Binding sites,singly bound states,and conformation coupling shape GABA-evoked currents

The time course of GABA-evoked currents is the main source of information on the GABA(A) receptor gating. Since the kinetics of these currents depends on the transitions between several receptor conformations, it is a major challenge to define the relations between current kinetics and the respective rate constants of the microscopic gating scheme. The aim of this study was to further explore the impact of different GABA(A) receptor conformations on the kinetics of currents elicited by ultra-fast GABA applications. We show that the rising phase and amplitude of GABA-evoked currents depend on desensitization and singly bound states. The occupancy of bound receptors depends not only on binding properties but also on opening/closing and desensitization. The impact of such functional coupling between channel states is critical in conditions of high non-equilibrium typical for synaptic transmission. The concentration dependence of the rising phase of the GABA-elicited current indicates positive cooperativity between agonist binding sites. We provide evidence that preequilibration at low GABA concentrations reduce GABA-evoked currents due to receptor trapping in a singly bound desensitized state.     

Characterization of L-homocysteate-induced currents in Purkinje cells from wild-type and NMDA receptor knockout mice

L-Homocysteic acid (HCA), an endogenous excitatory amino acid in the mammalian CNS, potently activates N-methyl-D-aspartate (NMDA) receptors in hippocampal neurons. However, the responses to HCA in Purkinje cells, which lack functional NMDA receptors, have been largely unexplored: HCA may activate conventional non-NMDA receptors by its mixed agonistic action on both NMDA and non-NMDA receptors, or it may activate a novel non-NMDA receptor that has high affinity for HCA. To test these possibilities, we compared the responses to HCA in cultured Purkinje cells with those in hippocampal neurons by using the whole cell patch-clamp technique. To clearly isolate HCA responses mediated by non-NMDA receptors, we complemented pharmacological methods by using neurons from mutant mice (NR(-/-)) that lack functional NMDA receptors. A moderate dose of HCA (100 microM) induced substantial responses in Purkinje cells. These responses were blocked by non-NMDA receptor antagonists but were insensitive to NMDA receptor antagonists. HCA also activated responses mediated by non-NMDA receptors in both wild-type and NR1(-/-) hippocampal neurons. HCA responses in Purkinje cells had a pharmacological profile (EC(50) and Hill coefficient) very similar to that of non-NMDA receptor components of HCA responses in hippocampal neurons. Moreover, the amplitude of the non-NMDA receptor component of HCA responses was directly correlated with that of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and kainate-induced responses in both types of neurons. Finally, in both types of neurons, HCA currents mediated by non-NMDA receptors were potently blocked by the AMPA receptor antagonist GYKI52466. These findings indicate that HCA-activated AMPA receptors in Purkinje cells are similar to those in hippocampal neurons and that there is no distinct HCA-preferring receptor in Purkinje cells. We also found that in hippocampal neurons, the EC(50)s of HCA for non-NMDA receptors and for NMDA receptors were more similar than originally reported; this finding indicates that HCA is similar to other mixed agonists, such as glutamate. HCA responses may appear to be selective at NMDA receptors in cells that express these receptors, such as hippocampal neurons; in cells that express few functional NMDA receptors, such as Purkinje cells, HCA may appear to be selective at non-NMDA receptors.     

Desensitization and resensitization rates of glutamate-activated channels may regulate motoneuron excitability.

1. Single-channel properties of desensitizing glutamate-activated channels were analyzed in outside-out patch-clamp recordings from a motoneuron-enriched cell fraction from embryonic chick. A piezo-driven device was used to achieve fast solution exchange at the electrode tip, resulting in maximum activation within 2 ms. 2. Quisqualate/AMPA receptors, with a 13-pS conductance, desensitized rapidly; the desensitization rate depended on agonist concentration but not on membrane potential. When quisqualate was applied slowly, the quisqualate-activated channels desensitized without prior channel opening, indicating desensitization from the closed state. After a 10-ms refractory period, resensitization of all channels required up to 300 ms; resensitization rate did not depend on the duration of the preceding quisqualate application. 3. At agonist concentrations less than or equal to 1 mM, kainate receptors, with a 20-pS conductance, did not desensitize. At kainate concentrations greater than or equal to 1 mM, though, kainate receptors desensitized to a low steady-state conductance within approximately 200 ms. Resensitization of all channels required as long as 3 s, which could render kainate receptors inexcitable during high-frequency activation. 4. Desensitization rates of whole-cell currents were similar to those observed in outside-out mode. Glutamate- and quisqualate-activated responses were similar, suggesting that the rapidly desensitizing quisqualate-sensitive receptor type may dominate the kinetics of whole-cell excitatory postsynaptic currents (EPSCs) in this preparation. 5. It may be concluded that the efficacy of glutamate-mediated synaptic transmission is modulated by differences in the rates of desensitization and resensitization.     

Gadolinium reduces AMPA receptor desensitization and deactivation in hippocampal neurons.

The actions of the trivalent cation Gd(3+) on whole cell AMPA receptor-mediated currents were studied in isolated hippocampal neurons, in nucleated or outside-out patches taken from cultured hippocampal neurons, and on miniature excitatory postsynaptic currents (mEPSCs) recorded in cultured hippocampal neurons. Glutamate, AMPA, or kainate was employed to activate AMPA receptors. Applications of relatively low concentrations of Gd(3+) (0.1-10 microM) substantially enhanced steady-state whole cell glutamate and kainate-evoked currents without altering peak currents, suggesting that desensitization was reduced. However, higher concentrations (>30 microM) depressed steady-state currents, indicating an underlying inhibition of channel activity. Lower concentrations of Gd(3+) also increased the potency of peak glutamate-evoked currents without altering that of steady-state currents. An ultrafast perfusion system and nucleated patches were then used to better resolve peak glutamate-evoked currents. Low concentrations of Gd(3+) reduced peak currents, enhanced steady-state currents, and slowed the onset of desensitization, providing further evidence that this cation reduces desensitization. In the presence of cyclothiazide, a compound that blocks desensitization, a low concentration Gd(3+) inhibited both peak and steady-state currents, indicating that Gd(3+) both reduces desensitization and inhibits these currents. Gd(3+) reduced the probability of channel opening at the peak of the currents but did not alter the single channel conductance calculated using nonstationary variance analysis. Recovery from desensitization was enhanced, and glutamate-evoked current activation and deactivation were slowed by Gd(3+). The Gd(3+)-induced reduction in desensitization did not require the presence of the GluR2 subunit as this effect was seen in hippocampal neurons from GluR2 null-mutant mice. Gd(3+) reduced the time course of decay of mEPSCs perhaps as a consequence of its slowing of AMPA receptor deactivation although an increase in the frequency of mEPSCs also suggested enhanced presynaptic release of transmitter. These results demonstrate that Gd(3+) potently reduces AMPA receptor desensitization and mimics a number of the properties of the positive modulators of AMPA receptor desensitization such as cyclothiazide.     

Substance P modulates glutamate-induced currents in acutely isolated rat spinal dorsal horn neurones.

The whole-cell patch-clamp technique was used to examine the effect of substance P (SP) on glutamate-induced currents in freshly dissociated rat spinal dorsal horn neurons (LI-III). In 48% of examined cells SP (10(-10)-10(-6) M) at -70 mV, induced in inward current that desensitized in the continued presence of SP. When applied simultaneously with, or prior to L-glutamate, SP caused a potentiation of L-glutamate-induced current in 65% of the tested cells. Since glutamate activates both N-methyl-D-aspartate (NMDA) and non-NMDA receptors in rat dorsal horn neurons, selective agonists, kainate, quisqualate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and NMDA were used to determine which subtype of excitatory amino acid receptors interacted with SP. We found that the responses to quisqualate, kainate, and AMPA were not significantly affected by SP (less than 20% increase). In contrast, the inward currents induced by NMDA (30-300 microM) appear to be reduced and potentiated after the administration of 2-200 nM of SP. These results suggest that post-synaptic mechanisms of action of tachykinins may contribute to the regulation of the strength of glutamate-mediated excitatory transmission in the rat spinal dorsal horn.     

Physiological evidence that D-aspartate activates a current distinct from ionotropic glutamate receptor currents in Aplysia californica neurons

D-Aspartate (D-Asp) activates an excitatory current in neurons of Aplysia californica. Although D-Asp is presumed to activate a subset of L-glutamate (L-Glu) channels, the identities of putative d-Asp receptors and channels are unclear. Whole cell voltage- and current-clamp studies using primary cultures of Aplysia buccal S cluster (BSC) neurons were executed to characterize D-Asp-activated ion channels. Both D-Asp and L-Glu evoked currents with similar current-voltage relationships, amplitudes, and relatively slow time courses of activation and inactivation when agonists were pressure applied. D-Asp-induced currents, however, were faster and desensitized longer, requiring 40 s to return to full amplitude. Of cells exposed to both agonists, 25% had D-Asp- but not L-Glu-induced currents, suggesting a receptor for D-Asp that was independent of l-Glu receptors. D-Asp channels were permeable to Na(+) and K(+), but not Ca2?, and were vulnerable to voltage-dependent Mg2? block similarly to vertebrate NMDA receptor (NMDAR) channels. d-Asp may activate both NMDARs and non-l-Glu receptors in the nervous system of Aplysia.     

How do tonic glutamatergic synapses evade receptor desensitization?

Photoreceptor output synapses are the best known tonic chemical synapses in the nervous system, in which glutamate is continuously released in darkness, activating AMPA/kainate receptors in postsynaptic neurons. It has been shown that glutamate receptors in certain types of second-order retinal cells are largely desensitized in darkness, leading to small postsynaptic currents and reduced response dynamic ranges. Here we show that the tonic glutamatergic synapses between photoreceptors and rod-dominated hyperpolarizing bipolar cells (HBC_Rs) in the salamander retina evade postsynaptic receptor desensitization by using (1) multiple invaginating ribbon junctions as releasing sites for low-frequency, synchronized multiquantal release at each site; and (2) the GluR4 AMPA receptors as the postsynaptic receptors. The multiquantal events exhibit faster decay time than the GluR4 receptor desensitization time constant and therefore self-desensitization is minimized, and the average inter-event duration in darkness is much longer than the GluR4 desensitization recovery time and thus mutual desensitization is avoided. Consequently, the HBC_Rs are not desensitized in darkness, allowing light signals to be encoded by the full operating range of the glutamate-gated postsynaptic currents. Our study illustrates for the first time how a tonic glutamatergic synapse avoids postsynaptic receptor desensitization, a strategy that may be shared by many other synapses in the nervous system that need extended operation capacity.     

Dual-component miniature excitatory synaptic currents in rat hippocampal CA3 pyramidal neurons.

1. Spontaneous miniature synaptic events were studied with tight-seal whole-cell recordings from CA3 neurons maintained in the hippocampal slice from immature rats (3-15 days). CA3 neurons suffer a constant, high-frequency barrage of inhibitory synaptic input. When inhibitory postsynaptic currents were suppressed by bicuculline, a smaller contribution from excitatory synapses was revealed. 2. Addition of tetrodotoxin (TTX) removed a persistent inward current and substantially reduced the baseline noise facilitating the detection of "miniature" excitatory currents. Addition of hyperosmotic media increased the frequency of spontaneous excitatory postsynaptic currents (EPSCs). 3. Under both physiological and elevated potassium conditions, individual spontaneous miniature EPSCs (10-30 pA amplitude) were composed of components mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors as determined by their voltage dependence, time course, and sensitivity to selective antagonists. 6-Cyano-7-nitro-quinoxaline-2,3-dione (CNQX) or D-2-amino-5-phosphonovaleric acid (D-APV) shifted the amplitude distribution of miniature EPSCs to a smaller mode at both +40 mV and -40 mV. Similar to EPSCs recorded in CA1 neurons, the rise and decay times of the NMDA receptor component were slower than those of the non-NMDA component. The time course of the non-NMDA component was voltage independent. 4. In 13 of 21 neurons, no correlation existed between individual EPSC rise times and their corresponding halfwidth, peak amplitude, or decay time constant. This suggests that the large range of EPSC kinetics observed in each individual neuron was not due solely to cable attenuation of EPSCs widely distributed over the dendritic tree. Plots of the mean EPSC rise time against mean halfwidth for each cell, however, revealed a striking correlation, suggesting that in neonates, active synapses may be grouped in a restricted region of the dendritic tree and as such are subject to similar amounts of dendritic filtering. 5. The electrotonic length of CA3 neurons (L = 0.52) predicted that at this maturity the electrotonic compactness of the neuron facilitated voltage control over all but the most distal synapses. The reversal potential of the fast component of spontaneous events was close to 0 mV, whereas the reversal potential of exogenously applied kainate and NMDA was more positive. This discrepancy likely reflects a compromise of the voltage clamp by the activation of conductances distributed over the entire cell.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in the time course of miniature endplate currents induced by bath-applied acetylcholine

Bath application of 0.5 and 2 microM acetylcholine (ACh) slowed the decay phase of miniature endplate currents (MEPC) recorded in isolated, voltage-clamped and prostigmine-treated frog sartorius muscle. Washout of ACh led to a decrease of the decay time constant of the MEPC to 72 +/- 5% (n = 5) and 51 +/- 3% (n = 6) of initial values, respectively, followed by very slow and incomplete recovery. MEPC amplitude changed slightly and recovered relatively fast. This discrepancy in the recovery rates is suggested to be due to a 'trapping' ability of desensitized receptors which can compete with the free receptors for ACh molecules and prevent repetitive binding. Thus the high affinity of desensitized receptors to ACh may partially compensate the absence of acetylcholinesterase activity.     

Microscopic kinetic determinants of macroscopic currents: insights from coupling and uncoupling of GABAA receptor desensitization and deactivation

The time course of inhibitory postsynaptic currents (IPSCs) reflects GABA_A receptor deactivation, the process of current relaxation following transient activation. Fast desensitization has been demonstrated to prolong deactivation, and these processes have been described as being 'coupled'. However, the relationship between desensitization and deactivation remains poorly understood. We investigated the 'uncoupling' of GABA_A receptor macroscopic desensitization and deactivation using experimental conditions that affected these two processes differently. Changing agonist affinity preferentially altered deactivation, changing agonist concentration preferentially altered macroscopic desensitization, and a pore domain mutation prolonged deactivation despite blocking fast desensitization. To gain insight into the mechanistic basis for coupling and uncoupling, simulations were used to systematically evaluate the interplay between agonist affinity, gating efficacy, and desensitized state stability in shaping macroscopic desensitization and deactivation. We found that the influence of individual kinetic transitions on macroscopic currents depended not only on model connectivity, but also on the relationship among transitions within a given model. In addition, changing single rate constants differentially affected macroscopic desensitization and deactivation, thus providing parsimonious kinetic explanations for experimentally observed uncoupling. Finally, these findings permitted development of an algorithmic framework for kinetic interpretation of experimental manipulations that alter macroscopic current properties.     

Monte Carlo simulation of spontaneous miniature excitatory postsynaptic currents in rat hippocampal synapse in the presence and absence of desensitization

 Using the Monte Carlo method, spontaneous fast excitatory postsynaptic currents (mEPSCs) at a hippocampal synapse were simulated by releasing 150–20,000 glutamate molecules from a point source centred 15 nm above a rectangular grid of 14 × 14 α-amino-3-hydroxy-methyl-isoxazole (AMPA) receptors and assuming the channel kinetics to be as reported by Jonas et al. [J Physiol (Lond) 472:615; 1993]. The relationship between the amplitudes of mEPSCs and their time constants of decay is positive, but not pronounced in physiological conditions (except when the number of molecules released is very high). It increases as desensitization is reduced and becomes highly pronounced when it is eliminated. mEPSCs are prolonged with repeated opening of AMPA channels due to enhancement of two concentration-dependent processes: (1) binding of glutamate molecules by AMPA receptors, and (2) occupancy of both activatable bound states. In contrast, the time constant of decay of the patch currents evoked by a short glutamate pulse is independent of glutamate concentration and current amplitude in control conditions, and only moderately concentration dependent in the absence of desensitization. The fast application protocol thus fails to reproduce synaptic currents reliably when there is repeated binding of glutamate molecules to AMPA receptors. During an mEPSC, the occupancy of desensitized states increases rapidly and it strongly depends on the number of glutamate molecules released. Desensitization reaches its maximum after an mEPSC decays to very low levels, and recovers very slowly (from tens to hundreds of milliseconds), and in a concentration-dependent manner. In conclusion, under physiological conditions the desensitization of AMPA receptors plays a major role in shaping the time course of mEPSCs by minimizing the repeated opening of AMPA channels. Received: 17 April 1997 / Received: after revision: 11 August 1997 / Accepted: 1 September 1997     

Membrane currents induced by L-homocysteic acid in mouse cultured hippocampal neurons.

The concentration-response relationship of membrane currents induced by L-homocysteic acid was studied on mouse embryonic hippocampal neurons in culture (n = 56). In the majority of neurons two phases in the dose-response relationship could be distinguished. The first was characterized by responses to 3-100 microM L-homocysteic acid which desensitized with a time-constant greater than 1 s in a concentration-dependent manner and were antagonized by 30 microM D-L-2-amino-5-phosphonovaleric acid indicating activation of the N-methyl-D-aspartate receptors. At higher concentrations of L-homocysteic acid this component was strongly depressed. The second phase was characterized by sustained responses that were concentration-dependent (1 mM L-homocysteic acid maximum concentration tested) and were not blocked by D-L-2-amino-5-phosphonovaleric acid indicating activation of non-N-methyl-D-aspartate receptors. Eight neurons did not exhibit these two-phase characteristics in the concentration-response relationship at the beginning of the recording. The magnitude of responses to L-homocysteic acid was positively related to concentration and the responses were partially blocked by D-L-2-amino-5-phosphonovaleric acid. In these neurons, however, repeated applications of L-homocysteic acid at concentrations 30 microM up to 300 microM resulted in a long-lasting, three- to four-fold increase of the membrane current. This increase was completely blocked by D-L-2-amino-5-phosphonovaleric acid (50-100 microM) suggesting that it was produced by activation of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopamine D1 receptors co-distribute with N-methyl-D-aspartic acid type-1 subunits and modulate synaptically-evoked N-methyl-D-aspartic acid currents in rat basolateral amygdala

Activation of dopamine D1 or glutamate, N-methyl-d-aspartic acid (NMDA) receptors in the basolateral amygdala (BLA) can potently influence affective behaviors and associative learning. Physical protein-protein interactions also can occur between C-terminal peptides of D1 receptors and the NMDA-receptor subunit-1 (NR1), suggesting intracellular associations of direct relevance to dopaminergic modulation of NMDA currents. We examined this possibility by combining electron microscopic immunolabeling of the D1 and NR1 C-terminal peptides with in vitro patch-clamp recording in the rat BLA. In the in vivo preparations, D1 and NR1 were localized to the surface or endomembranes of many of the same somata and dendrites as well as a few axon terminals, including those forming asymmetric, excitatory-type synapses. In vitro analysis of physiologically characterized projection neurons revealed an excitatory response to bath application of either dopamine or the preferential D1 receptor agonist, dihydrexidine. In these neurons, dopamine also selectively reduced stimulation-evoked isolated NMDA receptor-mediated currents, but not isolated non-NMDA receptor-mediated currents or the response to exogenous NMDA application. The selective reduction of the NMDA receptor-mediated currents suggests that this effect occurs at a postsynaptic locus. Moreover, both D1 and NR1 were localized to postsynaptic surfaces of biocytin-filled and physiologically characterized projection neurons. Our results provide ultrastructural evidence for D1/NR1 endomembrane associations that may dynamically contribute to the attenuation of NMDA receptor-mediated currents following prior activation of D1 receptors in BLA projection neurons. The potential for postsynaptic cross-talk between D1 and NMDA receptors in BLA projection neurons as well as a similar interaction in presynaptic terminals could have important implications for the formation and extinction of affective memories.     

Differential effects of extra- and intracellular anions on GABA-activated currents in bullfrog sensory neurons

1. Kinetic properties of gamma-aminobutyric acid (GABA)-gated inward and outward anion currents were investigated in the frog sensory neurons perfused internally and externally with various anions with the use of a rapid concentration-jump (termed as 'concentration-clamp') technique. 2. Extracellular Br- [( Br-]o) shifted the dose-response curves of GABA-induced inward anion currents to the left without affecting the maximum values, whereas [Cl-]o, [I-]o, [No3-]o, [HCOO-]o, and [CH3COO-]o altered the rate of desensitization differently without shifting the GABA dose-response curves, indicating that the kinetics of desensitization phase are affected differently by various extracellular anions. 3. [CH3COO-]o suppressed the maximum current of the dose-response curve of the GABA-induced inward ICl without affecting Kd. 4. Both activation and desensitization phases of GABA-induced ICl consisted of fast and slow components, respectively. [Br-]o, [I-]o, and [NO3-]o significantly prolonged the slow desensitization component, whereas both [HCOO-]o and [CH3COO-]o shortened it. The fast desensitization and the fast and slow activation components were also affected by these foreign anions. 5. GABA dose-response curves of inward currents carried by various intracellular anions (Cl-, Br-, NO3-, I-, SCN-, HCOO-, F-, CH3COO-, CH3CH2COO-, BrO3-, and ClO3-) while keeping a constant [Cl-]o had a constant Kd value but different saturating maximum currents. There were no marked differences among their current kinetics except in the case of SCN-, indicating that the current kinetics is not affected by replacing intracellular Cl- [( Cl-]i) with various foreign anions. 6. The configuration and amplitude of GABA-gated outward anion currents at a constant [Cl-]i reflected the extracellular action of individual anions on the anion-binding site of GABA receptor associated with the anion-selective channel. 7. The relative conductances of the various anions, calculated from the maximum peak currents in dose-response curves of the GABA-induced inward anion currents at a constant [Cl-]o, was in the sequence: I- greater than Br- greater than or equal to NO3- greater than ClO3- greater than SCN- greater than or equal to Cl- greater than HCOO- greater than BrO3- greater than CH3COO- greater than F- greater than CH3CH2COO-.(ABSTRACT TRUNCATED AT 400 WORDS)     

Miniature postsynaptic currents recorded from identified rat spinal dorsal horn projection neurons in thin-slice preparations.

Whole-cell voltage-clamp recordings were made from spinothalamic and spinomesencephalic tract neurons in thin-slice preparations of rat spinal cord. In the presence of tetrodotoxin, spontaneous inward and outward postsynaptic currents were observed near the resting membrane potential. These currents were divided into miniature excitatory postsynaptic currents (mEPSCs) mediated by glutamate, and miniature inhibitory postsynaptic currents (mIPSCs) mediated by glycine or gamma-aminobutyric acid (GABA). Glutamatergic mEPSCs had two components mediated by NMDA and non-NMDA receptors. Analyzing these miniature synaptic currents, valuable information concerning the pre- and postsynaptic mechanisms underlying modulation of synaptic transmission in the spinal dorsal horn could be obtained.     

Desensitization of GABA-induced currents in cultured rat hippocampal neurons.

The basic characteristics of desensitization of the GABAA receptor were investigated in cultured rat hippocampal neurons (three days to four weeks in vitro) using whole cell patch clamp techniques. GABA at 10-500 microM was perfused on to neurons for 30 or 60 s, with 60 s intervals of wash with control bath solution between perfusions. Desensitization, evaluated by peak-to-plateau ratio and time constants of current decay (tau), was dose-dependent and culture age-dependent. Desensitization was observed as early as three days in culture, the earliest time tested. At all ages, higher concentrations of GABA induced both larger and faster desensitization. Desensitization was markedly voltage-dependent and decreased with depolarization; peak-to-plateau ratio went from 6.3 to 1.4 and tau went from 4.6 to 26.8 s when holding potentials were changed from -80 mV to +30 mV. Low concentrations of GABA (1-2 microM) perfused for 2-60 s, which did not induce any current, had no effect on the maximal response nor desensitization produced by a subsequent application of 100 microM GABA. This finding suggests that GABA receptors were not desensitized without first being activated.     

P2X receptor-mediated ionic currents in dorsal root ganglion neurons.

Nociceptive neurons in the dorsal root ganglia (DRG) are activated by extracellular ATP, implicating P2X receptors as potential mediators of painful stimuli. However, the P2X receptor subtype(s) underlying this activity remain in question. Using electrophysiological techniques, the effects of P2X receptor agonists and antagonists were examined on acutely dissociated adult rat lumbar DRG neurons. Putative P2X-expressing nociceptors were identified by labeling neurons with the lectin IB4. These neurons could be grouped into three categories based on response kinetics to extracellularly applied ATP. Some DRG responses (slow DRG) were relatively slowly activating, nondesensitizing, and activated by the ATP analogue alpha,beta-meATP. These responses resembled those recorded from 1321N1 cells expressing recombinant heteromultimeric rat P2X2/3 receptors. Other responses (fast DRG) were rapidly activating and desensitized almost completely during agonist application. These responses had properties similar to those recorded from 1321N1 cells expressing recombinant rat P2X3 receptors. A third group (mixed DRG) activated and desensitized rapidly (P2X3-like), but also had a slow, nondesensitizing component that functionally prolonged the current. Like the fast component, the slow component was activated by both ATP and alpha, beta-meATP and was blocked by the P2X antagonist TNP-ATP. But unlike the fast component, the slow component could follow high-frequency activation by agonist, and its amplitude was potentiated under acidic conditions. These characteristics most closely resemble those of rat P2X2/3 receptors. These data suggest that there are at least two populations of P2X receptors present on adult DRG nociceptive neurons, P2X3 and P2X2/3. These receptors are expressed either separately or together on individual neurons and may play a role in the processing of nociceptive information from the periphery to the spinal cord.     

GABAB receptor activation desensitizes postsynaptic GABAB and A1 adenosine responses in rat hippocampal neurones

Whole-cell recordings of EPSCs and G-protein-activated inwardly rectifying (GIRK) currents were made from cultured hippocampal neurones to determine the effect of long-term agonist treatment on the presynaptic and postsynaptic responses mediated by GABA_B receptors (GABA_BRs). GABA_BR-mediated presynaptic inhibition was unaffected by agonist (baclofen) treatment for up to 48 h, and was desensitized by about one-half after 96 h. In contrast, GABA_BR-mediated GIRK currents were desensitized by a similar amount after only 2 h of agonist treatment. In addition, presynaptic inhibition mediated by A₁ adenosine receptors (A₁Rs) was unaffected by prolonged GABA_BR activation, whereas A₁R-mediated GIRK currents were desensitized. Desensitization of postsynaptic GABA_BR and A₁R responses was blocked by the GABA_BR antagonist (1-(S)-3,4-dichlorophenylethyl)amino-2-(S) hydroxypropyl-p-benzyl-phosphonic acid (CGP 55845A), but not by the A₁R antagonist cyclopentyldipropylxanthine (DPCPX). GIRK current amplitude could be partially restored after baclofen treatment by either coapplication of baclofen and adenosine, or intracellular infusion of the non-hydrolysable GTP analog 5'-guanylylimidodiphosphate (Gpp(NH)p). Short-term (4-24 h) baclofen treatment also significantly desensitized the inhibition of postsynaptic voltage-gated calcium channels by activation of GABA_BRs or A₁Rs. These results show that responses mediated by GABA_BRs and A₁Rs desensitize differently in presynaptic and postsynaptic compartments, and demonstrate the heterologous desensitization of postsynaptic A1R responses.     

ASIC3 and ASIC1 mediate FMRFamide-related peptide enhancement of H+-gated currents in cultured dorsal root ganglion neurons

The acid-sensing ion channels (ASICs) form cation channels that are transiently activated by extracellular protons. They are expressed in dorsal root ganglia (DRG) neurons and in the periphery where they play a function in nociception and mechanosensation. Previous studies showed that FMRFamide and related peptides potentiate H(+)-gated currents. To better understand this potentiation, we examined the effect of FMRFamide-related peptides on DRG neurons from wild-type mice and animals missing individual ASIC subunits. We found that FMRFamide and FRRFamide potentiated H(+)-gated currents of wild-type DRG in a dose-dependent manner. They increased current amplitude and slowed desensitization following a proton stimulus. Deletion of ASIC3 attenuated the response to FMRFamide-related peptides, whereas the loss of ASIC1 increased the response. The loss of ASIC2 had no effect on FMRFamide-dependent enhancement of H(+)-gated currents. These data suggest that FMRFamide-related peptides modulate DRG H(+)-gated currents through an effect on both ASIC1 and ASIC3 and that ASIC3 plays the major role. The recent discovery of RFamide-related peptides (RFRP) in mammals suggested that they might also modulate H(+)-gated current. We found that RFRP-1 slowed desensitization of H(+)-gated DRG currents, whereas RFRP-2 increased the peak amplitude. COS-7 cells heterologously expressing ASIC1 or ASIC3 showed similar effects. These results suggest that FMRFamide-related peptides, including the newly identified RFRPs, modulate H(+)-gated DRG currents through ASIC1 and ASIC3. The presence of several ASIC subunits, the diversity of FMRFamide-related peptides, and the distinct effects on H(+)-gated currents suggest the possibility of substantial complexity in modulation of current in DRG sensory neurons.     

Rapid drug application resolves two types of nicotinic receptors on rat sympathetic ganglion cells

 The properties of nicotinic acetylcholine receptors (AChRs) on cultured rat superior cervical ganglion (SCG) neurons were analysed. AChR agonists [1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), cytisine] were applied to whole cells within 70ms. The desensitization rate of whole-cell currents during constant application of DMPP varied between neurons. The time course of desensitization was fitted by double exponentials with time constants k _fast, of between 0.35 and 0.55s, and k _slow, of 3–5s. By exchanging intracellular chloride for caesium methanesulphonate, the possibility of interference by a calcium-activated chloride current was excluded. In cells that exhibited a slowly desensitizing current during the application 20 μM DMPP, equimolar cytisine induced a larger peak current compared to the response to DMPP, while in cells with rapidly desensitizing DMPP-induced currents the response to equimolar cytisine was smaller. The differences in desensitization rates and agonist potencies are due to different functional properties of AChR subtypes, as indicated by currents recorded from outside-out patches upon rapid agonist application and removal (2ms each). The results indicate the presence of two distinct AChR subtypes on SCG neurons: one with a fast and one with a slow activation/desensitization rate, but both with similar single-channel conductances. Slow activation/desensitization was found to be associated with a high potency of cytisine/low potency of DMPP. For AChRs with rapid activation/desensitization kinetics the agonist potencies were reversed. Received: 8 October 1996 / Received after revision: 13 December 1996 / Accepted: 23 January 1997