Slow Desensitization Research Articles

Enhancing Calcium Signaling

Tosetti et al. investigated the modulation of heterotrimeric guanosine 5′-triphosphate (GTP)-binding protein (G protein)-mediated inhibition of voltage-dependent Ca 2+ channels in dorsal root ganglion cells (DRGs) and discovered two Ca 2+ -dependent mechanisms that could preferentially enhance Ca 2+ signaling in particularly active neurons. Signaling by G proteins is terminated by the GTPase activity of the α subunit. Regulators of G protein signaling (RGS) accelerate this process; the mechanisms governing RGS activity, however, remain poorly understood. In chick DRGs, γ-aminobutyric acid (GABA) acts through Gα o to inhibit Ca 2+ channels. Tosetti et al. overexpressed variants of RGS3 and examined their effects on GABA-mediated inhibition of whole-cell Ca 2+ currents. RGS3s produced a rapid attenuation--or desensitization--of GABA-mediated inhibition of Ca 2+ currents. GABA-mediated inhibition of Ba 2+ flux through Ca 2+ channels was not affected, and an RGS3 mutant lacking an EF-hand (a Ca 2+ -binding domain) was ineffective. A gel mobility-shift assay indicated that Ca 2+ , but not Ba 2+ , bound intact RGS3. RGS3ss, an alternately spliced variant that lacked the EF hand did not elicit rapid desensitization, but did cause a slower form of Ca 2+ -dependent desensitization. Pharmacological analysis indicated that this slow desensitization depended on Ca 2+ indirectly through calmodulin, rather than directly through Ca 2+ binding. Thus, the two RGS variants are subject to different modes of Ca 2+ -dependent regulation. The authors suggest that the two isoforms could provide flexibility in Ca 2+ -dependent regulation of G protein-mediated inhibition of Ca 2+ channels, and that Ca 2+ regulation of RGS could provide a positive feedback mechanism to selectively enhance Ca 2+ influx into particularly active neurons. P. Tosetti, N. Pathak, M. H. Jacob, K. Dunlap, RGS3 mediates a calcium-dependent termination of G protein signaling in sensory neurons. Proc. Natl. Acad. Sci. U.S.A. 100 , 7337-7342 (2003). [Abstract] [Full Text]

A tyrosine residue in TM6 of the Vanilloid Receptor TRPV1 involved in desensitization and calcium permeability of capsaicin-activated currents

The Vanilloid Receptor TRPV1 is a non-selective cation channel with a high relative Ca 2+ permeability. TRPV1 exhibits slow desensitization, a potential mechanism regulating adaptation of peripheral sensory neurons to noxious stimuli. The predicted folding pattern of TRPV1 resembles that of voltage-gated channels. Sequence alignment of segments 6 of TRPV1 and voltage-gated Na + channels reveals a conserved aromatic amino acid that in Na + channels is involved in fast inactivation and pharmacological block. We found that replacing this tyrosine Y671 by positively charged lysine (K) completely abrogated Ca 2+-dependent desensitization. Y671K also exhibited significant reduction in Ca 2+ permeability that was not responsible for the lack in desensitization. Substitution of Y671 with negatively charged aspartate or uncharged alanine slightly altered desensitization but left Ca 2+ permeability unchanged. Substitution of Y671 with positively charged arginine produced a phenotype similar to Y671K. We propose that residue Y671 is critical for the high relative Ca 2+ permeability of TRPV1 and participates in the structural rearrangements of the channel protein leading to Ca 2+-dependent desensitization.

Long-term-desensitization of prostacyclin receptors is independent of the C-terminal tail

Persistent stimulation of the G s protein-coupled prostacyclin receptor (IP-R) causes its slow desensitization in a variety of cell types, a significant desensitization requiring several hours. To evaluate the role of the human IP-R C-terminus in desensitization and agonist-induced internalization, a C-terminally truncated hIP-receptor was generated. The C-terminal 68 amino acid residues were deleted by introduction of a stop codon for exchange of the original S319 codon (termed D318 mutant). Wild-type (WT) and truncated receptor were expressed in COS1 cells. Pretreatment of cells with the stable prostacyclin mimetic cicaprost (200 nM) desensitized cAMP production via WT and D318 receptors to similar extents. The cAMP response of WT and D318, respectively, was reduced by approximately 50% of maximal cAMP formation after 8 hr of continuous agonist stimulation, indicating significant long-term desensitization. Moreover, agonist-promoted sequestration of WT and D318 C-terminally tagged with green fluorescent protein was demonstrated, indicating that receptor internalization was not prevented by truncation of the C-terminus. These results demonstrated that long-term desensitization and sequestration of hIP-R did not depend on structures located in the hIP-R C-terminus.

Muscarinic Cholinergic Excitation of Smooth Muscle: Signal Transduction and Single Cationic Channel Properties

Acetylcholine, the principal neurotransmitter of the parasympathetic nervous system, evokes smooth muscle excitation and contraction by acting at the muscarinic receptors which, in many tissues, including the gastrointestinal tract, are comprised of the M2 and M3 subtypes. The opening of ion channels selective for monovalent cations (e.g., Na+ and K+) is the major mechanism of cholinergic excitation. We have studied signal transduction pathways and single cationic channel properties using patch-clamp recording and Ca2+ imaging techniques in guinea-pig single ileal myocytes. Cationic channels were found to couple to both M2 and M3 receptors via the GTP-bound Goα and phospholipase C activation, respectively. When these primarily signaling links are established, cationic channel opening can be further potentiated by membrane depolarization and an increase in the intracellular Ca2+ concentration. A strong synergism exists between the receptor occupancy by the agonist and intrinsic voltage dependence of the current as the former can effectively modulate the voltage range of cationic channel activation, while membrane depolarization produces a strong sensitizing effect. However, at potentials close to 0 mV ion flux is terminated by channel flickery block, while further depolarization induces long-lasting channel inactivation. Channel flicker is not caused by intracellular Mg2+, polyamines, or any other freely diffusible molecule and is confined to potentials around 0 mV irrespective of the driving force. Thus, it appears to be an intrinsic channel property of physiological importance as it improves conditions for the action potential discharge and propagation. Similarly, intracellular Ca2+-dependent facilitation of channel opening is counteracted by a slower desensitization. Further, the most intriguing negative control was discovered in the experiments whereby all cellular G proteins were non-selectively and persistently activated by GTPγS infusion, in which case, over time, carbachol instead of activation caused strong and almost irreversible inhibition of the cationic current. In cell-attached and outside-out membrane patches exposed to 50 μM carbachol or 200 μM internal GTPγS, the activity of three types of cationic channels was observed. They had dissimilar conductances (10, 50, and 130 pS), voltage dependence, and kinetics. The properties of the 50 pS channel are consistent with the whole-cell current behavior, at least when [Ca2+] i is “clamped” at 100 nM. The voltage-independent component of the cationic conductance, which appears at higher levels of [Ca2+] i , is likely mediated by the 130 pS channel, while the role of the 10 pS channel at present is unclear. Thus, smooth muscle cationic channels can uniquely detect and integrate many of the most important physiological signals such as the active conformation of two different muscarinic receptors, their associated G proteins and enzymes, as well as membrane potential and [Ca2+] i levels. Moreover, some signals act in synergy, while most of them, depending on the intensity, can be either stimulatory or inhibitory.

Intact synaptic GABAergic inhibition and altered neurosteroid modulation of thalamic relay neurons in mice lacking delta subunit.

Robust GABA-mediated inhibitory postsynaptic currents (IPSCs) in neurons of the thalamic relay (TC) nuclei are important in sustaining oscillatory activity within thalamic and thalamocortical circuits. The biophysical properties and pharmacological sensitivities of these IPSCs both depend on the subunit combination of postsynaptic gamma-aminobutyric acid-A (GABA(A)) receptors. Recombinant GABA(A) receptors containing the delta subunit (heavily expressed in TC nuclei) have been shown to exhibit slowed desensitization rates and high affinity for GABA in heterologous expression systems. We tested whether the GABA(A)-mediated synaptic inhibition in TC neurons would be affected by loss of the delta subunit. Spontaneous and evoked IPSCs were recorded from neurons in the ventral basal complex (VB) of the thalamus from brain slices of wild-type (delta(+/+)) and homozygous delta subunit deficient mice (delta(-/-)). Spontaneous IPSCs (sIPSCs) from delta(-/-) mice had no significant differences in amplitude, duration, or frequency compared with their delta(+/+) counterparts. However, baseline noise (63% of control) and the relative contribution of the slow component to overall decay (79% of control) were significantly lower in delta(-/-) VB recordings. Evoked IPSCs (eIPSCs) in delta(-/-) neurons showed no difference in peak amplitude, but had an accelerated slow decay component (40- vs. 55-ms time constant). We further tested whether neurosteroid modulation of GABA(A) receptors was dependent on the presence of the delta subunit, as previously reported in recombinant systems. Pregnenolone sulfate (PS) significantly reduced eIPSC peak amplitude (-30%) and increased duration in delta(-/-), but not in delta(+/+) mice. sIPSCs were not affected in any neurons, delta(-/-) or delta(+/+). In contrast, 3-alpha,5-alpha-tetrahydrodeoxycorticosterone (THDOC) increased the durations of eIPSCs and sIPSCs in both delta(-/-) and delta(+/+) VB neurons. Our findings show that although the delta subunit confers a striking PS insensitivity to eIPSCs in VB neurons, it plays only a minor role in the synaptic inhibition of VB neurons. This suggests delta subunit containing GABA(A) receptors may be functionally limited to an extrasynaptic locus in VB neurons.

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.

Disruption of coherent oscillations in inhibitory networks with anesthetics: role of GABA(A) receptor desensitization.

The effect of anesthetic drugs at central synapses can be described quantitatively by developing kinetic models of ligand-gated ion channels. Experiments have shown that the hypnotic propofol and the sedative benzodiazepine midazolam have similar effects on single inhibitory postsynaptic potentials (IPSPs) but very different effects on slow desensitization that are not revealed by examining single responses. Synchronous oscillatory activity in networks of interneurons connected by inhibitory synapses has been implicated in many hippocampal functions, and differences in the kinetics of the GABAergic response observed with anesthetics can affect this activity. Thus we have examined the effect of propofol and midazolam-enhanced IPSPs using mathematical models of self-inhibited one- and two-cell inhibitory networks. A detailed kinetic model of the GABA(A) channel incorporating receptor desensitization is used at synapses in our models. The most dramatic effect of propofol is the modulation of slow desensitization. This is only revealed when the network is driven at frequencies that are thought to be relevant to cognitive tasks performed in the hippocampus. The level of desensitization at synapses with propofol is significantly reduced compared to control synapses. In contrast, midazolam increases macroscopic desensitization at network synapses by altering receptor affinity without concurrently modifying desensitization rates. These differences in gating between the two drugs are shown to alter network activity in stereotypically different ways. Specifically, propofol dramatically increases the amount of excitatory drive necessary for synchronized behavior relative to control, which is not the case for midazolam. Moreover, the range of parameters for which synchrony occurs is larger for propofol but smaller for midazolam, relative to control. This is an important first step in linking alterations in channel kinetics with behavioral changes.

Slow phases of GABAA receptor desensitization: structural determinants and possible relevance for synaptic function

GABA(A) receptor fast desensitization is thought to shape the time course of individual IPSCs. Although GABA(A) receptors also exhibit slower phases of desensitization, the possible role of slow desensitization in modifying synaptic function is poorly understood. In transiently transfected human embryonic kidney (HEK293T) cells, rat alpha1beta3delta and alpha1beta3gamma2L GABA(A) receptors showed distinct desensitization patterns during long (28 s) concentration jumps using a saturating (1 mM) GABA concentration. alpha1beta3gamma2L receptors desensitized extensively (approximately 90%), with four phases (tau(1) approximately 20 ms, tau(2) approximately 400 ms, tau(3) approximately 2 s, tau(4) approximately 10 s), while alpha1beta3delta receptors desensitized slowly and less extensively (approximately 35 %), with one or two slow phases with time constants similar to tau(3) and tau(4) of alpha1beta3gamma2L receptors. To determine the structural basis of subunit-specific desensitization, delta-gamma2L chimera subunits were expressed with alpha1 and beta3 subunits. Replacing the entire N-terminus of the gamma2L subunit with delta subunit sequence did not alter the number of phases or the extent of desensitization. Although extension of delta subunit sequence into transmembrane domain 1 (TM1) abolished the fast and intermediate components of desensitization, the two slow phases still accounted for substantial current loss (approximately 65 %). However, when delta subunit sequence was extended through TM2, the extent of desensitization was significantly decreased and indistinguishable from that of alpha1beta3delta receptors. The importance of TM2 sequence was confirmed by introducing gamma2 subunit TM2 residues into the delta subunit, which significantly increased the extent of desensitization, without introducing either the fast or intermediate desensitization phases. However, introducing delta subunit TM2 sequence into the gamma2L subunit had minimal effect on the rates or extent of desensitization. The results suggest that distinct delta subunit structures are responsible for its unique desensitization properties: lack of fast and intermediate desensitization and small contribution of the slow phases of desensitization. Finally, to investigate the possible role of slow desensitization in synaptic function, we used a pulse train protocol. We observed inhibition of peak current amplitude that depended on the frequency and duration of GABA pulses for receptors exhibiting extensive desensitization, whether fast phases were present or not. The minimally desensitizing alpha1beta3delta receptor exhibited negligible inhibition during pulse trains. Because receptors that desensitized without the fast and intermediate phases showed pulse train inhibition, we concluded that receptors can accumulate in slowly equilibrating desensitized states during repetitive receptor activation. These results may indicate a previously unrecognized role for the slow phases of desensitization for synaptic function under conditions of repeated GABA(A) receptor activation.

Electrophysiological evidence for expression of glycine receptors in freshly isolated neurons from nucleus accumbens.

In the course of studying N-methyl-D-aspartate (NMDA) receptors of the nucleus accumbens (NAcc), we found that 20% of freshly isolated medium spiny neurons, as well as all interneurons, responded in an unexpected way to long (5-s) coapplication of NMDA and glycine, the coagonist of NMDA receptors. Whereas the reversal potential of the peak NMDA current of this subset of neurons was still around 0 mV, the desensitizing current became outward at hyperpolarized potentials around -30 mV. A Cl(-)-free solution shifted the equilibrium potentials of the desensitized currents to around 0 mV. This outward current was not blocked by a Ca(2+)-free, Ba(2+)-containing solution, suggesting that the anionic conductance was not activated by Ca(2+) influx through NMDA receptor channels. Interestingly, glycine alone also evoked a current with a similar hyperpolarized reversal potential in this subset of neurons. The glycine current reversed around -50 mV, rectified outwardly, and inactivated strongly. Its desensitization was best fitted with a double exponential. Only the slow desensitization showed clear voltage dependence. The glycine current was not blocked by 200 microM picrotoxin and 10 microM zinc, was weakly antagonized by 1 microM strychnine, and was not enhanced by 1 microM zinc. In addition, 1 mM taurine, but not GABA, inactivated glycine currents, and 1 mM glycine occluded 10 mM taurine-mediated currents. These data indicate that a subset of nucleus accumbens neurons expresses glycine receptors and that either glycine or taurine could be an endogenous agonist for these receptors.

Pharmacological characterization of a novel cell line expressing human alpha(4)beta(3)delta GABA(A) receptors.

1: The pharmacology of the stable cell line expressing human alpha(4)beta(3)delta GABA(A) receptor was investigated using whole-cell patch-clamp techniques. 2: alpha(4)beta(3)delta receptors exhibited increased sensitivity to GABA when compared to alpha(4)beta(3)gamma(2) receptors, with EC(50)'s of 0.50 (0.46, 0.53) microM and 2.6 (2.5, 2.6) microM respectively. Additionally, the GABA partial agonists piperidine-4-sulphonate (P4S) and 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP) displayed markedly higher efficacy at alpha(4)beta(3)delta receptors, indeed THIP demonstrated greater efficacy than GABA at these receptors. 3: The delta subunit conferred slow desensitization to GABA, with rate constants of 4.8+/-0.5 s for alpha(4)beta(3)delta and 2.5+/-0.2 s for alpha(4)beta(3)gamma(2). However, both P4S and THIP demonstrated similar levels of desensitization on both receptor subtypes suggesting this effect is agonist specific. 4: alpha(4)beta(3)delta and alpha(4)beta(3)gamma(2) demonstrated equal sensitivity to inhibition by the cation zinc (2-3 microM IC(50)). However, alpha(4)beta(3)delta receptors demonstrated greater sensitivity to inhibition by lanthanum. The IC(50) for GABA antagonists SR-95531 and picrotoxin, was similar for alpha(4)beta(3)delta and alpha(4)beta(3)gamma(2). Likewise, inhibition was observed on both subtypes at high and low pH. 5: alpha(4)beta(3)delta receptors were insensitive to modulation by benzodiazepine ligands. In contrast Ro15-4513 and bretazenil potentiated GABA responses on alpha(4)beta(3)gamma(2) cells, and the inverse agonist DMCM showed allosteric inhibition of alpha(4)beta(3)gamma(2) receptors. 6: The efficacy of neurosteroids at alpha(4)beta(3)delta receptors was greatly enhanced over that observed at alpha(4)beta(3)gamma(2) receptors. The greatest effect was observed using THDOC with 524+/-71.6% potentiation at alpha(4)beta(3)delta and 297.9+/-49.7% at alpha(4)beta(3)gamma(2) receptors. Inhibition by the steroid pregnenolone sulphate however, showed no subtype selectivity. The efficacy of both pentobarbitone and propofol was slightly augmented and etomidate greatly enhanced at alpha(4)beta(3)delta receptors versus alpha(4)beta(3)gamma(2) receptors. 7: We show that the alpha(4)beta(3)delta receptor has a distinct pharmacology and kinetic profile. With its restricted distribution within the brain and unique pharmacology this receptor may play an important role in the action of neurosteroids and anaesthetics. British Journal of Pharmacology (2002) 136, 965-974

Desensitization Protocols for Vancomycin Hypersensitivity

OBJECTIVE:To discuss the pathophysiology of vancomycin-induced immediate hypersensitivity reactions, review the process of vancomycin desensitization, and provide specific directions for ordering and preparing rapid and slow desensitization protocols.DATA SOURCES:A MEDLINE search (1966–February 2001) of English-language literature pertaining to vancomycin desensitization and hypersensitivity reactions was performed. Tertiary sources were also used.DATA EXTRACTION:Published clinical studies and case reports.DATA SYNTHESIS:The pathophysiology of vancomycin-induced hypersensitivity reactions is discussed along with the procedure of vancomycin desensitization. Desensitization should be considered in Red Man syndrome (RMS) that does not respond to the usual treatment measures, and in vancomycin-induced anaphylaxis. Rapid desensitization is preferred as it is effective in the majority of patients and enables therapeutic dosing of vancomycin within 24 hours. In patients who fail rapid desensitization, a slow desensitization protocol may be tried.CONCLUSIONS:Vancomycin-induced immediate hypersensitivity reactions include RMS and anaphylaxis. Vancomycin desensitization should be considered for severe RMS reactions not responding to usual measures and in anaphylactic reactions to vancomycin, when substitution of another antibiotic is not feasible.

Desensitization protocols for vancomycin hypersensitivity.

To discuss the pathophysiology of vancomycin-induced immediate hypersensitivity reactions, review the process of vancomycin desensitization, and provide specific directions for ordering and preparing rapid and slow desensitization protocols. A MEDLINE search (1966-February 2001) of English-language literature pertaining to vancomycin desensitization and hypersensitivity reactions was performed. Tertiary sources were also used. Published clinical studies and case reports. The pathophysiology of vancomycin-induced hypersensitivity reactions is discussed along with the procedure of vancomycin desensitization. Desensitization should be considered in Red Man syndrome (RMS) that does not respond to the usual treatment measures, and in vancomycin-induced anaphylaxis. Rapid desensitization is preferred as it is effective in the majority of patients and enables therapeutic dosing of vancomycin within 24 hours. In patients who fail rapid desensitization, a slow desensitization protocol may be tried. Vancomycin-induced immediate hypersensitivity reactions include RMS and anaphylaxis. Vancomycin desensitization should be considered for severe RMS reactions not responding to usual measures and in anaphylactic reactions to vancomycin, when substitution of another antbiotic is not feasible.

Dominance of the lurcher mutation in heteromeric kainate and AMPA receptor channels.

Homomeric glutamate receptor (GluR) channels become spontaneously active when the last alanine residue within the invariant SYTANLAAF-motif in the third membrane segment is substituted by threonine. The same mutation in the orphan GluRdelta2 channel is responsible for neurodegeneration in "Lurcher" (Lc) mice. Since most native GluRs are composed of different subunits, we investigated the effect of an Lc-mutated subunit in heteromeric kainate and AMPA receptors expressed in HEK293 cells. Kainate receptor KA2 subunits, either wild type or carrying the Lc mutation (KA2(Lc)), are retained inside the cell but are surface-expressed when assembled with GluR6 subunits. Importantly, KA2(Lc) dominates the gating of KA2(Lc)/GluR6(WT) channels, as revealed by spontaneous activation and by slowed desensitization and deactivation kinetics of ligand-activated whole-cell currents. Moreover, the AMPA receptor subunit GluR-B(Lc)(Q) which forms spontaneously active homomeric channels with rectifying current-voltage relationships, dominates the gating of heteromeric GluR-B(Lc)(Q)/GluR-A(R) channels. The spontaneous currents of these heteromeric AMPAR channels show linear current-voltage relationships, and the ligand-activated whole-cell currents display slower deactivation and desensitization kinetics than the respective wild-type channels. For heteromeric Lc-mutated kainate and AMPA receptors, the effects on kinetics were reduced relative to the homomeric Lc-mutated forms. Thus, an Lc-mutated subunit can potentially influence heteromeric channel function in vivo, and the severity of the phenotype will critically depend on the levels of homomeric GluR(Lc) and heteromeric GluR(Lc)/GluR(WT) channels.

Polar Residues of the Second Transmembrane Domain Influence Cation Permeability of the ATP-gated P2X2 Receptor

P2X receptors are simple polypeptide channels that mediate fast purinergic depolarizations in both nerve and muscle. Although the depolarization results mainly from the influx of Na(+), these channels also conduct a significant Ca(2+) current that is large enough to evoke transmitter release from presynaptic neurons. We sought to determine the molecular basis of this Ca(2+) conductance by a mutational analysis of recombinant P2X(2) receptors. Wild type and 31 mutant P2X(2) receptors were expressed in HEK-293 cells and studied under voltage-clamp. We found that the relative Ca(2+) permeability measured from the reversal potentials of ATP-gated currents was unaffected by neutralizing fixed charge (Asp(315), Asp(349)) near the mouths of the channel pore. By contrast, mutations that changed the character or side chain volume of three polar residues (Thr(336), Thr(339), Ser(340)) within the pore led to significant changes in P(Ca)/P(Cs). The largest changes occurred when Thr(339) and Ser(340) were replaced with tyrosine; these mutations almost completely abolished Ca(2+) permeability, reduced P(Li)/P(Cs) by about one-half, and shifted the relative permeability sequence of Cs(+), Rb(+), K(+), and Na(+) to their relative mobility in water. Our results suggest that the permeability sequence of the P2X(2) receptor arises in part from interactions of permeating cations with the polar side chains of three amino acids located in a short stretch of the second transmembrane domain.

Ω-conotoxin GVIA potently inhibits the currents mediated by P2X receptors in rat DRG neurons

We examined effects of ω-conotoxin previously known as a selective blocker of N-type calcium channels, on the adenosine triphosphate (ATP)-induced currents in the rat dorsal root ganglion neurons. These neurons express at least two types of ionotropic purinoreceptors: P2X3 receptors that have very rapid desensitization kinetics and P2X2/X3 heterooligomeric receptor, which exhibits slow desensitization. We have found that ω-conotoxin GVIA potently inhibits the inward currents mediated by both receptor types. This effect was specific for the receptor subtypes: the IC 50 value for responses evoked by 10 μM ATP was 21.2 ± 1.7 nM for the P2X3 receptor-mediated responses and 3.84 ± 0.43 μM for slower responses mediated by P2X2/X3 heteropolymers. The efficacy of another type of ω-conotoxin, MVIIC, is much lower: at 10 μM the latter toxin inhibited the rapidly desensitizing response by 65% and the slowly desensitizing response by 18%. The effects of both toxins were reversible and independent on the membrane potential. Ω-Conotoxin GVIA shifted the dose dependence for the agonistic action of ATP on P2X3 receptors to higher concentrations without producing any effect on the kinetics of the response. It is suggested that ω-conotoxin allosterically modulates the receptor properties, rather than competes for the agonist binding site.

Interaction of the neuroprotective drug riluzole with GABA A and glycine receptor channels

Riluzole is used as therapeutic agent in amyotrophic lateral slerosis. We investigated the interaction of riluzole with recombinant GABA (γ-aminobutyric acid) A receptor channels (α 1β 2γ 2-subunits) and glycine receptor channels (α 1β-subunits) transiently expressed in HEK293 cells. For electrophysiological experiments, the patch–clamp technique in combination with tools for ultrafast solution exchange was used. Saturating concentrations of GABA or glycine were applied with different concentrations of riluzole to outside-out patches containing α 1β 2γ 2 GABA A receptor channels or α 1β-glycine receptor channels on their surface, respectively. The current declined after application of GABA or glycine with three time constants of desensitization to a steady-state current amplitude. Application of riluzole resulted in a shift to fast desensitized states at both receptors. The proportion of the time constants of fast desensitization increased and the time constants of slow desensitization and the steady-state current decreased whereas the maximal current amplitudes were not affected by riluzole. The data of the study demonstrate for the first time interaction of GABAergic and glycinergic currents with riluzole under physiological conditions.

Assembly of functional alpha6beta3gamma2delta GABA(A) receptors in vitro.

Transgenic mice deficient in the alpha6 subunit of the GABA(A) receptor show reduced levels of the delta subunit protein and an altered GABA(A) receptor pharmacology, suggesting selective assembly mechanisms. Delta reduced the binding of [3H]Ro15-4513 or t-butylbicyclophosphoro[35S]thionate and, to a lesser extent, [3H]muscimol to recombinant alpha1beta1gamma2(delta), alpha4beta1gamma2(delta) and alpha6beta1gamma2(delta) receptors, paralleled by diminished GABA-evoked maximal currents in electrophysiological recordings for the latter one. The delta subunit gave rise to a lower EC50 for GABA and a slowed desensitization indicating its assembly in alpha6beta2delta, alpha6beta1gamma2delta and alpha6beta2gamma2delta receptors. The data show that the delta subunits assemble in various functional GABA(A) receptor subtypes in vitro to reduce GABA-evoked maximal currents and ligand binding, but increase the potency for GABA.

Slow Desensitization Regulates the Availability of Synaptic GABAAReceptors

At central synapses, a large and fast spike of neurotransmitter efficiently activates postsynaptic receptors. However, low concentrations of transmitter can escape the cleft and activate presynaptic and postsynaptic receptors. We report here that low concentrations of GABA reduce IPSCs in hippocampal neurons by preferentially desensitizing rather than opening GABA(A) channels. GABA transporter blockade also caused desensitization by locally elevating GABA to approximately 1 microm. Recovery of the IPSC required several seconds, mimicking recovery of the channel from slow desensitization. These results indicate that low levels of GABA can regulate the amplitude of IPSCs by producing a slow form of receptor desensitization. Accumulation of channels in this absorbing state allows GABA(A) receptors to detect even a few molecules of GABA in the synaptic cleft.

Subclassified acutely dissociated cells of rat DRG: histochemistry and patterns of capsaicin-, proton-, and ATP-activated currents.

We used a "current signature" method to subclassify acutely dissociated dorsal root ganglion (DRG) cells into nine subgroups. Cells subclassified by current signature had uniform properties. The type 1 cell had moderate capsaicin sensitivity (25.9 pA/pF), powerful, slowly desensitizing (tau = 2,300 ms), ATP-activated current (13.3 pA/pF), and small nondesensitizing responses to acidic solutions (5.6 pA/pF). Type 1 cells expressed calcitonin gene-related peptide immunoreactivity (CGRP-IR), manifested a wide action potential (7.3 ms), long duration afterhyperpolarization (57.0 ms), and were IB4 positive. The type 2 cell exhibited large capsaicin activated currents (134.9 pA/pF) but weak nondesensitizing responses to protons (15.3 pA/pF). Currents activated by ATP and alphabeta-m-ATP (51.7 and 44.6 pA/pF, respectively) had fast desensitization kinetics (tau = 214 ms) that were distinct from all other cell types. Type 2 cells were IB4 positive but did not contain either substance P (SP) or CGRP-IR. Similar to capsaicin-sensitive nociceptors in vivo, the afterhyperpolarization of the type 2 cell was prolonged (54.7 ms). The type 3 cell expressed, amiloride-sensitive, rapidly desensitizing (tau = 683 ms) proton-activated currents (127.0 pA/pF), and was insensitive to ATP or capsaicin. The type 3 cell was IB4 negative and contained neither CGRP nor SP-IR. The afterhyperpolarization (17.5 ms) suggested nonnociceptive function. The type 4 cell had powerful ATP-activated currents (17.4 pA/pF) with slow desensitization kinetics (tau = 2, 813 ms). The afterhyperpolarization was prolonged (46.5 ms), suggesting that this cell type might belong to a capsaicin-insensitive nociceptor population. The type 4 cell did not contain peptides. The type 7 cell manifested amiloride-sensitive, proton-activated currents (45.8 pA/pF) with very fast desensitization kinetics (tau = 255 ms) and was further distinct from the type 3 cell by virtue of a nondesensitizing amiloride-insensitive component (6.0 pA/pF). Capsaicin and ATP sensitivity were relatively weak (4.3 and 2.9 pA/pF, respectively). Type 7 cells were IB4 positive and contained both SP and CGRP-IR. They exhibited an exceptionally long afterhyperpolarization (110 ms) that was suggestive of a silent (mechanically insensitive) nociceptor. We concluded that presorting of DRG cells by current signatures separated them into internally homogenous subpopulations that were distinct from other subclassified cell types.

Functional and molecular characterization of neuronal nicotinic ACh receptors in rat CA1 hippocampal neurons.

The molecular and functional properties of neuronal nicotinic acetylcholine receptors (nAChRs) were characterized from CA1 neurons in rat hippocampal slices using single-cell reverse-transcription polymerase chain reaction (RT-PCR) in conjunction with whole-cell patch-clamp recordings. We analysed the presence of the neuronal nAChR subunit mRNAs alpha2-7 and beta2-4, along with the mRNA for the GABAergic markers GAD (glutamic acid decarboxylase) 65 and 67 isoforms, and VGAT (vesicular GABA transporter) in interneurons from the stratum radiatum and stratum oriens, and in CA1 pyramidal neurons. Functional nAChR-mediated currents were detected in both interneuron populations, but not in pyramidal neurons. The neuronal nAChR subunit mRNAs detected in > 20 % of the populations examined were alpha4, alpha5, alpha7 and beta2-4 in stratum radiatum interneurons; alpha2, alpha3, alpha4, alpha7, beta2 and beta3 subunits in stratum oriens interneurons; and beta2-4 in pyramidal neurons. High levels of GABAergic marker mRNAs were detected in both interneuron populations, but not in pyramidal neurons. Significant co-expression of nAChR subunits within individual neurons was detected for alpha3 + alpha5, alpha4 + beta2, alpha4 + beta3, alpha7 + beta2, beta2 + beta3 and beta3 + beta4. The kinetics of the nAChR-mediated currents in response to the application of 100 microM ACh were significantly correlated with the expression of particular nAChR subunits. The alpha3, alpha7 and beta2 nAChR subunits were individually correlated with a fast rise time, the alpha2 nAChR subunit was correlated with a medium rise time, and the alpha4 nAChR subunit was correlated with a slow rise time. The alpha2 and alpha4 nAChR subunits were also significantly correlated with slow desensitization kinetics.