Rate Of Desensitization Research Articles

In the developing rat hippocampus a tonic GABAA‐mediated conductance selectively enhances the glutamatergic drive of principal cells

In the adult hippocampus, two different forms of GABA(A) receptor-mediated inhibition have been identified: phasic and tonic. The first is due to the activation of GABA(A) receptors facing the presynaptic releasing sites, whereas the second is due to the activation of receptors localized away from the synapses. Because of their high affinity and low desensitization rate, extrasynaptic receptors are persistently able to sense low concentrations of GABA. Here we show that, early in postnatal life, between postnatal day (P) 2 and P6, CA1 and CA3 pyramidal cells but not stratum radiatum interneurons, express a tonic GABA(A)-mediated conductance. Block of the neuronal GABA transporter GAT-1 slightly enhanced the persistent GABA conductance in principal cells but not in GABAergic interneurons. However, in adulthood, a tonic GABA(A)-mediated conductance could be revealed in stratum radiatum interneurons, indicating that the ability of these cells to sense ambient GABA levels is developmentally regulated. Pharmacological analysis of the tonic conductance in principal cells demonstrated the involvement of beta2/beta 3, alpha 5 and gamma 2 GABA(A) receptor subunits. Removal of the tonic depolarizing action of GABA with picrotoxin, reduced the excitability and the glutamatergic drive of principal cells but did not modify the excitability of stratum radiatum interneurons. The increased cell excitability and synaptic activity following the activation of extrasynaptic GABA(A) receptors by ambient GABA would facilitate the induction of giant depolarizing potentials.

Desensitization of the Dopamine D1 and D2 Receptor Hetero-Oligomer Mediated Calcium Signal by Agonist Occupancy of Either Receptor

When dopamine D1 and D2 receptors were coactivated in D1-D2 receptor hetero-oligomeric complexes, a novel phospholipase C-mediated calcium signal was generated. In this report, desensitization of this Gq/11-mediated calcium signal was demonstrated by pretreatment with dopamine or with the D1-selective agonist (+/-)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-81297) or the D2-selective agonist quinpirole. Desensitization of the calcium signal mediated by D1-D2 receptor hetero-oligomers was initiated by agonist occupancy of either receptor subtype even though the signal was generated only by occupancy of both receptors. The efficacy, potency, and rate of calcium signal desensitization by agonist occupancy of the D1 receptor (t1/2, approximately 1 min) was far greater than by the D2 receptor (t1/2, approximately 10 min). Desensitization of the calcium signal was not mediated by depletion of calcium stores or internalization of the hetero-oligomer and was not decreased by inhibiting second messenger-activated kinases. The involvement of G protein-coupled receptor kinases 2 or 3, but not 5 or 6, in the desensitization of the calcium signal was shown, occurring through a phosphorylation independent mechanism. Inhibition of Gi protein function associated with D2 receptors increased D1 receptor-mediated desensitization of the calcium signal, suggesting that cross-talk between the signals mediated by the activation of different G proteins controlled the efficacy of calcium signal desensitization. Together, these results demonstrate the desensitization of a signal mediated only by hetero-oligomerization of two G protein-coupled receptors that was initiated by agonist occupancy of either receptor within the hetero-oligomer, albeit with differences in desensitization profiles observed.

Desensitization and binding properties determine distinct α1β2γ2 and α3β2γ2 GABAA receptor‐channel kinetic behavior

GABAA receptor subtypes comprising the α1 and α3 subunits change with development and have a specific anatomical localization in the adult brain. These receptor subtypes have been previously demonstrated to greatly differ in deactivation kinetics but the underlying gating mechanisms have not been fully elucidated. Therefore, we expressed rat α1β2γ2 and α3β2γ2 receptors in human embryonic kidney 293 cells and recorded current responses to ultrafast GABA applications at macroscopic and single-channel levels. We found that the slow deactivation of α3β2γ2-mediated currents is associated with a relatively small rate and extent of apparent desensitization. In contrast, responses mediated by α1β2γ2 receptors had faster deactivation and stronger desensitization. α3β2γ2 receptors had faster recovery in the paired-pulse agonist applications than α1β2γ2 channels. The onset of currents mediated by α3β2γ2 receptors was slower than that of α1β2γ2 for a wide range of GABA concentrations. Single-channel analysis did not reveal differences in the opening/closing kinetics of α1β2γ2 and α3β2γ2 channels but burst durations were longer in α3β2γ2 receptors. Simulation with a previously reported kinetic model was used to explore the differences in respective rate constants. Reproduction of major kinetic differences required a smaller desensitization rate as well as smaller binding and unbinding rates in α3β2γ2 compared with α1β2γ2 receptors. Our work describes the mechanisms underlying the kinetic differences between two major GABAA receptor subtypes and provides a framework to interpret data from native GABA receptors.

A Conformation Change in the Extracellular Domain that Accompanies Desensitization of Acid-sensing Ion Channel (ASIC) 3

Acid-sensing ion channels (ASICs) are thought to trigger some forms of acid-induced pain and taste, and to contribute to stroke-induced neural damage. After activation by low extracellular pH, different ASICs undergo desensitization on time scales from 0.1 to 10 s. Consistent with a substantial conformation change, desensitization slows dramatically when temperature drops (Askwith, C.C., C.J. Benson, M.J. Welsh, and P.M. Snyder. 2001. PNAS. 98:6459–6463). The nature of this conformation change is unknown, but two studies showed that desensitization rate is altered by mutations on or near the first transmembrane domain (TM1) (Coric, T., P. Zhang, N. Todorovic, and C.M. Canessa. 2003. J. Biol. Chem. 278:45240–45247; Pfister, Y., I. Gautschi, A.-N. Takeda, M. van Bemmelen, S. Kellenberger, and L. Schild. 2006. J. Biol. Chem. 281:11787–11791). Here we show evidence of a specific conformation change associated with desensitization. When mutated from glutamate to cysteine, residue 79, which is some 20 amino acids extracellular to TM1, can be altered by cysteine-modifying reagents when the channel is closed, but not when it is desensitized; thus, desensitization appears to conceal the residue from the extracellular medium. D78 and E79 are a pair of adjacent acidic amino acids that are highly conserved in ASICs yet absent from epithelial Na+ channels, their acid-insensitive relatives. Despite large effects on desensitization by mutations at positions 78 and 79—including a shift to 10-fold lower proton concentration with the E79A mutant—there are not significant effects on activation.

Structure and Mechanism of Kainate Receptor Modulation by Anions

L-glutamate, the major excitatory neurotransmitter in the human brain, activates a family of ligand-gated ion channels, the major subtypes of which are named AMPA, kainate, and NMDA receptors. In common with many signal transduction proteins, glutamate receptors are modulated by ions and small molecules, including Ca(2+), Mg(2+), Zn(2+), protons, polyamines, and steroids. Strikingly, the activation of kainate receptors by glutamate requires the presence of both Na(+) and Cl(-) in the extracellular solution, and in the absence of these ions, receptor activity is abolished. Here, we identify the site and mechanism of action of anions. Surprisingly, we find that Cl(-) ions are essential structural components of kainate receptors. Cl(-) ions bind in a cavity formed at the interface between subunits in a dimer pair. In the absence of Cl(-), dimer stability is reduced, the rate of desensitization increases, and the fraction of receptors competent for activation by glutamate drops precipitously.

Regulation of Human Melanocortin 1 Receptor Signaling and Trafficking by Thr-308 and Ser-316 and Its Alteration in Variant Alleles Associated with Red Hair and Skin Cancer

The melanocortin 1 receptor (MC1R), a G protein-coupled receptor (GPCR) positively coupled to adenylyl cyclase, is a key regulator of melanocyte proliferation and differentiation and a determinant of pigmentation, skin phototype, and skin cancer risk. MC1R activation stimulates melanogenesis and increases the ratio of black, strongly photoprotective eumelanins to yellowish and poorly photoprotective pheomelanin pigments. Desensitization and internalization are key regulatory mechanisms of GPCR signaling. Agonist-induced desensitization usually depends on phosphorylation by a GPCR kinase (GRK) followed by receptor internalization in endocytic vesicles. We have shown that MC1R desensitization is mediated by two GRKs expressed in melanocytes and melanoma cells, GRK2 and GRK6. Here we show that in contrast with this dual specificity for desensitization, GRK6 but not GRK2 mediated MC1R internalization. Mutagenesis studies suggested that the targets of GRK6 are two residues located in the MC1R cytosolic C terminus, Thr-308 and Ser-316. A T308D/S316D mutant mimicking their phosphorylated state was constitutively desensitized and associated with endosomes, whereas a T308A/S316A mutant was resistant to desensitization and internalization. We studied the desensitization and internalization of three variant MC1R forms associated with red hair and increased skin cancer risk: R151C, R160W, and D294H. These variants showed a less efficient desensitization. Moreover, D294H was resistant to internalization, thus accounting for its abnormally high surface expression. Co-expression of variant and wild type MC1R modified its desensitization and internalization behavior. These data suggest that MC1R might be regulated by novel mechanisms including differential effects of GRKs and altered desensitization rates of certain allelic combinations.

Block of Kainate Receptor Desensitization Uncovers a Key Trafficking Checkpoint

A prominent feature of ionotropic glutamate receptors from the AMPA and kainate subtypes is their profound desensitization in response to glutamate-a process thought to protect the neuron from overexcitation. In AMPA receptors, it is well established that desensitization results from rearrangements of the interface formed between agonist-binding domains of adjacent subunits; however, it is unclear how this mechanism applies to kainate receptors. Here we show that stabilization of the binding domain dimer by the generation of intermolecular disulfide bonds apparently blocked desensitization of the kainate receptor GluR6. This result establishes a common desensitization mechanism in both AMPA and kainate receptors. Surprisingly, however, surface expression of these nondesensitizing mutants was drastically reduced and did not depend on channel activity. Therefore, in addition to its role at the synapse, we now propose an intracellular role for desensitization in controlling maturation and trafficking of glutamate receptors.

G Protein-coupled Receptor Kinase and β-Arrestin-mediated Desensitization of the Angiotensin II Type 1A Receptor Elucidated by Diacylglycerol Dynamics

Receptor desensitization progressively limits responsiveness of cells to chronically applied stimuli. Desensitization in the continuous presence of agonist has been difficult to study with available assay methods. Here, we used a fluorescence resonance energy transfer-based live cell assay for the second messenger diacylglycerol to measure desensitization of a model seven-transmembrane receptor, the Gq-coupled angiotensin II type 1(A) receptor, expressed in human embryonic kidney 293 cells. In response to angiotensin II, we observed a transient diacylglycerol response reflecting activation and complete desensitization of the receptor within 2-5 min. By utilizing a variety of approaches including graded tetracycline-inducible receptor expression, mutated receptors, and overexpression or short interfering RNA-mediated silencing of putative components of the cellular desensitization machinery, we conclude that the rate and extent of receptor desensitization are critically determined by the following: receptor concentration in the plasma membrane; the presence of phosphorylation sites on the carboxyl terminus of the receptor; kinase activity of G protein-coupled receptor kinase 2, but not of G protein-coupled receptor kinases 3, 5, or 6; and stoichiometric expression of beta-arrestin. The findings introduce the use of the biosensor diacylglycerol reporter as a powerful means for studying Gq-coupled receptor desensitization and document that, at the levels of receptor overexpression commonly used in such studies, the properties of the desensitization process are markedly perturbed and do not reflect normal cellular physiology.

In Search of Novel AMPA Potentiators

Glutamate is the major excitatory neurotransmitter in the brain. Amongst ionotropic receptors responding to glutamate, the AMPA subtype has been considered as essential for the fast excitatory neurotransmission in the central nervous system and the expression and maintenance of long-term potentiation. As glutamate is known to be involved in many neurological and psychiatric disorders, AMPA receptors seem to represent interesting targets to develop therapeutic drugs. Hence, the enhancement of AMPA signals is an approach currently investigated for the management of Alzheimer's disease, schizophrenia or mood disorders. In particular, many efforts are being conducted in the development of AMPA positive allosteric modulators ("potentiators"), which alter the rate of receptor desensitization. The major chemical families developed as AMPA potentiators are aniracetam derivatives, cyclothiazide derivatives and biarylpropylsulfonamides derivatives.

GABAAReceptor Function is Regulated by Lipid Bilayer Elasticity

Docosahexaenoic acid (DHA) and other polyunsaturated fatty acids (PUFAs) promote GABA(A) receptor [(3)H]-muscimol binding, and DHA increases the rate of GABA(A) receptor desensitization. Triton X-100, a structurally unrelated amphiphile, similarly promotes [(3)H]-muscimol binding. The mechanism(s) underlying these effects are poorly understood. DHA and Triton X-100, at concentrations that affect GABA(A) receptor function, increase the elasticity of lipid bilayers measured as decreased bilayer stiffness using gramicidin channels as molecular force transducers. We have previously shown that membrane protein function can be regulated by amphiphile-induced changes in bilayer elasticity and hypothesized that GABA(A) receptors could be similarly regulated. We therefore studied the effects of four structurally unrelated amphiphiles that decrease bilayer stiffness (Triton X-100, octyl-beta-glucoside, capsaicin, and DHA) on GABA(A) receptor function in mammalian cells. All the compounds promoted GABA(A) receptor [(3)H]-muscimol binding by increasing the binding capacity of high-affinity binding without affecting the associated equilibrium binding constant. A semiquantitative analysis found a similar quantitative relation between the effects on bilayer stiffness and [(3)H]-muscimol binding. Membrane cholesterol depletion, which also decreases bilayer stiffness, similarly promoted [(3)H]-muscimol binding. In whole-cell voltage-clamp experiments, Triton X-100, octyl-beta-glucoside, capsaicin, and DHA all reduced the peak amplitude of the GABA-induced currents and increased the rate of receptor desensitization. The effects of the amphiphiles did not correlate with the expected changes in monolayer spontaneous curvature. We conclude that GABA(A) receptor function is regulated by lipid bilayer elasticity. PUFAs may generally regulate membrane protein function by affecting the elasticity of the host lipid bilayer.

An Interaction Involving an Arginine Residue in the Cytoplasmic Domain of the 5-HT3A Receptor Contributes to Receptor Desensitization Mechanism

A large cytoplasmic domain accounts for approximately one-third of the entire protein of one superfamily of ligand-gated membrane ion channels, which includes nicotinic acetylcholine (nACh), gamma-aminobutyric acid type A (GABA(A)), serotonin type 3 (5-HT3), and glycine receptors. Desensitization is one functional feature shared by these receptors. Because most molecular studies of receptor desensitization have focused on the agonist binding and channel pore domains, relatively little is known about the role of the large cytoplasmic domain (LCD) in this process. To address this issue, we sequentially deleted segments of the LCD of the 5-HT3A receptor and examined the function of the mutant receptors. Deletion of a small segment that contains three amino acid residues (425-427) significantly slowed the desensitization kinetics of the 5-HT3A receptor. Both deletion and point mutation of arginine 427 altered desensitization kinetics in a manner similar to that of the (425-427) deletion without significantly changing the apparent agonist affinity. The extent of receptor desensitization was positively correlated with the polarity of the amino acid residue at 427: the desensitization accelerates with increasing polarity. Whereas the R427L mutation produced the slowest desensitization, it did not significantly alter single channel conductance of 5-HT3A receptor. Thus, the arginine 427 residue in the LCD contributes to 5-HT3A receptor desensitization, possibly through forming an electrostatic interaction with its neighboring residues. Because the polarity of the amino acid residue at 427 is highly conserved, such a desensitization mechanism may occur in other members of the Cys-loop family of ligand-gated ion channels.

Different Domains of the AMPA Receptor Direct Stargazin-mediated Trafficking and Stargazin-mediated Modulation of Kinetics

Stargazin is an accessory protein of AMPA receptors that enhances surface expression and also affects the biophysical properties of the receptor. AMPA receptor domains necessary for either of these two processes have not yet been identified. Here, we used confocal imaging and electrophysiology of heterologously expressed, fluorophore-tagged GluR1, GluR2, and stargazin to study surface expression and desensitization kinetics. Stargazin-mediated trafficking was sensitive to the nature of the AMPA receptor cytoplasmic domain. The insertion of YFP after residue 15 of the truncated cytoplasmic tail of GluR1i perturbed stargazin-mediated trafficking of the receptor but not its modulation of desensitization kinetics. This construct also failed to permit fluorescence resonance energy transfer (FRET) with stargazin in the endoplasmic reticulum (ER), whereas FRET between fluorophore-tagged stargazin and non-truncated AMPA receptors demonstrated a specific interaction between these proteins, both in the ER and the plasma membrane. Rather than encoding a specific binding site, the fluorophore-tagged C terminus may restrict access to one or more ER retention sites. Although perturbations of the C terminus impeded stargazin-mediated trafficking to the plasma membrane, the effects of stargazin on the biophysical properties of AMPA receptors (i.e. modulation of desensitization) remained intact. These data provide strong evidence that the AMPA receptor domains required for stargazin modulation of gating and trafficking are separable.

Interaction between cyclodextrin and neuronal membrane results in modulation of GABAA receptor conformational transitions

Cyclodextrins (CDs) are nanostructures widely applied in biotechnology and chemistry. Owing to partially hydrophobic character, CDs interact with biological membranes. While the mechanisms of CDs interactions with lipids were widely studied, their effects on proteins are less understood. In the present study we investigated the effects of beta cyclodextrin (betaCD) on GABA(A) receptor (GABA(A)R) gating. To reliably resolve the kinetics of conformational transitions, currents were elicited by ultrafast gamma-aminobutyric acid (GABA) applications to outside-out patches from rat cultured hippocampal neurons. betaCD increased the amplitude of responses to saturating GABA concentration ([GABA]) in a dose-dependent manner and this effect was accompanied by profound alterations in the current kinetics. Current deactivation was slowed down by betaCD but this effect was biphasic with a maximum at around 0.5 mM betaCD. While the fast deactivation time constant was monotonically slowed down within considered betaCD concentration range, the slow component first increased and then, at millimolar betaCD concentration, decreased. The rate and extent of desensitization was decreased by betaCD in a dose-dependent manner. The analysis of current responses to nonsaturating [GABA] indicated that betaCD affected the GABA(A)R agonist binding site by slowing down the unbinding rate. Modulation of GABA(A)R desensitization and binding showed different concentration-dependence suggesting different modualtory sites with higher affinity of the latter one. All the betaCD effects were fully reversible indicating that cholesterol uptake into betaCD was not the primary mechanism. We conclude that betaCD is a strong modulator of GABA(A)R conformational transitions.

Peripherally applied neuropeptide SF is equally algogenic in wild type and ASIC3−/− mice

RFa-related peptides play a significant role in the processing of pain in the CNS of mammals. Recently it has been found that, when applied subcutaneously, these peptides elicit a powerful algogenic effect. The question arises whether this peripheral effect can be connected with the ability of RFa-related peptides to decrease the rate of desensitization of acid sensing ionic channels (ASICs) expressed in primary sensory neurons. We have addressed this question by comparing the effects of neuropeptide SF (NPSF), mammalian RFa peptide, in ASIC3−/− and wild-type C57BL/6J mice. Knockout of ASIC3 gene results in the changes in some of the behavioral parameters. However, subcutaneous injections of the NPSF into the n.saphenous innervation area result in a clearly nociceptive behavior in both strains of mice. There is no significant difference in the total time of licking of injected paw in the ASIC3−/− (194 ± 22 s) and C57BL/6J (227 ± 25 s) animals. Thus peripheral algogenic effects of NPSF cannot be explained only in terms of their action on the ASIC3 channels and involves some other, still unidentified mechanism.

Effect of extracellular pH on recombinant α1β2γ2 and α1β2 GABA A receptors

Recently, we have reported that extracellular protons allosterically modulated neuronal GABA A receptors [Mozrzymas, J.W., Zarnowska, E.D., Pytel, M., Mercik, K., 2003a. Modulation of GABA(A) receptors by hydrogen ions reveals synaptic GABA transient and a crucial role of desensitiztion process. Journal of Neuroscience 23, 7981–7992]. However, GABA ARs in neurons are heterogeneous and the effect of hydrogen ions depends on the receptor subtype. In particular, γ2 subunit sets the receptor sensibility to several modulators including protons. However, the mechanisms whereby protons modulate γ2-containing and γ2-free GABA ARs have not been fully elucidated. To this end, current responses to ultrafast GABA applications were recorded for α1β2γ2 and α1β2 receptors at different pH values. For both receptor types, increase in pH induced a decrease in amplitudes of currents elicited by saturating [GABA] but this effect was stronger for α1β2 receptors. In the case of α1β2γ2 receptors, protons strongly affected the current time course due to a down regulation of binding and desensitization rates. This effect was qualitatively similar to that described in neurons. Protons strongly influenced the amplitude of α1β2 receptor-mediated currents but the effect on their kinetics was weak suggesting a predominant direct non-competitive inhibition with a minor allosteric modulation. In conclusion, we provide evidence that extracellular protons strongly affect GABA A receptors and that, depending on the presence of the γ2 subunit, the modulatory mechanisms show profound quantitative and qualitative differences.

Mutations of L293 in transmembrane two of the mouse 5‐hydroxytryptamine3A receptor alter gating and alcohol modulatory actions

1 The goal of this study was to determine whether mutations of L293 at the 15' position of TM2 in the 5-HT(3A) receptor alter macroscopic current kinetics, and if these changes could account for alterations in alcohol modulation. Receptor function was assessed in Xenopus oocytes under voltage-clamp and in HEK293 cells with whole-cell patch-clamp recording and rapid drug application. 2 Examination of responses of L293C and L293S receptors to agonist alone revealed enhanced activation, deactivation, and desensitization rates relative to the wild-type receptor. The L293G mutation produced marked slowing of deactivation and desensitization rates. Increased potency of 5-HT and increased efficacy of the partial agonist, DA, was also observed in these mutant receptors. 3 Ethanol and trichloroethanol (TCEt) enhancement of receptor function was reduced or eliminated in receptors containing L293 mutations to C, G, or S. The L293I mutant receptor retained ethanol and TCEt sensitivity. Ethanol and TCEt enhanced activation rate in the wild-type, but not the L293G and L293S receptors. No relationship was observed between any physicochemical property of the substituted amino acids and the change in alcohol potentiation of function. 4 The changes in receptor-channel properties in the mutant receptors support the idea that the L293 residue has important roles in channel gating. Our findings indicate that loss of allosteric modulation by alcohols is not related in any simple way to changes in channel kinetic properties brought about by L293 mutants. We did not observe any evidence that L293 is part of an alcohol binding site.

Experimental and Modeling Studies of Desensitization of P2X3 Receptors

The function of ATP-activated P2X3 receptors involved in pain sensation is modulated by desensitization, a phenomenon poorly understood. The present study used patch-clamp recording from cultured rat or mouse sensory neurons and kinetic modeling to clarify the properties of P2X3 receptor desensitization. Two types of desensitization were observed, a fast process (t1/2 = 50 ms; 10 microM ATP) following the inward current evoked by micromolar agonist concentrations, and a slow process (t1/2 = 35 s; 10 nM ATP) that inhibited receptors without activating them. We termed the latter high-affinity desensitization (HAD). Recovery from fast desensitization or HAD was slow and agonist-dependent. When comparing several agonists, there was analogous ranking order for agonist potency, rate of desensitization and HAD effectiveness, with 2-methylthioadenosine triphosphate the strongest and beta,gamma-methylene-ATP the weakest. HAD was less developed with recombinant (ATP IC50 = 390 nM) than native P2X3 receptors (IC50 = 2.3 nM). HAD could also be induced by nanomolar ATP when receptors seemed to be nondesensitized, indicating that resting receptors could express high-affinity binding sites. Desensitization properties were well accounted for by a cyclic model in which receptors could be desensitized from either open or closed states. Recovery was assumed to be a multistate process with distinct kinetics dependent on the agonist-dependent dissociation rate from desensitized receptors. Thus, the combination of agonist-specific mechanisms such as desensitization onset, HAD, and resensitization could shape responsiveness of sensory neurons to P2X3 receptor agonists. By using subthreshold concentrations of an HAD-potent agonist, it might be possible to generate sustained inhibition of P2X3 receptors for controlling chronic pain.

Dual Potentiating and Inhibitory Actions of a Benz[e]indene Neurosteroid Analog on Recombinant α1β2γ2 GABAA Receptors

Benz[e]indenes are tricyclic analogs of neuroactive steroids and can be modulators of GABA(A) receptor activity. We have examined the mechanisms of action of the benz[e]indene compound [3S-(3alpha,3aalpha,5abeta,7beta,9aalpha,9bbeta)]-dodecahydro-7-(2-hydroxyethyl)-3a-methyl-1H-benz[e]indene-3-carbonitrile (BI-2) using single-channel patch-clamp and whole-cell recordings from human embryonic kidney cells transfected with rat GABA(A) receptor alpha1, beta2, and gamma2L subunits. The data demonstrate that BI-2 is a positive modulator of GABA(A) receptor activity with a peak effect at 2 microM. The mechanism of modulation is similar but not identical to that of neuroactive steroids. Similar to steroids, BI-2 acts by prolonging the mean open time duration through an effect on the duration and prevalence of the longest open time component. However, in contrast to many steroids, BI-2 does not selectively reduce the channel closing rate. The potentiating action of BI-2 seems to be mediated through interactions with the classic neuroactive steroid binding site. Mutation to the membrane-spanning region in the alpha1 subunit Q242W and the double mutation alpha1N408A/Y411F, previously shown to abolish potentiation by neurosteroids, also diminish potentiation by BI-2. At higher concentrations (>5 microM), BI-2 inhibits receptor function by enhancing the apparent rate of desensitization. From single-channel recordings, we estimate that the entry rate into the inhibited or blocked state, k(+B), is 0.50 microM(-1) s(-1). Based on the kinetic mechanism of action, and the finding that this effect is blocked by the alpha1V256S mutation, we propose that BI-2 acts through an inhibitory site first postulated for the inhibitory neurosteroid pregnenolone sulfate.

Roles of Purinergic P2X Receptors as Pacemaking Channels and Modulators of Calcium-Mobilizing Pathway in Pituitary Gonadotrophs

Anterior pituitary cells release ATP and express several subtypes of purinergic P2 receptors, but their biophysical properties and roles in spontaneous and receptor-controlled electrical activity have not been characterized. Here we focused on extracellular ATP actions in gonadotrophs from embryonic, neonatal, and adult rats. In cells from all three age groups, the Ca2+-mobilizing agonist GnRH induced oscillatory, hyperpolarizing, nondesensitizing, and slow deactivating currents. In contrast, ATP induced nonoscillatory, depolarizing, slowly desensitizing, and rapidly deactivating current, indicating that these cells express cation-conducting P2X channels but not Ca2+-mobilizing P2Y receptors. The amplitudes of P2X current response and the rates of receptor desensitization were dependent on ATP concentration. The biophysical and pharmacological properties of P2X currents were consistent with the expression of P2X2 subtype of channels in these cells. ATP-induced rapid depolarization of gonadotrophs lead to initiation of firing in quiescent cells, an increase in the frequency of action potentials in spontaneously active cells, and a transient stimulation of LH release. ATP also influenced GnRH-induced current and membrane potential oscillations and LH release in an extracellular Ca2+-dependent manner. These inositol 1,4,5-triphosphate-dependent oscillations were facilitated, slowed, or stopped, depending of ATP concentration, the time of its application, and the level of Ca2+ content in intracellular stores. These results indicate that, in gonadotrophs, P2X receptors could operate as pacemaking channels and modulators of GnRH-controlled electrical activity and secretion.

Cloning and functional identification of a novel rat endothelin receptor type A isoform

Endothelins (ET) act via two receptors, ETAR and ETBR, to regulate variable cellular functions in numerous tissues. In anterior pituitary, the BQ123-sensitive ETAR are present in all secretory cell types and are involved in the control of calcium signaling and hormone secretion. However, in gonadotrophs but not in lactotrophs these receptors rapidly desensitize, raising the possibility of the existence of pituitary cell type-specific ETAR variants. Here, we report about the cloning and expression of a novel ETAR isoform from rat anterior pituitary, termed ETAR-C13. The cloned cDNA has an inserted fragment between the exons 6 and 7 of wild type ETAR, resulting in substitution of C-terminal 382S-426N sequence of wild type receptor with shorter 382A-399L sequence of spliced receptor. The putative domain of C-terminal sequence responsible for coupling to Gq/11 proteins was altered, and the predicted protein kinase C and casein kinase 2 phosphorylation sites were deleted in ETAR-C13. The mRNA transcripts for ETAR-C13 were identified in immortalized and cultured pituitary cells and pituitary tissue, but not in other tissues. The 125I-ET-1 binding at ETAR-C13 expressed in HEK293 cells was completely displaced by BQ123, the coupling to Gq/11 proteins was preserved but was shifted rightward, and the coupling to Gs proteins was significantly attenuated. Furthermore, the rate of ETAR-C13 desensitization and internalization was lower compared to wild type ETAR. These results indicate that the expression of ETAR-C13 in lactotrophs and somatotrophs probably accounts for the long-lasting signaling. Supported by the Intramural Research Program of the NICHD, NIH.