Receptor Population Research Articles

Stochastic Induction of Long-Term Potentiation and Long-Term Depression.

Long-term depression (LTD) and long-term potentiation (LTP) of granule-Purkinje cell synapses are persistent synaptic alterations induced by high and low rises of the intracellular calcium ion concentration ([Ca2+]), respectively. The occurrence of LTD involves the activation of a positive feedback loop formed by protein kinase C, phospholipase A2, and the extracellular signal-regulated protein kinase pathway, and its expression comprises the reduction of the population of synaptic AMPA receptors. Recently, a stochastic computational model of these signalling processes demonstrated that, in single synapses, LTD is probabilistic and bistable. Here, we expanded this model to simulate LTP, which requires protein phosphatases and the increase in the population of synaptic AMPA receptors. Our results indicated that, in single synapses, while LTD is bistable, LTP is gradual. Ca2+ induced both processes stochastically. The magnitudes of the Ca2+ signals and the states of the signalling network regulated the likelihood of LTP and LTD and defined dynamic macroscopic Ca2+ thresholds for the synaptic modifications in populations of synapses according to an inverse Bienenstock, Cooper and Munro (BCM) rule or a sigmoidal function. In conclusion, our model presents a unifying mechanism that explains the macroscopic properties of LTP and LTD from their dynamics in single synapses.

Subsecond Regulation of Synaptically Released Dopamine by COMT in the Olfactory Bulb.

The efficacy of neurotransmission depends on multiple factors, including presynaptic vesicular release of transmitter, postsynaptic receptor populations and clearance/inactivation of the transmitter. In the olfactory bulb (OB), short axon cells (SACs) form an interglomerular circuit that uses GABA and dopamine (DA) as cotransmitters. Selective optical activation of SACs causes GABA and DA co-release, resulting in a fast, postsynaptic GABA inhibitory response and a slower G-protein-coupled DA rebound excitation. In most systems, vesicular release of DA is cleared by the dopamine transporter (DAT). However, in the OB, high levels of specific DA metabolites suggest that enzymatic catalysis by catechol-O-methyl-transferase (COMT) predominates over DAT re-uptake. To assess this possibility we measured the amount of the DA breakdown enzyme, COMT, present in the OB. Compared with the striatum, the brain structure richest in DA terminals, the OB contains 50% more COMT per unit of tissue. Furthermore, the OB has dramatically less DAT compared with striatum, supporting the idea that COMT enzymatic breakdown, rather than DAT recycling, is the predominant mechanism for DA clearance. To functionally assess COMT inactivation of vesicular release of DA we used fast-scan cyclic voltammetry and pharmacological blockade of COMT. In mice expressing ChR2 in tyrosine hydroxylase-containing neurons, optical activation of SACs evoked robust DA release in the glomerular layer. The COMT inhibitor, tolcapone, increased the DA signal ∼2-fold, whereas the DAT inhibitor GBR12909 had no effect. Together, these data indicate that the OB preferentially employs COMT enzymatic inactivation of vesicular release of DA. In the olfactory bulb (OB), odors are encoded by glomerular activation patterns. Dopaminergic short axon neurons (SACs) form an extensive network of lateral connections that mediate cross talk among glomeruli, releasing GABA and DA onto sensory nerve terminals and postsynaptic neurons. DA neurons are ∼10-fold more numerous in OB than in ventral tegmental areas that innervate the striatum. We show that OB has abundant expression of the DA catalytic enzyme catechol-O-methyl-transferase (COMT), but negligible expression of the dopamine transporter. Using optogenetics and fast-scan cyclic voltammetry, we show that inhibition of COMT increases DA signals ∼2-fold. Thus, in contrast to the striatum, which has the brain's highest proportion of DAergic synapses, the DA catalytic pathway involving COMT predominates over re-uptake in OB.

Interaction of Trypanosoma evansi with the plasminogen-plasmin system

Trypanosoma evansi is a widely-distributed haemoflagellated parasite of veterinary importance that infects a variety of mammals including horses, mules, camels, buffalos, cattle and deer. It is the causal agent of a trypanosomiasis known as Surra which produces epidemics of great economic importance in Africa, Asia and South America. The main pathology includes an enlarged spleen with hypertrophy of lymphoid follicles, congested lungs, neuronal degeneration and meningoencephalitis, where migration of the parasites from the blood to the tissues is essential. Most cells, including pathogenic cells, use diverse strategies for tissue invasion, such as the expression of surface receptors to bind plasminogen or plasmin. In this work, we show that T. evansi is able to bind plasminogen and plasmin on its surface. The analysis of this binding revealed a high affinity dissociation constant (Kd of 0.080±0.009μM) and 1×105 plasminogen binding sites per cell. Also a second population of receptors with a Kd of 0.255±0.070μM and 3.2×104 plasminogen binding sites per cell was determined. Several proteins with molecular masses between ∼18 and ∼70kDa are responsible for this binding. This parasite-plasminogen interaction may be important in the establishment of the infection in the vertebrate host, where the physiological concentration of available plasminogen is around 2μM.

Soil Cleanup Goal for Dioxin Using Probabilistic Risk Assessment Techniques

ABSTRACTState regulators in Florida recently approved a first-of-its-kind probabilistic risk assessment (PRA) for determining an alternative residential Soil Cleanup Target Level (SCTL) for dioxin (32 ng/kg TEQ). The default residential SCTL (7 ng/kg TEQ) is based on a single, deterministic calculation with numerous conservative assumptions, resulting in an overly conservative value far beyond the regulatory mandate (i.e., 10−6 increase in cancer risk). Conversely, this PRA used a Monte Carlo simulation to estimate risk for all members of a large population using a combination of scientific data and professional judgment, with final details developed during negotiations with regulators. The simulation parameters were defined probabilistically and reflect the ranges of values for the following exposure variables: body weight, exposure duration, exposure frequency, fraction from contaminated source, soil ingestion rate, and relative bioavailability. Other variable and uncertain parameters were treated deterministically per direction from the regulators. The state also required that a pre-supposed high-risk subpopulation be analyzed separate from the full receptor population. Despite the conservativeness of the alternative SCTL, this PRA represents a significant step toward more realistic estimates of human health risks caused by environmental contaminant exposure.

Detection of transient synchrony across oscillating receptors by the central electrosensory system of mormyrid fish.

Recently, we reported evidence for a novel mechanism of peripheral sensory coding based on oscillatory synchrony. Spontaneously oscillating electroreceptors in weakly electric fish (Mormyridae) respond to electrosensory stimuli with a phase reset that results in transient synchrony across the receptor population (Baker et al., 2015). Here, we asked whether the central electrosensory system actually detects the occurrence of synchronous oscillations among receptors. We found that electrosensory stimulation elicited evoked potentials in the midbrain exterolateral nucleus at a short latency following receptor synchronization. Frequency tuning in the midbrain resembled peripheral frequency tuning, which matches the intrinsic oscillation frequencies of the receptors. These frequencies are lower than those in individual conspecific signals, and instead match those found in collective signals produced by groups of conspecifics. Our results provide further support for a novel mechanism for sensory coding based on the detection of oscillatory synchrony among peripheral receptors.

Bacterial Chemoreceptor Dynamics: Helical Stability in the Cytoplasmic Domain Varies with Functional Segment and Adaptational Modification

Dynamics are thought to be important features of structure and signaling in the cytoplasmic domain of bacterial chemoreceptors. However, little is known about which structural features are dynamic. For this largely helical domain, comprising a four-helix bundle and an extended four-helix coiled coil, functionally important structural dynamics likely involves helical mobility and stability. To investigate, we used continuous wave EPR spectroscopy and site-specific spin labels that directly probed, in essentially physiological conditions, the mobility of helical backbones in the cytoplasmic domain of intact chemoreceptor Tar homodimers inserted into lipid bilayers of Nanodiscs. We observed differences among functional regions, between companion helices in helical hairpins of the coiled coil and between receptor conformational states generated by adaptational modification. Increased adaptational modification decreased helical dynamics while preserving dynamics differences among functional regions and between companion helices. In contrast, receptor ligand occupancy did not have a discernable effect on dynamics to which our approach was sensitive, implying that the two sensory inputs alter different chemoreceptor features. Spectral fitting indicated that differences in helical dynamics we observed for ensemble spin-label mobility reflected differences in proportions of a minority receptor population in which the otherwise helical backbone was essentially disordered. We suggest that our measurements provided site-specific snapshots of equilibria between a majority state of well-ordered helix and a minority state of locally disordered polypeptide backbone. Thus, the proportion of polypeptide chain that is locally and presumably transiently disordered is a structural feature of cytoplasmic domain dynamics that varies with functional region and modification-induced signaling state.

In silico Exploration of the Conformational Universe of GPCRs.

The structural plasticity of G protein coupled receptors (GPCRs) leads to a conformational universe going from inactive to active receptor states with several intermediate states. Many of them have not been captured yet and their role for GPCR activation is not well understood. The study of this conformational space and the transition dynamics between different receptor populations is a major challenge in molecular biophysics. The rational design of effector molecules that target such receptor populations allows fine-tuning receptor signalling with higher specificity to produce drugs with safer therapeutic profiles. In this minireview, we outline highly conserved receptor regions which are considered determinant for the establishment of distinct receptor states. We then discuss in-silico approaches such as dimensionality reduction methods and Markov State Models to explore the GPCR conformational universe and exploit the obtained conformations through structure-based drug design.

Nuclear grade and ER/PR expression level on residual tumor after preoperative chemotherapy to predict late recurrence in estrogen receptor-positive breast cancer.

e12049Background: It is not known which population of estrogen receptor (ER)-positive/HER2-negative breast cancer patients should continue hormonal therapy beyond 5 years to overcome late recurrenc...

Limit theorems for empirical Rényi entropy and divergence with applications to molecular diversity analysis

Quantitative methods for studying biodiversity have been traditionally rooted in the classical theory of finite frequency tables analysis. However, with the help of modern experimental tools, like high-throughput sequencing, we now begin to unlock the outstanding diversity of genomic data in plants and animals reflective of the long evolutionary history of our planet. This molecular data often defies the classical frequency/contingency tables assumptions and seems to require sparse tables with very large number of categories and highly unbalanced cell counts, e.g., following heavy-tailed distributions (for instance, power laws). Motivated by the molecular diversity studies, we propose here a frequency-based framework for biodiversity analysis in the asymptotic regime where the number of categories grows with sample size (an infinite contingency table). Our approach is rooted in information theory and based on the Gaussian limit results for the effective number of species (the Hill numbers) and the empirical Renyi entropy and divergence. We argue that when applied to molecular biodiversity analysis, our methods can properly account for the complicated data frequency patterns on one hand and the practical sample size limitations on the other. We illustrate this principle with two specific RNA sequencing examples: a comparative study of T-cell receptor populations and a validation of some preselected molecular hepatocellular carcinoma (HCC) markers.

Synthesis and evaluation of 18F-labeled 5-HT2A receptor agonists as PET ligands

IntroductionThe serotonin 2A receptor (5-HT2AR) is the most abundant excitatory 5-HT receptor in the human brain and implicated in various brain disorders such as schizophrenia, depression, and Alzheimer's disease. Positron emission tomography (PET) can be used to image specific proteins and processes in the human brain and several 5-HT2AR PET antagonist radioligands are available. In contrast to an antagonist radioligand, an agonist radioligand should be able to image the population of functional receptors, i.e., those capable of inducing neuroreceptor signaling. Recently, we successfully developed and validated the first 5-HT2AR agonist PET tracer, [11C]Cimbi-36, for neuroimaging in humans and herein disclose some of our efforts to develop an 18F-labeled 5-HT2AR agonist PET-ligand. Methods and resultsThree fluorine containing derivatives of Cimbi-36 were synthesized and found to be potent 5-HT2A agonists. 18F-labeling of the appropriate precursors was performed using [18F]FETos, typically yielding 0.2–2.0GBq and specific activities of 40–120GBq/μmol. PET studies in Danish landrace pigs revealed that [18F]1 displayed brain uptake in 5-HT2AR rich regions. However, high uptake in bone was also observed. No blocking effect was detected during a competition experiment with a 5-HT2AR selective antagonist. [18F]2 and [18F]3 showed very low brain uptake. ConclusionNone of the investigated 18F-labeled Cimbi-36 derivatives [18F]1, [18F]2 and [18F]3 show suitable tracer characteristics for in vivo PET neuroimaging of the 5-HT2AR. Although for [18F]1 there was reasonable brain uptake, we suggest that a large proportion radioactivity in the brain was due to radiometabolites, which would explain why it could not be displaced by a 5-HT2AR antagonist.

A meta-analysis of the relationship between brain dopamine receptors and obesity: a matter of changes in behavior rather than food addiction?

Addiction to a wide range of substances of abuse has been suggested to reflect a ‘Reward Deficiency Syndrome'. That is, drugs are said to stimulate the reward mechanisms so intensely that, to compensate, the population of dopamine D2 receptors (DD2R) declines. The result is that an increased intake is necessary to experience the same degree of reward. Without an additional intake, cravings and withdrawal symptoms result. A suggestion is that food addiction, in a similar manner to drugs of abuse, decrease DD2R. The role of DD2R in obesity was therefore examined by examining the association between body mass index (BMI) and the Taq1A polymorphism, as the A1 allele is associated with a 30–40% lower number of DD2R, and is a risk factor for drug addiction. If a lower density of DD2R is indicative of physical addiction, it was argued that if food addiction occurs, those with the A1 allele should have a higher BMI. A systematic review found 33 studies that compared the BMI of those who did and did not have the A1 allele. A meta-analysis of the studies compared those with (A1/A1 and A1/A2) or without (A2/A2) the A1 allele; no difference in BMI was found (standardized mean difference 0.004 (s.e. 0.021), variance 0.000, Z=0.196, P<0.845). It was concluded that there was no support for a reward deficiency theory of food addiction. In contrast, there are several reports that those with the A1 allele are less able to benefit from an intervention that aimed to reduce weight, possibly a reflection of increased impulsivity.

P.2.004 Nonsedative doses of a GABA-B positive allosteric modulator normalise VTA neuroadaptations and self-administration induced by drugs of abuse in mice

We tested novel positive allosteric modulators (PAMs) of the γ-aminobutyric acid receptor B (GABAB), ADX71943 and ADX71441in the monosodium iodoacetate model of chronic osteoarthritis pain in rats with the objective to delineate the role of peripheral versus central GABAB receptor populations in modulation of chronic pain. Anesthetized Sprague-Dawley rats received an injection of monosodium iodoacetate into the knee and were tested for hyperalgesia starting post-MIA day 14. Effects of compounds on ipsilateral joint compression threshold were evaluated on post-MIA day 14 (after acute treatment), as well as after repeated, daily treatment on days 21 and 28 (ADX71943 only) and were compared to those of celecoxib (30 mg/kg, p.o.). The PAMs were also tested in the rat rotarod test for potential muscle-relaxant effects. Acutely, ADX71943 (1–30 mg/kg, p.o.), the peripherally restricted PAM, resulted in similar increases in pain threshold across the doses on day 14, while showing reduced efficacy on day 21 and no efficacy on day 28. A clear reduction in the efficacy of celecoxib across testing was also noted in this experiment. Acutely ADX71441 (0.3–15 mg/kg, p.o.), the central-peripheral PAM, resulted in over 2-fold increases in pain threshold at 15 mg/kg (but not at lower doses) on day 14, while causing more modest effects on day 21. Celecoxib increased pain threshold after both acute and daily treatment, showing overall similar efficacy. Thus, early, presumably more inflammatory phase of osteoarthritis pain in more sensitive to GABAB PAMs with peripherally restricted profile, while later, presumably more neuropathic phase is more sensitive to PAMs with central-peripheral profile.

Abstract P5-12-12: Hormone receptor expression level and nuclear grade associated with late recurrence in estrogen receptor-positive breast cancer patients

Abstract Background: It is not known which population of estrogen receptor (ER)-positive breast cancer patients should continue endocrine treatment beyond 5 years to overcome late recurrences. The aim of this study was to examine a combination of nuclear grade (NG) and expression level of ER and progesterone receptor (PR) to predict late recurrences. Methods: We assessed retrospectively 1677 consecutive ER-positive/HER2-negative patients who underwent surgical resection between 2004 and 2009. Patients with T2 or larger tumor and/or node-positive received pre- or postoperative chemotherapy following the international consensus panel from the St Gallen Conference, 2003. All patients had received adjuvant endocrine treatment. NG, ER and PR statuses were determined by immunohistochemistory on surgical specimen. We classified the patients into 3 groups as follows; ER-high (+++ or Allred score 7, 8)/PR-high (++ to +++ or 5-8) (n=212), ER-high / PR-low (- to + or 3-6) (n=208), and ER-low (+ to ++ or 3-6) / PR-any (n=255). We compared distant disease-free survival (DDFS) in each cohort based on the NG (1:low, and 2 or 3: high). Results: A median follow-up period was 77.0 months. Four hundred sixty seven patients (27.8%) received neoadjuvant chemotherapy, 208 patients (12.4%) received adjuvant chemotherapy, and 1002 patients (59.8%) did not received chemotherapy. Of the 467 patients with neoadjuvant chemotherapy, 65 patients (13.9%) had developed distant metastasis during study period (before 5 years in 51 (11.0%); and after 5 years in 14 (3.0%)). NG-low had significantly higher risk of late recurrence after 5 years than that of NG-high (p=0.005). According to hormonal receptor expression levels, in patients with NG-low, ER-low/PR-any had significantly higher overall DDFS rate than ER-high/PR-low (p=0.016). A similar trend was found before 5 years (p=0.077). However, ER-high/PR-high turned to have significantly high risk of recurrence after 5 years compared to ER-low/PR-any (p=0.024). Of the 208 patients with adjuvant chemotherapy, 16 patients (7.6%) had developed distant metastasis during study period (before 5 years in 5 (2.4%), and after 5 years in 11 (5.2%)). there was no association between a risk of recurrence and hormone receptor statuses at any study period. In the patients did not received chemotherapy, ER-high/PR-high had a trend of higher DDFS rate than others before 5 years (p=0.067). Of the 1002 patients without chemotherapy, only 36 patients (3.6%) had developed distant metastasis during study period (before 5 years in 27 (2.7%), and after 5 years in 9 (0.9%)). There was no difference of late recurrence after 5 years among the patients regardless of ER and PR expression level and NG with only low recurrence rate (0.9%). Conclusions: Our results demonstrated that, in ER-positive/HER2-negative patients who underwent neoadjuvant chemotherapy, NG-low/ER-high/PR-high should receive extend hormonal treatment over 5 years because of the high risk of late recurrence but NG-high/ER-high might not need. Furthermore, patients with T1 and node-negative may not need extend hormonal treatment because of the extremely low risk of late recurrence regardless of NG and hormone receptor statuses. Citation Format: Iwase M, Hayashi N, Yoshida A, Kajiura Y, Takahashi Y, Takei J, Suzuki K, Nakamura S, Yamauchi H. Hormone receptor expression level and nuclear grade associated with late recurrence in estrogen receptor-positive breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-12-12.

Probing Binding Interactions of Agonists with the α6β2 Nicotinic Acetylcholine Receptor

The nicotinic acetylcholine receptor (nAChR) is a ligand gated ion channel and a member of the Cys-loop family, which also contains receptors for serotonin, glycine, and GABA. There are twelve known neuronal subunits which pentamerize in different combinations to form various subtypes, each with a unique function, pharmacology and distribution in the brain. The α6β2-containing subtypes are found mainly in dopaminergic neurons and are thus important targets in the study of Parkinson's disease and addiction. Advanced knowledge of the binding site, which sits at the interface of α6 and β2, could lead toward design of agonists that specifically target this subtype. The α6L9'sβ2_(LFM/AAQA)L9's (α6β2‡) construct enabled heterologous expression and activation of a pure and stoichiometrically controlled population of α6β2 receptors in X. laevis oocytes. Currents were high enough in the α6β2‡ system to tolerate nonsense suppression-based non-canonical amino acid mutagenesis, which allowed for structure function studies between the receptor and several agonists. Initial structure-function studies probed for a cation-π interaction between agonists and the indole side chain of α6 TrpB. This is accomplished using an analog-tryptophan series whereby the negative electrostatic potential on the surface the indole ring was incrementally decreased via electron-withdrawing substituents such as fluorine and measuring the effect that has on binding. Results show that ACh makes a cation-π interaction, but surprisingly nicotine and TC-299423 do not. However, the latter two agonists do make a hydrogen bond interaction with the carbonyl of TrpB. This conclusion was made by substituting the adjacent amino acid with an alpha-hydroxy acid and measuring the corresponding shift in binding due to that amide-to-ester backbone mutation. The result was further quantified for nicotine in a double-mutant cycle analysis utilizing N’-methylnicotinium, which showed the interaction to have a coupling energy of ∼2 kcal/mol.

Orexinergic system in the locus coeruleus modulates the CO2 ventilatory response.

The orexins are hypothalamic neuropeptides involved in an array of functions such as regulation of sleep/wake states and chemoreception to CO2/pH. The locus coeruleus (LC) is a chemosensitive site and expresses an extensive population of orexin receptor 1 (OX1R). We tested the hypothesis that OX1Rs located in the LC participate in the ventilatory response to hypercapnia in a vigilance state and diurnal cycle-dependent manner. For this, we performed unilateral injections of SB-334867 (OX1R antagonist, 5mM) into the LC of male Wistar rats and evaluated the ventilatory response to 7% CO2 during wakefulness and sleep in the dark and light phases of the diurnal cycle. Hypercapnia induced an increase in ventilation (V E) in all groups compared to normocapnic values. However, during the dark phase, but not in the light phase, SB-334867 injection promoted an attenuation of the hypercapnic chemoreflex during wakefulness (V E: vehicle, 1502.6 ± 100mLkg(-1)min(-1) vs SB-334867, 1200.3 ± 70.0mLkg(-1)min(-1)) but not during sleep (V E: vehicle, 1383.0 ± 113.9 vs SB-334687, 1287.6 ± 92.1mLkg(-1)min(-1)), due to changes in tidal volume (V T). We suggest that projections of orexin-containing neurons to the LC contribute, via OX1Rs, to the hypercapnic chemoreflex during wakefulness in the dark phase.

Blast from the past! Phrenic motor memory of antecedent episodic hypercapnia is serotonin dependent: relevance to respiratory rehabilitation and sleep-disordered breathing?

Blast from the past! Phrenic motor memory of antecedent episodic hypercapnia is serotonin dependent: relevance to respiratory rehabilitation and sleep-disordered breathing?

ATP-Evoked Intracellular Ca²⁺ Signaling of Different Supporting Cells in the Hearing Mouse Hemicochlea.

Hearing and its protection is regulated by ATP-evoked Ca(2+) signaling in the supporting cells of the organ of Corti, however, the unique anatomy of the cochlea hampers observing these mechanisms. For the first time, we have performed functional ratiometric Ca(2+) imaging (fura-2) in three different supporting cell types in the hemicochlea preparation of hearing mice to measure purinergic receptor-mediated Ca(2+) signaling in pillar, Deiters' and Hensen's cells. Their resting [Ca(2+)]i was determined and compared in the same type of preparation. ATP evoked reversible, repeatable and dose-dependent Ca(2+) transients in all three cell types, showing desensitization. Inhibiting the Ca(2+) signaling of the ionotropic P2X (omission of extracellular Ca(2+)) and metabotropic P2Y purinergic receptors (depletion of intracellular Ca(2+) stores) revealed the involvement of both receptor types. Detection of P2X2,3,4,6,7 and P2Y1,2,6,12,14 receptor mRNAs by RT-PCR supported this finding and antagonism by PPADS suggested different functional purinergic receptor population in pillar versus Deiters' and Hensen's cells. The sum of the extra- and intracellular Ca(2+)-dependent components of the response was about equal with the control ATP response (linear additivity) in pillar cells, and showed supralinearity in Deiters' and Hensen's cells. Calcium-induced calcium release might explain this synergistic interaction. The more pronounced Ca(2+) leak from the endoplasmic reticulum in Deiters' and Hensen's cells, unmasked by cyclopiazonic acid, may also suggests the higher activity of the internal stores in Ca(2+) signaling in these cells. Differences in Ca(2+) homeostasis and ATP-induced Ca(2+) signaling might reflect the distinct roles these cells play in cochlear function and pathophysiology.

High and low GABA sensitivity α4β2δ GABAA receptors are expressed in Xenopus laevis oocytes with divergent stoichiometries

GABAA receptors that contain the α4 and δ subunits are thought to be located extrasynaptically, mediating tonic currents elicited by low concentrations of GABA. These α4βδ receptors are modulated by neurosteroids and certain anesthetics, identifying them as important drug targets in research. However, pharmacological studies on these receptors have often yielded variable results, possibly due to the expression of receptors in different stoichiometries or arrangements.In this study, we injected different ratios of α4, β2 and δ cRNA into Xenopus oocytes and measured the sensitivity to GABA and DS2 activation of the resulting receptor populations. By creating a matrix of RNA injection ratios from stock RNA concentrations, we were able to compare the changes in pharmacology between injection ratios where the ratio of only one subunit was altered.We identified two distinct populations of receptors, the first with an EC50 value of approximately 100nM to GABA, a low Hill slope of approximately 0.3 and substantial direct activation by DS2. The second population had an EC50 value of approximately 1μM to GABA, a steeper Hill slope of 1 and little direct activation, but substantial potentiation, by DS2. The second population was formed with high α4 ratios and low β2 ratios, but altering the ratio of δ subunit injected had little effect. We propose that receptors with high sensitivity to GABA and direct activation by DS2 are the result of a greater number of β2 subunits being incorporated into the receptor.

Differential regulation of synaptic and extrasynaptic α4 GABA(A) receptor populations by protein kinase A and protein kinase C in cultured cortical neurons

The GABAA α4 subunit exists in two distinct populations of GABAA receptors. Synaptic GABAA α4 receptors are localized at the synapse and mediate phasic inhibitory neurotransmission, while extrasynaptic GABAA receptors are located outside of the synapse and mediate tonic inhibitory transmission. These receptors have distinct pharmacological and biophysical properties that contribute to interest in how these different subtypes are regulated under physiological and pathological states. We utilized subcellular fractionation procedures to separate these populations of receptors in order to investigate their regulation by protein kinases in cortical cultured neurons. Protein kinase A (PKA) activation decreases synaptic α4 expression while protein kinase C (PKC) activation increases α4 subunit expression, and these effects are associated with increased β3 S408/409 or γ2 S327 phosphorylation respectively. In contrast, PKA activation increases extrasynaptic α4 and δ subunit expression, while PKC activation has no effect. Our findings suggest synaptic and extrasynaptic GABAA α4 subunit expression can be modulated by PKA to inform the development of more specific therapeutics for neurological diseases that involve deficits in GABAergic transmission.

Differential regulation of synaptic and extrasynaptic α4 GABA(A) receptor populations by protein kinase A and protein kinase C in cultured cortical neurons

Differential regulation of synaptic and extrasynaptic α4 GABA(A) receptor populations by protein kinase A and protein kinase C in cultured cortical neurons