RESUMEN
Retinal sensitivity to a variety of artificial sweeteners was tested by monitoring changes in internal free calcium in isolated retinal neurons using Fluo3. Several ligands, including aspartame and saccharin elevated internal free calcium. The effects of these ligands were mediated by both ligand-gated membrane channels and G-protein coupled receptors. We explored the receptors responsible for this phenomenon. Surprisingly, mRNA for subunits of the sweet taste receptor dimer (T1R2 and T1R3) were found in retina. Interestingly, knockdown of T1R2 reduced the response to saccharin but not aspartame. But TRPV1 channel antagonists suppressed the responses to aspartame. The results indicate that artificial sweeteners can increase internal free calcium in the retinal neurons through multiple pathways. Furthermore, aspartame reduced the b-wave, but not the a-wave, of the electroretinogram, indicating disruption of communication between photoreceptors and second order neurons.
RESUMEN
The skate retina provides a native all-rod retina suited for investigating a single type of photoreceptor regarding its properties and signaling to second order cells. Using the aspartate-induced isolated A-wave of the skate eyecup electroretinogram (ERG), it has been shown that adaptation in rods remains Weber-Fechner-like over a 6-log unit increase in background light intensity. Zinc, which can block calcium channels, has been found in the rod synaptic terminal and the synaptic cleft. Histidine is a zinc chelator. Voltage signals from neurons post-synaptic to rods indicate that histidine increases the dark release of glutamate and increases the horizontal cell light response. In histidine, the A-wave response to various light intensities in the dark-adapted retina increased more than fifty percent, corresponding to the effect on horizontal cells. In the presence of background light, although histidine-treated rod light responses remained Weber-Fechner-like, their increment threshold was raised significantly. This indicates that endogenous zinc feedback serves to increase rod sensitivity in a light-adapted retina, despite a corresponding reduction of threshold sensitivity in the dark. We propose that the increase in A-wave amplitude is a result of the increased conductance at the synaptic terminal and that the A-wave can be used to monitor changes in rod transmitter release. Furthermore, endogenous zinc may also provide the benefit of reducing metabolic stress and the risk of glutamate toxicity in the dark.
Asunto(s)
Electrorretinografía , Retina , Adaptación a la Oscuridad , Luz , Células Fotorreceptoras , Células Fotorreceptoras de VertebradosRESUMEN
KEY POINTS: Starburst amacrine cells release GABA and ACh. This study explores the coordinated function of starburst-mediated cholinergic excitation and GABAergic inhibition to bistratified retinal ganglion cells, predominantly direction-selective ganglion cells (DSGCs). In rat retina, under our recording conditions, starbursts were found to provide the major excitatory drive to a sub-population of ganglion cells whose dendrites co-stratify with starburst dendrites (putative DSGCs). In mouse retina, recordings from genetically identified DSGCs at physiological temperatures reveal that ACh inputs dominate the response to small spot-high contrast light stimuli, with preferential addition of bipolar cell input shifting the balance towards glutamate for larger spot stimuli In addition, starbursts also appear to gate glutamatergic excitation to DSGCs by postsynaptic and possibly presynaptic inhibitory processes ABSTRACT: Starburst amacrine cells release both GABA and ACh, allowing them to simultaneously mediate inhibition and excitation. However, the precise pre- and postsynaptic targets for ACh and GABA remain under intense investigation. Most previous studies have focused on starburst-mediated postsynaptic GABAergic inhibition and its role in the formation of directional selectivity in ganglion cells. However, the significance of postsynaptic cholinergic excitation is only beginning to be appreciated. Here, we found that light-evoked responses measured in bi-stratified rat ganglion cells with dendrites that co-fasciculate with ON and OFF starburst dendrites (putative direction-selective ganglion cells, DSGCs) were abolished by the application of nicotinic receptor antagonists, suggesting ACh could act as the primary source of excitation. Recording from genetically labelled DSGCs in mouse retina at physiological temperatures revealed that cholinergic synaptic inputs dominated the excitation for high contrast stimuli only when the size of the stimulus was small. Canonical glutamatergic inputs mediated by bipolar cells were prominent when GABA/glycine receptors were blocked or when larger spot stimuli were utilized. In mouse DSGCs, bipolar cell excitation could also be unmasked through the activation of mGluR2,3 receptors, which we show suppresses starburst output, suggesting that GABA from starbursts serves to inhibit bipolar cell signals in DSGCs. Taken together, these results suggest that starbursts amplify excitatory signals traversing the retina, endowing DSGCs with the ability to encode fine spatial information without compromising their ability to encode direction.
Asunto(s)
Acetilcolina/farmacología , Células Amacrinas/fisiología , Ácido Glutámico/metabolismo , Células Ganglionares de la Retina/fisiología , Sinapsis/fisiología , Vías Visuales/fisiología , Células Amacrinas/citología , Células Amacrinas/efectos de los fármacos , Animales , Células Cultivadas , Agonistas Colinérgicos/farmacología , Ratones , Inhibición Neural , Estimulación Luminosa , Ratas , Ratas Sprague-Dawley , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/efectos de los fármacos , Sinapsis/efectos de los fármacos , Transmisión Sináptica , Vías Visuales/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Ionotropic receptors are tightly regulated by second messenger systems and are often present along with their metabotropic counterparts on a neuron's plasma membrane. This leads to the hypothesis that the two receptor subtypes can interact, and indeed this has been observed in excitatory glutamate and inhibitory GABA receptors. In both systems the metabotropic pathway augments the ionotropic receptor response. However, we have found that the metabotropic GABAB receptor can suppress the ionotropic GABAA receptor current, in both the in vitro mouse retina and in human amygdala membrane fractions. Expression of amygdala membrane microdomains in Xenopus oocytes by microtransplantation produced functional ionotropic and metabotropic GABA receptors. Most GABAA receptors had properties of α-subunit containing receptors, with ~5% having ρ-subunit properties. Only GABAA receptors with α-subunit-like properties were regulated by GABAB receptors. In mouse retinal ganglion cells, where only α-subunit-containing GABAA receptors are expressed, GABAB receptors suppressed GABAA receptor currents. This suppression was blocked by GABAB receptor antagonists, G-protein inhibitors, and GABAB receptor antibodies. Based on the kinetic differences between metabotropic and ionotropic receptors, their interaction would suppress repeated, rapid GABAergic inhibition.
Asunto(s)
Neuronas/fisiología , Receptores de GABA-A/fisiología , Receptores de GABA-B/fisiología , Células Ganglionares de la Retina/fisiología , Potenciales de Acción/fisiología , Animales , Femenino , Humanos , Masculino , Ratones , Técnicas de Placa-Clamp , XenopusRESUMEN
KEY POINTS: GABA is an inhibitory transmitter but can sometimes produce paradoxical excitatory effects through synaptic networks. We found a novel GABA-mediated excitation within a single retinal cell. It involves a chain of events from receptor stimulation to the sequential modulation of two associated channels, resulting in enhanced neuroexcitability. GABAB receptor activation selectively suppresses N-type calcium channels. The BK-type potassium channels are exclusively linked to the N-type calcium channel. Thus, stimulation of GABAB receptors suppresses an outward current, increasing the excitatory range of single neurons. ABSTRACT: GABAB receptors (GABAB Rs) suppress voltage-gated calcium channels and activate G-protein coupled potassium channels (GIRK and TREK channels), both mechanisms serving to inhibit neurons. In isolated rat retinal spiking neurons, GABAB Rs produce both actions but the net effect is to enhance excitatory signals. This is because GABAB Rs selectively suppress N-type calcium channels, which in turn are specifically linked to BK channels. Consequently, when GABAB Rs are stimulated there is a reduction in outward current, allowing neurons to extend their level of depolarization. Whereas many retinal neurons use L-type channels to stimulate vesicle fusion, the suppression of N-type channels augments dynamic range without affecting transmitter release.
Asunto(s)
Canales de Calcio/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Receptores de GABA-B/fisiología , Células Ganglionares de la Retina/fisiología , Animales , Femenino , Ratas Sprague-DawleyRESUMEN
One general categorization of retinal ganglion cells is to segregate them into tonically or phasically responding neurons, each conveying discrete aspects of the visual scene. Although best identified in the output signals of the retina, this distinction is initiated at the first synapse: between photoreceptors and the dendrites of bipolar cells. In this study we found that the output synapses of bipolar cells also contribute to separate these pathways. Both transient and sustained ganglion cells can produce maintained spike activity, but bipolar cell glutamate release exhibits a divergence that corresponds to the response characteristics of the ganglion cells. Comparing light intensity coding in the sustained and transient ON pathways revealed that they shared the intensity spectrum. The transient pathway had greater sensitivity but smaller dynamic range, and switched from intensity coding to event detection at light levels where sustained pathway sensitivity began to rise. The distinctive properties of the sustained pathway depended upon inhibition and shifted toward those of the transient pathway in the absence of inhibition. The transient system was comparatively unaffected by the loss of inhibition and this was due to the concomitant activation of perisynaptic NMDA receptors. Overall, the properties of bipolar cell dendritic and axon terminals both contribute to the formation of key aspects of the sustained/transient dichotomy normally associated with ganglion cells.
Asunto(s)
Glutamatos/metabolismo , Células Bipolares de la Retina/fisiología , Sinapsis/metabolismo , Transmisión Sináptica , Animales , Neurotransmisores/metabolismo , Neurotransmisores/farmacología , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/metabolismo , Potenciales Sinápticos , UrodelosRESUMEN
Glutamate release at bipolar to ganglion cell synapses activates NMDA and AMPA/kainic acid (KA) ionotropic glutamate receptors. Their relative strength determines the output signals of the retina. We found that this balance is tightly regulated by presynaptic inhibition that preferentially suppresses NMDA receptor (NMDAR) activation. In transient ON-OFF neurons, block of GABA and glycine feedback enhanced total NMDAR charge by 35-fold in the ON response and 9-fold in the OFF compared with a 1.7-fold enhancement of AMPA/KA receptors. Blocking only glycine receptors enhanced the NMDAR excitatory postsynaptic current 10-fold in the ON and 2-fold in the OFF pathway. Blocking GABA(A) or GABA(C) receptors (GABA(C)Rs or GABA(A)Rs) produced small changes in total NMDAR charge. When both GABA(A)Rs and GABA(C)Rs were blocked, the total NMDAR charge increased ninefold in the ON and fivefold in the OFF pathway. This exposed a strong GABA(C)R feedback to bipolar cells that was suppressed by serial amacrine cell synapses mediated by GABA(A)Rs. The results indicate that NMDAR currents are large but latent, held in check by dual GABA and glycine presynaptic inhibition. One example of this controlled NMDAR activation is the cross talk between ON and OFF pathways. Blocking the ON pathway increased NMDAR relative strength in the OFF pathway. Stimulus prolongation similarly increased the NMDAR relative strength in the OFF response. This NMDAR enhancement was produced by a diminution in GABA and glycine feedback. Thus the retinal network recruits NMDAR pathways through presynaptic disinhibition.
Asunto(s)
Potenciales Postsinápticos Excitadores , Receptores de N-Metil-D-Aspartato/metabolismo , Células Bipolares de la Retina/metabolismo , Células Ganglionares de la Retina/metabolismo , Células Amacrinas/metabolismo , Células Amacrinas/fisiología , Ambystoma , Animales , Retroalimentación Fisiológica , Agonistas del GABA/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Glicina/farmacología , Receptores de Glicina/antagonistas & inhibidores , Células Bipolares de la Retina/fisiología , Células Ganglionares de la Retina/fisiología , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Sinapsis/fisiología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Picrotoxin is a pore blocker that can differentiate ligand-gated inhibitory chloride channels. Even within one receptor type, such as the glycine receptor, picrotoxin block differs between subunits. The effect of subunit gating properties on block of the inhibitory glycine receptor (GlyR) was explored using heteromeric α subunit expression in voltage-clamped HEK293 cells. The α2 GlyR is more sensitive to picrotin block than the α1 GlyR, and this difference was used to explore whether mutations that interfered with gating of the α2 subunit would also interfere with picrotin block. Two mutations were used: one that decreased the glycine sensitivity of α2 by almost two log units and the other that was unresponsive to glycine. In both cases, the sensitivity to picrotin was essentially unaltered. The results indicated that α2 subunits can determine the picrotin sensitivity of α1α2-heteromeric receptors and that direct gating of the α2 subunit is not required for this picrotin inhibition.
Asunto(s)
Cloruros/metabolismo , Glicina/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Picrotoxina/análogos & derivados , Receptores de Glicina/antagonistas & inhibidores , Sustitución de Aminoácidos , Animales , Arginina/química , Canales de Cloruro/antagonistas & inhibidores , Dimerización , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Concentración 50 Inhibidora , Lisina/química , Mutación Missense , Técnicas de Placa-Clamp , Picrotoxina/farmacología , Mutación Puntual , Estructura Terciaria de Proteína , Ratas , Receptores de Glicina/química , Receptores de Glicina/genética , Receptores de Glicina/fisiología , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/fisiología , Sesterterpenos , TransfecciónRESUMEN
GABAergic amacrine cell feedback to bipolar cells in retina has been described, activating both GABA(A) and GABA(C) receptors. We explored whether metabotropic GABA(B) receptors also participate in this feedback pathway. CGP55845, a potent GABA(B) receptor antagonist, was employed to determine the endogenous role of these receptors. Ganglion cell EPSCs and IPSCs were monitored to measure the output of bipolar and amacrine cells. Using the tiger salamander slice preparation, we found that GABA(B) receptor pathways regulate bipolar cell release directly and indirectly. In the direct pathway, the GABA(B) receptor antagonist reduces EPSC amplitude, indicating that GABA(B) receptors cause enhanced glutamate release from bipolar cells to one set of ganglion cells. In the indirect pathway, the GABA(B) receptor antagonist reduces EPSC amplitude in another set of ganglion cells. The indirect pathway is only evident when GABA(A) receptors are inhibited, and is blocked by a glycine receptor antagonist. Thus, this second feedback pathway involves direct glycine feedback to the bipolar cell and this glycinergic amacrine cell is suppressed by GABAergic amacrine cells, through both GABA(A) and GABA(B) but not GABA(C) receptors. Overall, GABA(B) receptors do contribute to feedback regulation of bipolar cell transmitter release. However, unlike the ionotropic GABA receptor pathways, the metabotropic GABA receptor pathways act to enhance bipolar cell transmitter release. Furthermore, there are three discrete subsets of bipolar cell output regulated by GABA(B) receptor feedback (direct, indirect and null), implying three distinct, non-overlapping bipolar cell to ganglion cell circuits.
Asunto(s)
Ambystoma/fisiología , Retroalimentación Fisiológica/fisiología , Ácidos Fosfínicos/farmacología , Propanolaminas/farmacología , Receptores de GABA-B/fisiología , Células Bipolares de la Retina/fisiología , Animales , Antagonistas del GABA/farmacologíaRESUMEN
We found that caffeine is a structural analogue of strychnine and a competitive antagonist at ionotropic glycine receptors (GlyRs). Docking simulations indicate that caffeine and strychnine may bind to similar sites at the GlyR. The R131A GlyR mutation, which reduces strychnine antagonism without suppressing activation by glycine, also reduces caffeine antagonism. GlyR subtypes have differing caffeine sensitivity. Tested against the EC(50) of each GlyR subtype, the order of caffeine potency (IC(50)) is: alpha2beta (248 +/- 32 microm) alpha3beta (255 +/- 16 microm) > alpha4beta (517 +/- 50 microm) > alpha1beta(837 +/- 132 microm). However, because the alpha3beta GlyR is more than 3-fold less sensitive to glycine than any of the other GlyR subtypes, this receptor is most effectively blocked by caffeine. The glycine dose-response curves and the effects of caffeine indicate that amphibian retinal ganglion cells do not express a plethora of GlyR subtypes and are dominated by the alpha1beta GlyR. Comparing the effects of caffeine on glycinergic spontaneous and evoked IPSCs indicates that evoked release elevates the glycine concentration at some synapses whereas summation elicits evoked IPSCs at other synapses. Caffeine serves to identify the pharmacophore of strychnine and produces near-complete inhibition of glycine receptors at concentrations commonly employed to stimulate ryanodine receptors.
Asunto(s)
Cafeína/farmacología , Riñón/metabolismo , Receptores de Glicina/antagonistas & inhibidores , Receptores de Glicina/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Riñón/efectos de los fármacosRESUMEN
Glycine is the lone fast neurotransmitter for which a metabotropic pathway has not been identified. In retina, we found a strychnine-insensitive glycine response in bipolar and ganglion cells. This glycine response reduced high voltage-activated calcium current. It was G-protein mediated and protein kinase A dependent. The EC(50) of the metabotropic glycine response is 3 mum, an order of magnitude lower than the ionotropic glycine receptor in the same retina. The bipolar cell glutamatergic input to ganglion cells was suppressed by metabotropic glycine action. The synaptic output of about two-thirds of bipolar cells and calcium current in two-thirds of ganglion cells are sensitive to the action of glycine at metabotropic receptors, suggesting this signal regulates specific synaptic pathways in proximal retina. This study resolves the curious absence of a metabotropic glycine pathway in the nervous system and reveals that the major fast inhibitory neurotransmitters, GABA and glycine, both activate G-protein-coupled pathways as well.
Asunto(s)
Glicina/metabolismo , Inhibición Neural/fisiología , Receptores de Glicina/metabolismo , Células Ganglionares de la Retina/fisiología , Transmisión Sináptica/fisiología , Urodelos/fisiología , Animales , Células Cultivadas , Retina/citología , Retina/fisiologíaRESUMEN
Retinal rod photoreceptors are depolarized in darkness to approximately -40 mV, a state in which they maintain sustained glutamate release despite low levels of calcium channel activation. Blocking voltage-gated calcium channels or ryanodine receptors (RyRs) at the rod presynaptic terminal suppressed synaptic communication to bipolar cells. Spontaneous synaptic events were also inhibited when either of these pathways was blocked. This indicates that both calcium influx and calcium release from internal stores are required for the normal release of transmitter of the rod. RyR-independent release can be evoked by depolarization of a rod to a supraphysiological potential (-20 mV) that activates a large fraction of voltage-gated channels. However, this calcium channel-mediated release depletes rapidly if RyRs are blocked, indicating that RyRs support prolonged glutamate release. Thus, the rod synapse couples a small transmembrane calcium influx with a RyR-dependent amplification mechanism to support continuous vesicle release.
Asunto(s)
Señalización del Calcio/fisiología , Células Fotorreceptoras/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Transmisión Sináptica/fisiología , Ambystoma , Animales , Bloqueadores de los Canales de Calcio/farmacología , Electrofisiología , Técnicas In Vitro , Potenciales de la Membrana/fisiología , Rianodina/farmacología , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Sinapsis/efectos de los fármacos , Sinapsis/fisiologíaRESUMEN
In the dark-adapted salamander retina, spikes could be elicited from rods under normal physiological conditions. Spike activity was observed in rods during the recovery phase of the response to saturating light. These action potentials were calcium spikes, blocked by cadmium and L-type calcium channel blockers. In response to light stimuli that saturate the rod peak response, calcium action potentials occurred with a delay that depended on light intensity, with stronger light increasing spike latency. Therefore, these spikes encode rod visual information at light intensities beyond rod saturation. Postsynaptic currents of similar time course were observed in second and third order neurones. Since rods exposed to brighter light stimuli produced more delayed spike activity, these signals might contribute to negative afterimages.
Asunto(s)
Adaptación Ocular/fisiología , Postimagen/fisiología , Calcio/metabolismo , Luz , Células Fotorreceptoras Retinianas Bastones/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Adaptación Ocular/efectos de los fármacos , Ambystoma , Animales , Cadmio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Neuronas Aferentes/fisiología , Canales de Potasio Calcio-Activados/fisiología , Factores de TiempoRESUMEN
Large-conductance calcium-activated potassium (BK) channels are colocalized with calcium channels at sites of exocytosis at the presynaptic terminals throughout the nervous system. It is expected that their activation would provide negative feedback to transmitter release, but the opposite is sometimes observed. Attempts to resolve this apparent paradox based on alterations in action potential waveform have been ambiguous. In an alternative approach, we investigated the influence of this channel on neurotransmitter release in a nonspiking neuron, the salamander rod photoreceptors. Surprisingly, the BK channel facilitates calcium-mediated transmitter release from rods. The two presynaptic channels form a positive coupled loop. Calcium influx activates the BK channel current, leading to potassium efflux that increases the calcium current. The normal physiological voltage range of the rod is well matched to the dynamics of this positive loop. When the rod is further depolarized, then the hyperpolarizing BK channel current exceeds its facilitatory effect, causing truncation of transmitter release. Thus, the calcium channel-BK channel linkage performs two functions at the synapse: nonlinear potentiator and safety brake.
Asunto(s)
Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Neurotransmisores/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Sinapsis/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Canales de Calcio/metabolismo , Técnicas In Vitro , Canales de Potasio de Gran Conductancia Activados por el Calcio/antagonistas & inhibidores , Neurotransmisores/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/farmacología , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Sinapsis/efectos de los fármacos , UrodelosRESUMEN
Glycinergic and GABAergic inhibition are juxtaposed at one retinal synaptic layer yet likely perform different functions. These functions have usually been evaluated using receptor antagonists. In examining retinal glycine receptors, we were surprised to find that commonly used concentrations of GABA antagonists blocked significant fractions of the glycine current. In retinal amacrine and ganglion cells, the competitive GABAA receptor antagonists (bicuculline and SR95531) were also competitive GlyR antagonists. Picrotoxinin produced a noncompetitive inhibition of retinal GlyRs. [1,2,5,6-tetrahydropyridine-4-yl] methylphosphinic acid, the GABACR antagonist, did not inhibit glycine receptors. All three GABAA receptor antagonists were competitive inhibitors of homomeric alpha1 or alpha2 GlyRs expressed in human embryonic kidney cells (HEK293) cells. Interestingly, bicuculline was much more effective at alpha2 GlyRs and might be used to separate glycine receptor subtypes. Thus commonly used concentrations of GABA antagonists do not unambiguously differentiate GABA and glycine pathways. Picrotoxinin inhibition of GABAC receptors requires two amino acids in the second transmembrane region (TM2): 2' serine and 6' threonine. Although TM2 regions in GABA and glycine receptors are highly homologous, neither 2' serine nor 6' threonine is essential for picrotoxinin sensitivity in glycine receptors.
Asunto(s)
Antagonistas del GABA/farmacología , Neuronas/efectos de los fármacos , Receptores de Glicina/metabolismo , Retina/citología , Ambystoma , Secuencia de Aminoácidos , Animales , Células Cultivadas , Estimulantes del Sistema Nervioso Central/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Glicina/farmacología , Humanos , Mutagénesis/fisiología , Neuronas/metabolismo , Técnicas de Placa-Clamp/métodos , Ratas , Receptores de Glicina/química , Transfección/métodosRESUMEN
Glutamate receptor currents were examined in horizontal cells from cultured human retina using whole-cell recording procedures. Horizontal cells possess both AMPA and kainate receptors and both produce significant sustained currents. The kainate-induced current did not show significant desensitization and was not enhanced by concanavalin A. The sustained AMPA current was smaller than the kainate current, but the difference was almost entirely due to pronounced desensitization. The horizontal cell AMPA current was enhanced by cyclothiazide but not by PEPA, indicating the presence of the flip receptor variant. GYKI-52466 blocked the AMPA response (IC50 = 5 microM against 100 microM AMPA) but also blocked the kainate response (IC50 = 45 microM against 100 microM kainate). The diversity of glutamate receptors in human horizontal cells suggests that synaptic input to these neurons may be multiplexed through both kainate and AMPA channels.
Asunto(s)
Neuronas/metabolismo , Receptores AMPA/metabolismo , Receptores de GABA/metabolismo , Receptores de Glutamato/metabolismo , Receptores de Ácido Kaínico/metabolismo , Retina/metabolismo , Benzodiazepinas/farmacología , Clorotiazida/farmacología , Concanavalina A/farmacología , Técnicas de Cultivo , Conductividad Eléctrica , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Humanos , Ácido Kaínico/farmacología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/fisiología , Receptores de GABA/fisiología , Receptores de Glutamato/fisiología , Receptores de Ácido Kaínico/antagonistas & inhibidores , Receptores de Ácido Kaínico/fisiología , Retina/citología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Retinal ganglion cells exhibit fast and slow inhibitory synaptic glycine currents that can be selectively inhibited by strychnine and 5,7-dichlorokynurenic acid (DCKA), respectively. In this study we examined whether strychnine and DCKA selectivity correlated with the subunit composition of the glycine receptor. Homomeric alpha1, alpha2 or alpha2* glycine subunits were in vitro expressed in human embryonic kidney cells (HEK 293). In cells expressing the alpha1 subunit, responses to 200 microm glycine were blocked by 1 microm strychnine but not by 500 microm DCKA. In cells expressing the alpha2 subunit, both 1 microm strychnine and 500 microm DCKA were effective antagonists of 200 microm glycine. In cells expressing alpha2* subunits, which are much less glycine-sensitive, 10 mm glycine was inhibited by 500 microm DCKA but not by 1 microm strychnine. A single amino acid mutation in the alpha1 subunit (R196G), converted this subunit from DCKA-insensitive to DCKA-sensitive. In conclusion, the comparative effectiveness of strychnine and DCKA can be used to distinguish between the alpha1, alpha2 and alpha2* receptor responses. Furthermore, a single amino acid near the glycine receptor's putative agonist binding site may account for differences in DCKA sensitivity amongst the alpha subunits.
Asunto(s)
Antagonistas de Aminoácidos Excitadores/farmacología , Glicinérgicos/farmacología , Ácido Quinurénico/análogos & derivados , Ácido Quinurénico/farmacología , Receptores de Glicina/antagonistas & inhibidores , Estricnina/farmacología , Animales , Arginina , Línea Celular , Relación Dosis-Respuesta a Droga , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Glicina/administración & dosificación , Glicina/antagonistas & inhibidores , Glicina/farmacología , Glicinérgicos/administración & dosificación , Humanos , Ácido Quinurénico/administración & dosificación , Mutación , Concentración Osmolar , Ratas , Receptores de Glicina/genética , Estricnina/administración & dosificaciónRESUMEN
Although there are many glutamate receptors in the retina, 2-amino-4-phosphonobutyrate (L-AP4) is an agonist that acts selectively at metabotropic glutamate receptors (mGluR6) of ON bipolar cells. We explored the properties of agonists that activate this receptor. The effects of various glutamate analogs on the b-wave of the electroretinogram (ERG) were used as a measure of their activity. Conformational comparisons among agonists suggest that ligands in an extended conformation preferentially bind to the ON bipolar synaptic receptor. But this property is insufficient to explain the selectivity of mGluR6 because some inactive glutamate analogs could also match this extended conformation. Comparative molecular field analysis (CoMFA) was used to compare the electrostatic and steric potentials of agonists with their action at the ON bipolar synapse. Steric potentials beneath a plane defined by the three putative binding sites plays a key role in determining agonist activity. The CoMFA model was used to predict the activity of glutamate analogs and correlations between predicted and measured activity support the model.