solutions.     

Modulation of P2X3 receptors by Mg2+ on rat DRG neurons in culture

On nociceptive neurons the commonest response to ATP is a rapidly desensitizing current mediated by P2X(3) receptors and believed to be involved in certain forms of pain. P2X(3) receptor recovery from desensitization is a slow process. We studied whether Mg(2+) might modulate such ATP-evoked currents on rat cultured DRG neurons, and thus account for its analgesic action in vivo. Transient increases in extracellular Mg(2+) strongly and reversibly depressed ATP currents which had not recovered from desensitization. Ca(2+)-free solution had the same action as Mg(2+). High Mg(2+) or Ca(2+)-free modulation depended on exposure length to modified divalent cation solutions, whereas it was independent from membrane potential or intracellular Ca(2+) buffering. Paired-pulse protocols showed that high Mg(2+) or Ca(2+)-free medium delayed ATP receptor recovery from desensitization, while leaving desensitization onset apparently unchanged. Tests with various concentrations of Ca(2+) and Mg(2+) showed that the depressant action by Mg(2+) was primarily due to functional antagonism of a facilitatory effect of Ca(2+) on ATP receptor function. The present results suggest that, on sensory neurons, P2X(3) receptors could be inhibited by high Mg(2+) or lack of Ca(2+), representing a negative feedback process to limit ATP-mediated nociception.     

Kinetic and pharmacological properties of the GABA-induced chloride current in Aplysia neurones: a 'concentration clamp' study

1. gamma-Aminobutyric acid (GABA) was applied by the 'concentration clamp' technique to isolated neurones of Aplysia. GABA induced a chloride current (ICl) due to activation of a single class of chloride-channel. 2. The concentration-response curve for the peak ICl gave an apparent dissociation constant of 6.4 X 10(-5) M and a Hill coefficient of 0.88. The current-voltage relationship was linear in the voltage range examined (-40 to +10 mV). 3. The activation phase of the ICl could be fitted to a single exponential function and desensitization followed the sum of two exponential functions. The time constants of activation and desensitization decreased with increasing concentrations of GABA but were voltage-independent. The recovery process from desensitization also followed the sum of two exponential functions. 4. As for the rate-limiting step of the channel activation, the hyperbolic relationship between the activation rate and GABA concentration showed that the rapid binding assumption holds, suggesting that the isomerization step is rate-limiting. The apparent channel closing rate constant was estimated to be 10 s-1 from the ordinate intercept of the linear part of the above relationship at lower concentrations. 5. Muscimol and beta-alanine induced a ICl, which cross-desensitized with that evoked by GABA. The GABA-ICl was not enhanced by diazepam (10(-6) M) or alpha-chloralose (10(-3) M), in fact depressant effects were evident. 6. Pentobarbitone decreased the GABA-ICl non-competitively without altering activation or desensitization kinetics. The concentration-inhibition curve gave a KD value of 8.9 x 10(-5) M and a Hill coefficient of 1.0. 7. These results suggest that GABA activates a single class of Cl channel in Aplysia neurones, which have one binding site for the agonist. The GABA receptor-Cl channel complex in Aplysia is pharmacologically and perhaps structurally different from that in vertebrates.     

The properties of the ATP-induced depolarization and current in single cells isolated from the guinea-pig urinary bladder.

1. The actions of exogenously applied ATP were investigated with the whole-cell patch clamp method in single cells isolated from guinea-pig urinary bladder with a modified concentration jump technique. 2. Rapid application of ATP (threshold ca. 100 nM) depolarized the cell membrane with superimposition of action potentials which was followed by transient hyperpolarization. In the presence of D600, the amplitude of the ATP-induced depolarization was a function of the ATP concentration (EC50: 0.5-1 microM). 3. ATP activated a dose-dependent inward current with a short latency (18 ms with 10 microM ATP; measured as the time between the start of application and 10% of the peak). The relationship of the peak current versus ATP concentration was well fitted by a Michaelis-Menten equation with a Hill coefficient (n) of 1.7 and a dissociation constant (Kd) of 2.3 microM. The current desensitized rapidly and the time course of desensitization was a function of the ATP concentration and could be fitted by two exponentials. 4. The reversal potential of the ATP-activated current was near 0 mV. Replacement of extracellular Na by other monovalent or divalent cations indicated that the current flows through nonselective cation channels. 5. alpha,beta-Methylene ATP also produced a dose-dependent inward current but was less potent than ATP (n: 1.6, Kd: 10.4 microM). alpha,beta-Methylene ATP blocked the response to ATP by desensitization of the receptor. 6. alpha,beta-Methylene ATP was 50-100 times more potent than ATP at eliciting a contractile response of strips of detrusor smooth muscle. 7. The relevance of the above results to the possible role of ATP as the fast excitatory transmitter is discussed.     

Characterization of voltage-dependent gating of P2X2 receptor/channel

The role of a voltage-dependent gate of recombinant P2X2 receptor/channel was investigated in Xenopus oocytes. When a voltage step to -110 mV was applied from a holding potential of -50 mV, a gradual increase was observed in current evoked by 30 microM ATP. Contribution of this voltage-dependent component to total ATP-evoked current was greater when the current was evoked by lower concentrations of ATP. The voltage-dependent gate closed upon depolarization, and half the gates were closed at -80 mV. On the other hand, a potential at which half the gates opened was about -30 mV or more positive, which was determined using a series of hyperpolarization steps. The results of the present study suggest that the voltage-dependent gate behavior of P2X2 receptor is not due to simple activation and deactivation of a single gate, but rather due to transition from a low to a high ATP affinity state.     

Meproadifen reaction with the ionic channel of the acetylcholine receptor: potentiation of agonist-induced desensitization at the frog neuromuscular junction

The actions of the nicotinic noncompetitive antagonist meproadifen on both the acetylcholine (ACh) receptor-ion channel complex and electrically excitable membrane were examined in frog sciatic-nerve sartorius muscle preparations. Meproadifen (10-25 microM) blocked the nerve-evoked twitch without affecting the directly evoked twitch, the threshold, overshoot, amplitude, rate of rise, or falling phase of the directly elicited action potential in muscle. This suggests that this agent, at the concentrations that affect the nicotinic receptor, had negligible effect on the excitable membrane. In addition, the drug did not affect either the quantal content or quantal size of the end-plate potential. Meproadifen caused a voltage- and time-dependent decrease in the peak amplitude of the end-plate current (EPC) without significantly shortening the time constant of EPC decay. The voltage- and time-dependent effects of meproadifen were more pronounced at more negative potentials, as evidenced by hysteresis loops and nonlinearity in the current-voltage relationship of the EPC. Both hysteresis and nonlinearity in the current-voltage relationship of the EPC were eliminated when brief conditioning pulses were used for stepwise changes of membrane potentials. The decay time constant of the EPC in the presence of meproadifen remained an exponential function of time. Meproadifen blocked iontophoretically elicited EPCs but did not affect single-channel lifetime, conductance, or the decay time constant of the miniature EPC. Thus, the blockade was more marked on iontophoretically elicited EPCs than on miniature EPCs. Meproadifen also caused desensitization of both the junctional and extrajunctional ACh receptors, but, more important, meproadifen accelerated steady-state desensitization by several-fold (compared with the agonist). The marked depression of peak EPC amplitude and miniature EPC, its high affinity for the binding sites in the presence of the agonist, and acceleration of agonist-induced desensitization suggest that meproadifen interacts with the ACh-bound but nonconducting state of the ACh receptor-ion channel complex. Therefore, it appears that meproadifen interacts with the closed ionic channel of the ACh receptor in its resting and activated but nonconducting states, and only slightly affects the open conformation of the ionic channel.     

The acetylcholine receptor of the neuromuscular junction recognizes mecamylamine as a noncompetitive antagonist

The secondary amine, mecamylamine, interacts with the nicotinic receptor ionic channel complex as a noncompetitive antagonist. Mecamylamine (1-10 microM) blocked indirect muscle twitches with no discernible effect on the membrane potential, overshoot, or amplitude of the action potential. It also produced a voltage- and concentration-dependent depression of the peak amplitude of the endplate currents (EPC) and induced nonlinearity in the current-voltage relationship. The decay time constant of the EPC (TEPC) was significantly shortened. The linear relationship between the reciprocal of TEPC and the drug concentration suggested an open channel blockade. Patch-clamp studies, in agreement with the noise analysis results, revealed that mecamylamine (1-8 microM) shortened the lifetime of the open channels. Further, the single channel studies showed that at high concentrations mecamylamine reduced the double exponential nature of the distribution of open times characteristic of channels recorded from myoballs. Closed times had a complex distribution that could not be fitted to a single exponential function because of the presence of short closures or "flickers" during the open state. Although the frequency of channel openings progressively decreased with increasing drug concentration, the single channel conductance remained unchanged at all the concentrations tested. Biochemical studies showed that mecamylamine (up to 100 microM) did not block [3H]acetylcholine binding to the nicotinic receptor of the Torpedo electroplax, but inhibited the binding of [3H]perhydrohistionicotoxin to its channel site, both in the resting and the activated state. These results suggested that, at the nicotinic receptors of the neuromuscular junction, mecamylamine acted as a noncompetitive blocker, binding primarily to the receptor's open channel conformation. Most of the alterations of EPCs were consistent with the predictions of a sequential model for open channel blockade. Biochemical and patch-clamp results, however, could not be fully explained by this model and provided some evidence of the existence of additional blocked states most likely through pathways into desensitized species. In contrast to a competitive antagonism of acetylcholine receptors reported at autonomic ganglia, there was no such action of the drug at the neuromuscular junction; thus, mecamylamine is a useful tool to characterize the nicotinic receptors from different synapses.     

Cibacron blue allosterically modulates the rat P2X4 receptor

We have used whole-cell patch clamp electrophysiology to characterise the actions of the P2 antagonist, cibacron blue, on the rat recombinant P2X₄ receptor, stably expressed in human embryonic kidney 293 (HEK293) cells. In single cells, adenosine triphosphate (ATP) evoked inward currents, but the response was subject to considerable run down which precluded obtaining quantitative data. However, when recordings were made from cells that were part of a group of 20–40 electrically coupled cells (cell rafts), run-down of current was not observed and reproducible responses could be obtained. When studied using cell rafts, cibacron blue was a weak antagonist of the rat P2X₄ receptor (IC₅₀>300 μM) when co-applied with ATP. However, when cell rafts were preincubated with low concentrations of cibacron blue (3–30 μM) for 5 min prior to ATP addition, cibacron blue increased responses to ATP by increasing its potency (up to 4-fold) without affecting the maximum current. Potentiation of ATP-evoked currents was also observed following washout of high, inhibitory concentrations of cibacron blue (300 μM). In contrast to these effects on P2X₄ receptors, cibacron blue inhibited the ATP-induced response in both single cells and rafts of HEK293 cells expressing the P2X₂ receptor (IC₅₀600–800 nM). The effects of cibacron blue on the P2X₄ receptor were quantitatively similar to those of Zn²⁺ which also increased ATP-evoked currents by decreasing the EC₅₀ of ATP (up to 3.5-fold). These data are consistent with the concept that cibacron blue, like zinc, allosterically regulates the function of the P2X₄ receptor.     

Expression level dependent changes in the properties of P2X2 receptors

The currents of P2X(2) receptors expressed in Xenopus oocytes or HEK293 cells show significant cell-to-cell variation in many properties including the rate of desensitization and the magnitude of potentiation by zinc or acidic pH. In this study, we examined whether differences in expression levels underlie this variability. We injected Xenopus oocytes with different concentrations of RNA encoding rat P2X(2) to give a wide range of maximum current amplitudes, and then measured the potentiation of responses to 10 micro M adenosine 5'-triphosphate (ATP) by zinc or acidic pH.Individual oocytes showed potentiation ratios that ranged from 1.4- to 25-fold. Oocytes with small amplitude responses to a saturating concentration of ATP tended to have larger potentiation ratios than oocytes with large amplitude responses. This phenomenon was explained by an inverse correlation between the EC(50) for ATP and the maximum current amplitude, with the EC(50) decreasing from about 37 to 7 micro M as expression level increased. In contrast, the Hill coefficient was not correlated with the maximum current amplitude. Truncated receptors lacking the last 76 amino acids also showed an inverse correlation between the EC(50) and the maximum current amplitude. Thus, the interactions that cause expression-dependent changes in P2X(2) receptor properties must involve domains proximal to position H397.     

Purine receptor-mediated endocannabinoid production and retrograde synaptic signalling in the cerebellar cortex

BACKGROUND AND PURPOSE

Presynaptic CB₁ cannabinoid receptors can be activated by endogenous cannabinoids (endocannabinoids) synthesized by postsynaptic neurones. The hypothesis of the present work was that activation of calcium-permeable transmitter-gated ion channels in postsynaptic neurones, specifically of P2X purine receptors, can lead to endocannabinoid production and retrograde synaptic signalling.

EXPERIMENTAL APPROACH

GABAergic inhibitory postsynaptic currents (IPSCs) were recorded with patch-clamp techniques in Purkinje cells in mouse cerebellar slices. Purine receptors on Purkinje cells were activated by pressure ejection of ATP from a pipette.

KEY RESULTS

ATP evoked an inward current in Purkinje cells, most likely due to P2X receptor activation. The ATP-evoked currents were accompanied by currents via voltage-gated calcium channels. ATP suppressed electrical stimulation-evoked IPSCs and miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin, and these effects were prevented by the CB₁ antagonist rimonabant and the calcium chelator BAPTA (applied into the Purkinje cell). ATP also suppressed mIPSCs when voltage-gated calcium channels were blocked by cadmium, and intracellular calcium stores were depleted by thapsigargin. However, ATP failed to suppress mIPSCs when the extracellular calcium concentration was zero.

CONCLUSIONS AND IMPLICATIONS

ATP elicits CB₁ receptor-dependent retrograde synaptic suppression, which is probably mediated by an endocannabinod released by the postsynaptic neurone. An increase in intracellular calcium concentration in the postsynaptic neurone is necessary for this retrograde signalling. We propose that ATP increases the calcium concentration by two mechanisms: calcium enters into the neurone via the P2X receptor ion channel and the ATP-evoked depolarization triggers voltage-gated calcium channels.     

The mechanism of the anti-desensitizing action of sodium fluoride at the amphibian neuromyal junction

Sodium fluoride was shown in these laboratories to facilite neuromyal transmission and to be a unique antagonist of desensitization. Possible mechanisms of action of sodium fluoride include a receptor effect and chelating activity. This study concerns the mechanism of the effect of sodium fluoride (0.1–2 mM) on the desensitization of the amphibian neuromyal junction and the interaction between this mechanism and calcium. Desensitization was obtained either by repetitive (10–50 Hz), brief (2 msec) iontophoretic pulses of acetylcholine (ACh), prolonged (40 sec) iontophoretic or bath application of ACh, or repetitive (10–50 Hz) presynaptic stimulation. Microelectrode and voltage-clamp methods were employed. The rate constant of desensitization induced by repetitive pulses of ACh was calculated in terms of the decay of current or potential. It depended on the frequency of pulses and on calcium ion concentration. In low calcium solution, a high frequency of pulses had to be employed to induce desensitization with a measurable rate constant. Sodium fluoride slowed down the rate of desensitization, and increased the amplitude of ACh current or potential during desensitization. The effect of sodium fluoride on the desensitization rate was pronounced at normal and low calcium ion concentrations and was slight in high calcium solutions. Desensitization did not affect reversal potential whether the latter was evaluated in terms of ACh potentials or ACh currents, and whether desensitization was induced by repeated ACh pulses or ACh perfusion. Nor did sodium fluoride (0.5–2 mM) affect the reversal potential of either a normal or desensitized endplate.Desensitization prolonged the decay time of the excitatory postsynaptic current (EPC) without affecting the slope of the decay time-membrane voltage relationship; sodium fluoride (0.1–2 mM) further prolonged decay time without affecting membrane voltage-decay time relation. The relationship between EPC or ACh current on the one hand and membrane voltage on the other was studied with respect to desensitization, sodium fluoride, and calcium concentration. Desensitization induced by repetitive (30 Hz) presynaptic stimulation did not affect the linearity of EPC-membrane voltage relationship; this remained true in different calcium solutions and when desensitization was induced in the presence of sodium fluoride. However, desensitization attenuated the slope of this relationship, the effect being similar in all three calcium solutions. This lack of effect of calcium on the slope must be considered with respect to calcium dependence of ACh release and, thus, on the EPC amplitude. The attenuation by desensitization of the slope of the EPC-voltage relationship was antagonized by sodium fluoride. Different frequencies of ACh pulses had to be employed to induce desensitization in the three calcium solutions in order to elicit desensitization with measurable rate constant. Also in this case, the ACh current-membrane voltage remained linear before and following desensitization in the three concentrations of calcium. Again, the slope of the current-voltage relationship was attenuated by desensitization at all concentrations of calcium. Sodium fluoride antagonized this effect to a significant degree at all concentrations of calcium; the antagonism was more pronounced at low than at high concentration of calcium. Desensitization induced by ACh perfusion did not affect the linearity of ACh current-membrane voltage at either of the three calcium concentrations. In this case also, desensitization attenuated the slope of the relationship in a calcium-dependent manner. Again, sodium fluoride restored significantly the amplitude of ACh current, the recovery being well-nigh complete in low, and amounting to 44% in high calcium solution. The EGTA (see Methods) did not affect the linearity of EPC-voltage or ACh current-voltage relationship, but decreased its slope; EGTA further diminished the slope of these relationships after the slope was attenuated by desensitization induced by repetitive presynaptic stimulation or ACh pulses. At that time, sodium fluoride restored to a significant degree the slope of ACh current-membrane voltage relationship; thus, sodium fluoride and EGTA exerted opposite effects on the slope of this relationship. These results support the notion that both desensitization and the effect of sodium fluoride concern the receptor rather than the channel. They also show that while calcium is involved in desensitization and the action of sodium fluoride, the anti-desensitizing effect of sodium fluoride does not depend on its chelating effect.     

Potentiation by cadmium ion of ATP-evoked dopamine release in rat phaeochromocytoma cells.

1. The effects of cadmium ion (Cd2+) on release of dopamine and on an inward current evoked by extracellular ATP were investigated in rat phaeochromocytoma PC12 cells. 2. Cd2+ (100 microM-3 mM) potentiated the dopamine release evoked by 30 microM ATP from the cells. Cd2+ (100 microM) shifted the concentration-response curve of ATP-evoked dopamine release to the left without affecting the maximal response. 3. Suramin (30 microM) completely abolished the dopamine release evoked by 30 microM ATP but only partially inhibited the release evoked by 100 microM ATP consistent with its role as a competitive antagonist. The response evoked by 30 microM ATP in the presence of Cd2+ (300 microM) was comparable to that observed with 100 microM ATP alone; however, only the former was almost completely inhibited by suramin. 4. Cd2+ (100 microM) potentiated an inward current activated by 30 microM ATP alone. A higher concentration of Cd2+ (300 microM) had a smaller effect on amplitude potentiation but significantly prolonged the duration of the current. 5. The time-course of the ATP-evoked dopamine release was investigated using a real-time monitoring system for dopamine release. Although Cd2+ (300 microM) had little effect on the time-course of activation the ATP-evoked dopamine release, it produced a long-lasting dopamine release which slowly returned to the baseline. 6. Taken together, these observations suggest that Cd2+ enhances ATP-evoked dopamine release by affecting P2-purinoceptor/channels. The enhancement may be attributed to a Cd(2+)-dependent increase in sensitivity to ATP.     

Effects of nucleotide analogs at the P2X3 receptor and its mutants identify the agonist binding pouch

In this study, we investigated the effects of single alanine substitutions of amino acid residues in the supposed ATP binding site of the human P2X3 receptor on the agonistic effect of nucleotide analogs. The wild-type and mutant receptors were expressed in HEK293 cells, and the nucleotide effects were measured by means of the whole-cell patch-clamp method. Modifications in the receptor binding site changed the concentration-response relationship, the current kinetics, and the recovery from desensitization during fast, pulsed, local agonist applications. On the basis of this fact, we were able to distinguish binding from other effects, such as gating/desensitization, by using a hidden Markov model that describes the complete channel behavior with a matrix of rate constants. The binding energies of the nucleotide analogs were calculated and compared with the binding energies of ATP at both the wild-type receptor and its alanine mutants. Changes in the binding energies caused by alterations in the receptor and/or agonist structures were concluded to be attributable to the preferential binding of certain structural constituents of ATP to certain amino acid moieties of the receptor. The results were also checked for consistency with a P2X3 homology model that we developed from the known zebrafish P2X4 crystal structure in the closed state. The functional data correlated well with the predictions of the agonist dockings to the structural model.     

Kinetic analysis of glutamate-induced chloride current in Aplysia neurones: a ''concentration clamp'' study

L-Glutamate (Glu) applied by the ‘concentration clamp’ technique to isolated neurones of Aplysia induced a chloride current (I_Cl) by activating a single population of the channel. The concentration-response curve for the peak I_Cl gave a dissociation constant of 1.3 × 10⁻⁴ M and a Hill coefficient of 1.8. The current-voltage relationship was linear in the voltage range examined (−60 to +10 mV). The activation phase of the I_Cl followed a single-exponential time course and desensitization was complete with a double-exponential time course. The time constant for activation and desensitization decreased with increasing concentrations of Glu but were voltage-independent. The process of recovery from desensitization was also double-exponential. The single-channel conductance estimated by ensemble noise analysis was 50±4.7 pS (n=4). These results suggest that the Glu receptor-Cl channel complex in Aplsia neurones consists of a single population with two binding sites for the agonist.     

Verapamil, neuromuscular transmission and the nicotinic receptor

The effect of verapamil on neuromuscular transmission was studied in the frog by analysing ionophoretic endplate current (iEPC) trains and the growth and decay phases of miniature endplate currents (mepcs). In addition, single channel data on the interaction of verapamil with the nicotinic acetylcholine receptor were obtained from cultured embryonic chick skeletal muscle cells. Verapamil caused both open and closed channel blockade in the iEPC trains. Mepc amplitude was decreased at low micromolar concentrations, and at higher concentrations there was also accelerated mepc decay indicating open channel blockade. The latter effect could not be explained by a sequential channel occlusion mechanism. Analysis of the mepc rising phase showed that low micromolar concentrations of the drug decreased the pool of receptors which could be activated. Single channel data confirmed the specific interaction of verapamil with the nicotinic receptor, showing closed channel blockade at low concentrations, and at higher levels the shortening of open channel lifetime. It is suggested that both forms of blockade may involve desensitization processes.     

The penultimate arginine of the carboxyl terminus determines slow desensitization in a P2X receptor from the cattle tick Boophilus microplus

P2X ion channels have been functionally characterized from a range of eukaryotes. Although these receptors can be broadly classified into fast and slow desensitizing, the molecular mechanisms underlying current desensitization are not fully understood. Here, we describe the characterization of a P2X receptor from the cattle tick Boophilus microplus (BmP2X) displaying extremely slow current kinetics, little desensitization during ATP application, and marked rundown in current amplitude between sequential responses. ATP (EC(50), 67.1 μM) evoked concentration-dependent currents at BmP2X that were antagonized by suramin (IC(50), 4.8 μM) and potentiated by the antiparasitic drug amitraz. Ivermectin did not potentiate BmP2X currents, but the mutation M362L conferred ivermectin sensitivity. To investigate the mechanisms underlying slow desensitization we generated intracellular domain chimeras between BmP2X and the rapidly desensitizing P2X receptor from Hypsibius dujardini. Exchange of N or C termini between these fast- and slow-desensitizing receptors altered the rate of current desensitization toward that of the donor channel. Truncation of the BmP2X C terminus identified the penultimate residue (Arg413) as important for slow desensitization. Removal of positive charge at this position in the mutant R413A resulted in significantly faster desensitization, which was further accentuated by the negatively charged substitution R413D. R413A and R413D, however, still displayed current rundown to sequential ATP application. Mutation to a positive charge (R413K) reconstituted the wild-type phenotype. This study identifies a new determinant of P2X desensitization where positive charge at the end of the C terminal regulates current flow and further demonstrates that rundown and desensitization are governed by distinct mechanisms.     

Blockade by local anaesthetics of the single Ca(2+)-activated K+ channel in rat hippocampal neurones.

1. Effects of local anaesthetics on single Ca(2+)-activated K+ channels were investigated using the inside-out configuration of the patch-clamp technique in single pyramidal neurones, which were freshly dissociated from rat hippocampus by use of proteolytic enzymes. 2. No significant effect was observed when 2 mM benzocaine was applied on either side of the membrane patch, or when 2 mM lignocaine or QX-314 was applied to the external surface of the membrane. 3. Lignocaine 1 mM, applied to the internal surface, slightly reduced the amplitude of the single K+ channel current. When applied to the internal surface QX-314 reduced the amplitude of the K+ channel current, accompanied by an increase in noise in the open channel current, suggesting a fast flickering block. The blocking effect of QX-314 on the outward current increased with depolarization, suggesting a binding site for the drug at an electrical distance of about 0.5 across the membrane field. 4. The open time histogram showed one exponential component and the closed time histogram showed at least two components. The mean open time of the outward current was increased when the amplitude was reduced by the drugs. 5. The ionized form of the local anaesthetics had a similar action on the Ca(2+)-activated K+ channels to that on Na+ channels, that is, they enter into the channel from the cytoplasmic side to induce open channel block. The blocking kinetics, however, might be so fast that they were beyond the frequency response of our recording apparatus, thus the recorded current amplitude was decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reversible cholinesterase inhibitor physostigmine has channel-blocking and agonist effects on the acetylcholine receptor-ion channel complex

The actions of the carbamate cholinesterase inhibitors, physostigmine (Phy) and physostigmine methiodide (MetPhy), were studied on the acetylcholine receptor-ion channel complex (AChR) of skeletal muscles. Low concentrations of these agents produced cholinesterase inhibition which resulted in potentiation of nerve-elicited muscle twitches and an increased peak amplitude and prolongation of the decay time constant (tau EPC) of endplate currents (EPCs) elicited in frog (Rana pipiens) sartorius muscles. However, increasing concentrations of Phy depressed the peak amplitude and shortened the decay phase of the EPC with an apparent loss in the voltage dependence of tau EPC. At higher concentrations and depolarized potentials, EPC decays were double exponential. The effects of both Phy and MetPhy on the postsynaptic AChR complex were also evident in preparations pretreated with diisopropylfluorophosphate. Under these conditions, a linear relationship between the reciprocal of tau EPC and the concentration of these agents was observed. Single channel studies revealed that Phy (20-600 microM) shortened channel lifetime and decreased channel conductance at very high concentrations. In addition, Phy (0.5 microM) induced the appearance of channel openings with conductance similar to that of acetylcholine. High concentrations (greater than 50 microM) of this agent activated channel openings with decreased conductance. Similar results were obtained with MetPhy. Thus, the reversible cholinesterase inhibitors Phy and MetPhy altered the properties of the AChR by interacting as agonists capable of inducing desensitization and blockade.     

Regulation of alpha4beta2 nicotinic receptor desensitization by calcium and protein kinase C

Neuronal nicotinic acetylcholine receptor (nAChR) desensitization is hypothesized to be a trigger for long-term changes in receptor number and function observed after chronic administration of nicotine at levels similar to those found in persons who use tobacco. Factors that regulate desensitization could potentially influence the outcome of long-lasting exposure to nicotine. The roles of Ca2+ and protein kinase C (PKC) on desensitization of alpha4beta2 nAChRs expressed in Xenopus laevis oocytes were investigated. Nicotine-induced (300 nM; 30 min) desensitization of alpha4beta2 receptors in the presence of Ca2+ developed in a biphasic manner with fast and slow exponential time constants of tauf = 1.4 min (65% relative amplitude) and taus = 17 min, respectively. Recovery from desensitization was reasonably well described by a single exponential with taurec = 43 min. Recovery was largely eliminated after replacement of external Ca2+ with Ba2+ and slowed by calphostin C (taurec = 48 min), an inhibitor of PKC. Conversely, the rate of recovery was enhanced by phorbol-12-myristate-13-acetate (taurec = 14 min), a PKC activator, or by cyclosporin A (with taurec = 8 min), a phosphatase inhibitor. alpha4beta2 receptors containing a mutant alpha4 subunit that lacks a consensus PKC phosphorylation site exhibited little recovery from desensitization. Based on a two-desensitized-state cyclical model, it is proposed that after prolonged nicotine treatment, alpha4beta2 nAChRs accumulate in a "deep" desensitized state, from which recovery is very slow. We suggest that PKC-dependent phosphorylation of alpha4 subunits changes the rates governing the transitions from "deep" to "shallow" desensitized conformations and effectively increases the overall rate of recovery from desensitization. Long-lasting dephosphorylation may underlie the "permanent" inactivation of alpha4beta2 receptors observed after chronic nicotine treatment.     

Kinetic properties of alpha 1 beta 1 gamma-aminobutyric acidA receptor channels expressed in Chinese hamster ovary cells: regulation by pentobarbital and picrotoxin.

Single-channel recordings from excised outside-out patches were obtained from Chinese hamster ovary cells stably transfected with plasmids containing bovine gamma-aminobutyric acid (GABA) type A (GABAA) receptor channel alpha 1 and beta 1 subunit cDNAs. The predominant or main conductance level recorded had a 17-pS chord conductance. There were minor contributions made from 25-pS and 11-pS conductance levels. Average open duration, burst duration, and openings/burst did not change as the GABA concentration was increased from 5 to 25 microM. However, opening frequency increased from 11.0 to 19.5 openings/sec. Pentobarbital increased average channel open duration without increasing opening frequency, whereas picrotoxin slightly reduced average channel open duration and reduced opening frequency. Open duration frequency distributions were fitted best with the sum of two exponential functions, suggesting that the alpha 1 beta 1 GABAA receptor channel had at least two open states. The time constants and relative proportions of the two components did not vary when GABA concentration was increased from 5 to 25 microM. Closed duration distributions of closures between main conductance level openings were fitted best with multiple exponential functions, suggesting that the alpha 1 beta 1 GABAA receptor channel had several closed states. Burst duration frequency distributions were fitted best with two exponential functions whose time constants and relative proportions did not change with GABA concentration. A gating kinetic scheme for the alpha 1 beta 1 GABAA receptor channel was proposed that consisted of a single binding site for GABA and at least two open and five closed states. The kinetic properties of the alpha 1 beta 1 main conductance level differed from those of the spinal cord neuron (native) 27-pS main conductance level and the 19-pS subconductance level. The native main conductance and subconductance levels were characterized by longer openings and at least three open states. Based on the aforementioned observations, it appears that different subunit combinations produce receptor channels with different kinetic properties, but the basic mechanism of regulation by pentobarbital and picrotoxin may be similar for the different receptor channels. Also, it is unlikely that the 19-pS substate of the native GABAA receptor is produced by an alpha 1 beta 1 dimer.