RESUMEN
In vertebrate retina, light responses generated by the rod photoreceptors are transmitted to the second-order neurons, the ON-bipolar cells (ON-BC), and this communication is indispensible for vision in dim light. In ON-BCs, synaptic transmission is initiated by the metabotropic glutamate receptor, mGluR6, that signals via the G-protein Go to control opening of the effector ion channel, TRPM1. A key role in this process belongs to the GTPase Activating Protein (GAP) complex that catalyzes Go inactivation upon light-induced suppression of glutamate release in rod photoreceptors, thereby driving ON-BC depolarization to changes in synaptic input. The GAP complex has a striking molecular complexity. It contains two Regulator of G-protein Signaling (RGS) proteins RGS7 and RGS11 that directly act on Go and two adaptor subunits: RGS Anchor Protein (R9AP) and the orphan receptor, GPR179. Here we examined the organizational principles of the GAP complex in ON-BCs. Biochemical experiments revealed that RGS7 binds to a conserved site in GPR179 and that RGS11 in vivo forms a complex only with R9AP. R9AP and GPR179 are further integrated via direct protein-protein interactions involving their cytoplasmic domains. Elimination of GPR179 prevents postsynaptic accumulation of R9AP. Furthermore, concurrent knock-out of both R9AP and RGS7 does not reconfigure the GAP complex and completely abolishes synaptic transmission, resulting in a novel mouse model of night blindness. Based on these results, we propose a model of hierarchical assembly and function of the GAP complex that supports ON-BCs visual signaling.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteínas RGS/metabolismo , Retina/citología , Células Bipolares de la Retina/fisiología , Oxidorreductasas de Alcohol , Animales , Cloruro de Cadmio/farmacología , Proteínas Co-Represoras , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Luz , Sustancias Macromoleculares/metabolismo , Masculino , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Modelos Moleculares , Fosfoproteínas/metabolismo , Proteínas RGS/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
The time course of signaling via heterotrimeric G proteins is controlled through their activation by G-protein coupled receptors and deactivation through the action of GTPase accelerating proteins (GAPs). Here we identify RGS7 and RGS11 as the key GAPs in the mGluR6 pathway of retinal rod ON bipolar cells that set the sensitivity and time course of light-evoked responses. We showed using electroretinography and single cell recordings that the elimination of RGS7 did not influence dark-adapted light-evoked responses, but the concurrent elimination of RGS11 severely reduced their magnitude and dramatically slowed the onset of the response. In RGS7/RGS11 double-knockout mice, light-evoked responses in rod ON bipolar cells were only observed during persistent activation of rod photoreceptors that saturate rods. These observations are consistent with persistently high G-protein activity in rod ON bipolar cell dendrites caused by the absence of the dominant GAP, biasing TRPM1 channels to the closed state.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Fototransducción/fisiología , Proteínas RGS/metabolismo , Células Bipolares de la Retina/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Transducción de Señal/fisiología , Animales , Western Blotting , Electrorretinografía , Proteínas Activadoras de GTPasa/genética , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Técnicas de Placa-Clamp , Estimulación Luminosa , Proteínas RGS/genética , Células Bipolares de la Retina/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismoRESUMEN
Cooling temperatures may modify action potential firing properties to alter sensory modalities. Herein, we investigated how cooling temperatures modify action potential firing properties in two groups of rat dorsal root ganglion (DRG) neurons, tetrodotoxin-sensitive (TTXs) Na(+) channel-expressing neurons and tetrodotoxin-resistant (TTXr) Na(+) channel-expressing neurons. We found that multiple action potential firing in response to membrane depolarization was suppressed in TTXs neurons but maintained or facilitated in TTXr neurons at cooling temperatures. We showed that cooling temperatures strongly inhibited A-type K(+) currents (IA) and TTXs Na(+) channels but had fewer inhibitory effects on TTXr Na(+) channels and non-inactivating K(+) currents (IK). We demonstrated that the sensitivity of A-type K(+) channels and voltage-gated Na(+) channels to cooling temperatures and their interplay determine somatosensory neuron excitability at cooling temperatures. Our results provide a putative mechanism by which cooling temperatures modify different sensory modalities including pain.
Asunto(s)
Frío/efectos adversos , Canales de Potasio con Entrada de Voltaje/metabolismo , Células Receptoras Sensoriales/fisiología , Canales de Sodio/metabolismo , Animales , Células Cultivadas , Femenino , Masculino , Dolor/etiología , Dolor/metabolismo , Dolor/fisiopatología , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacologíaRESUMEN
The TRPM8 channel is a principal cold transducer that is expressed on some primary afferents of the somatic and cranial sensory systems. However, it is uncertain whether TRPM8-expressing afferent neurons have the ability to convey innocuous and noxious cold stimuli with sensory discrimination between the two sub-modalities. Using rat dorsal root ganglion (DRG) neurons and the patch-clamp recording technique, we characterized membrane and action potential properties of TRPM8-expressing DRG neurons at 24°C and 10°C. TRPM8-expressing neurons could be classified into TTX-sensitive (TTXs/TRPM8) and TTX-resistant (TTXr/TRPM8) subtypes based on the sensitivity to tetrodotoxin (TTX) block of their action potentials. These two subtypes of cold-sensing cells displayed different membrane and action potential properties. Voltage-activated inward Na(+) currents were highly susceptible to cooling temperature and abolished by ~95% at 10°C in TTXs/TRPM8 DRG neurons, but remained substantially large at 10°C in TTXr/TRPM8 cells. In both TTXs/TRPM8 and TTXr/TRPM8 cells, voltage-activated outward K(+) currents were substantially inhibited at 10°C, and the cooling-sensitive outward currents resembled A-type K(+) currents. TTXs/TRPM8 neurons and TTXr/TRPM8 neurons were shown to fire action potentials at innocuous and noxious cold temperatures respectively, demonstrating sensory discrimination between innocuous and noxious cold by the two subpopulations of cold-sensing DRG neurons. The effects of cooling temperatures on voltage-gated Na(+) channels and A-type K(+) currents are likely to be contributing factors to sensory discrimination of cold by TTXs/TRPM8 and TTXr/TRPM8 afferent neurons.
Asunto(s)
Frío , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/patología , Canales Catiónicos TRPM/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Femenino , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiopatología , Activación del Canal Iónico/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/efectos de los fármacos , Tetrodotoxina/toxicidadRESUMEN
The cold-sensing channel transient receptor potential melastatin 8 (TRPM8) features Ca(2+)-dependent downregulation, a cellular process underlying somatosensory accommodation in cold environments. The Ca(2+)-dependent functional downregulation of TRPM8 is manifested with two distinctive phases, acute desensitization and tachyphylaxis. Here we show in rat dorsal root ganglion neurons that TRPM8 acute desensitization critically depends on phosphatidylinositol 4,5-bisphosphate (PIP(2)) availability rather than PIP(2) hydrolysis and is triggered by calmodulin activation. Tachyphylaxis, on the other hand, is mediated by phospholipase hydrolysis of PIP(2) and protein kinase C/phosphatase 1,2A. We further demonstrate that PIP(2) switches TRPM8 channel gating to a high-open probability state with short closed times. Ca(2+)-calmodulin reverses the effect of PIP(2), switching channel gating to a low-open probability state with long closed times. Thus, through gating modulation, Ca(2+)-calmodulin provides a mechanism to rapidly regulate TRPM8 functions in the somatosensory system.
Asunto(s)
Calmodulina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Neuronas/efectos de los fármacos , Fosfatidilinositol 4,5-Difosfato/farmacología , Canales Catiónicos TRPM/metabolismo , Taquifilaxis/fisiología , Análisis de Varianza , Animales , Biofisica , Calcio/metabolismo , Células Cultivadas , Quelantes/farmacología , Frío , Interacciones Farmacológicas , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Femenino , Ganglios Espinales/citología , Humanos , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/genética , Isotiocianatos/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Mentol/farmacología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Canales Catiónicos TRPM/genética , Taquifilaxis/genética , Factores de Tiempo , TransfecciónRESUMEN
Menthol-sensitive/capsaicin-insensitive neurons (MS/CI) and menthol-sensitive/capsaicin-sensitive neurons (MS/CS) are thought to represent two functionally distinct populations of cold-sensing neurons that use TRPM8 receptors to convey innocuous and noxious cold information respectively. However, TRPM8-mediated responses have not been well characterized in these two neuron populations. Using rat dorsal root ganglion neurons, here we show that MS/CI neurons had larger menthol responses with greater adaptation. In contrast, MS/CS neurons had smaller menthol responses with less adaptation. All menthol-sensitive neurons showed significant reduction of menthol responses following the treatment of cells with the protein kinase C (PKC) activator PDBu (Phorbol 12,13-dibutyrate). PDBu-induced reduction of menthol responses was completely abolished in the presence of PKC inhibitors BIM (bisindolylmaleimide) or staurosporine. When menthol responses were examined in the presence of protein kinase inhibitors, it was found that the adaptation was significantly attenuated by either BIM or staurosporine and also by the Ca2+/calmodulin-dependent protein kinase (CamKII) inhibitor KN62 (N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine) in MS/CI neurons. In contrast, in MS/CS neurons menthol response was not affected significantly by BIM, staurosporine or KN62. In both MS/CI and MS/CS neurons, the menthol responses were not affected by PKA activators forskolin and 8-Br-cAMP (8-Bromoadenosine-3', 5'-cyclic monophosphate) or by protein kinase A (PKA) inhibitor Rp-cAMPs (Rp-Adenosine-3',5'-cyclic monophosphorothioate). Taken together, these results suggest that TRPM8-mediated responses are significantly different between non-nociceptive-like and nociceptive-like neurons.
Asunto(s)
Adaptación Fisiológica/efectos de los fármacos , Mentol/farmacología , Neuronas/efectos de los fármacos , Neuronas/enzimología , Nociceptores/metabolismo , Proteínas Quinasas/metabolismo , Animales , Calcio/metabolismo , Capsaicina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activadores de Enzimas/farmacología , Femenino , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Isotiocianatos/farmacología , Masculino , Forbol 12,13-Dibutirato/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Cone photoreceptors scale dynamically the sensitivity of responses to maintain responsiveness across wide range of changes in luminance. Synaptic changes contribute to this adaptation, but how this process is coordinated at the molecular level is poorly understood. Here, we report that a cell adhesion-like molecule, LRIT1, is enriched selectively at cone photoreceptor synapses where it engages in a trans-synaptic interaction with mGluR6, the principal receptor in postsynaptic ON-bipolar cells. The levels of LRIT1 are regulated by the neurotransmitter release apparatus that controls photoreceptor output. Knockout of LRIT1 in mice increases the sensitivity of cone synaptic signaling while impairing its ability to adapt to background light without overtly influencing the morphology or molecular composition of photoreceptor synapses. Accordingly, mice lacking LRIT1 show visual deficits under conditions requiring temporally challenging discrimination of visual signals in steady background light. These observations reveal molecular mechanisms involved in scaling synaptic communication in the retina.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Sinapsis/metabolismo , Animales , Línea Celular , Células HEK293 , Humanos , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Receptores de Glutamato Metabotrópico/metabolismoRESUMEN
Neural circuit wiring relies on selective synapse formation whereby a presynaptic release apparatus is matched with its cognate postsynaptic machinery. At metabotropic synapses, the molecular mechanisms underlying this process are poorly understood. In the mammalian retina, rod photoreceptors form selective contacts with rod ON-bipolar cells by aligning the presynaptic voltage-gated Ca2+ channel directing glutamate release (CaV1.4) with postsynaptic mGluR6 receptors. We show this coordination requires an extracellular protein, α2δ4, which complexes with CaV1.4 and the rod synaptogenic mediator, ELFN1, for trans-synaptic alignment with mGluR6. Eliminating α2δ4 in mice abolishes rod synaptogenesis and synaptic transmission to rod ON-bipolar cells, and disrupts postsynaptic mGluR6 clustering. We further find that in rods, α2δ4 is crucial for organizing synaptic ribbons and setting CaV1.4 voltage sensitivity. In cones, α2δ4 is essential for CaV1.4 function, but is not required for ribbon organization, synaptogenesis, or synaptic transmission. These findings offer insights into retinal pathologies associated with α2δ4 dysfunction.
Asunto(s)
Axones/fisiología , Canales de Calcio Tipo L/fisiología , Canales de Calcio/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Transmisión Sináptica/fisiología , Animales , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Células Cultivadas , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Mutantes , Proteínas del Tejido Nervioso/metabolismo , Receptores de Glutamato Metabotrópico/fisiología , Células Bipolares de la Retina/fisiología , Células Fotorreceptoras Retinianas Conos/fisiología , Sinapsis/metabolismoRESUMEN
In the retina, synaptic transmission between photoreceptors and downstream ON-bipolar neurons (ON-BCs) is mediated by a GPCR pathway, which plays an essential role in vision. However, the mechanisms that control signal transmission at this synapse and its relevance to behavior remain poorly understood. In this study we used a genetic system to titrate the rate of GPCR signaling in ON-BC dendrites by varying the concentration of key RGS proteins and measuring the impact on transmission of signal between photoreceptors and ON-BC neurons using electroretinography and single cell recordings. We found that sensitivity, onset timing, and the maximal amplitude of light-evoked responses in rod- and cone-driven ON-BCs are determined by different RGS concentrations. We further show that changes in RGS concentration differentially impact visually guided-behavior mediated by rod and cone ON pathways. These findings illustrate that neuronal circuit properties can be modulated by adjusting parameters of GPCR-based neurotransmission at individual synapses.
Asunto(s)
Células Bipolares de la Retina/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Animales , Comunicación Celular , Electrorretinografía , Potenciales Evocados Visuales/fisiología , Regulación de la Expresión Génica , Cinética , Luz , Ratones , Ratones Noqueados , Estimulación Luminosa , Proteínas RGS/deficiencia , Proteínas RGS/genética , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Células Bipolares de la Retina/ultraestructura , Células Fotorreceptoras Retinianas Conos/ultraestructura , Células Fotorreceptoras Retinianas Bastones/ultraestructura , Análisis de la Célula Individual , Sinapsis/ultraestructura , Visión Ocular/fisiologíaRESUMEN
In the retina, rod and cone photoreceptors form distinct connections with different classes of downstream bipolar cells. However, the molecular mechanisms responsible for their selective connectivity are unknown. Here we identify a cell-adhesion protein, ELFN1, to be essential for the formation of synapses between rods and rod ON-bipolar cells in the primary rod pathway. ELFN1 is expressed selectively in rods where it is targeted to the axonal terminals by the synaptic release machinery. At the synapse, ELFN1 binds in trans to mGluR6, the postsynaptic receptor on rod ON-bipolar cells. Elimination of ELFN1 in mice prevents the formation of synaptic contacts involving rods, but not cones, allowing a dissection of the contributions of primary and secondary rod pathways to retinal circuit function and vision. We conclude that ELFN1 is necessary for the selective wiring of rods into the primary rod pathway and is required for high sensitivity of vision.