RESUMEN
Protein kinase A (PKA) has diverse functions in neurons. At rest, the subcellular localization of PKA is controlled by A-kinase anchoring proteins (AKAPs). However, the dynamics of PKA upon activation remain poorly understood. Here, we report that elevation of cyclic AMP (cAMP) in neuronal dendrites causes a significant percentage of the PKA catalytic subunit (PKA-C) molecules to be released from the regulatory subunit (PKA-R). Liberated PKA-C becomes associated with the membrane via N-terminal myristoylation. This membrane association does not require the interaction between PKA-R and AKAPs. It slows the mobility of PKA-C and enriches kinase activity on the membrane. Membrane-residing PKA substrates are preferentially phosphorylated compared to cytosolic substrates. Finally, the myristoylation of PKA-C is critical for normal synaptic function and plasticity. We propose that activation-dependent association of PKA-C renders the membrane a unique PKA-signaling compartment. Constrained mobility of PKA-C may synergize with AKAP anchoring to determine specific PKA function in neurons.
Asunto(s)
Membrana Celular/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Ácido Mirístico/metabolismo , Proteínas de Anclaje a la Quinasa A/metabolismo , Animales , Citosol/metabolismo , Activación Enzimática , Células HEK293 , Humanos , Plasticidad Neuronal , Neuronas/metabolismo , Fosforilación , Unión Proteica , Ratas , Especificidad por Sustrato , Sinapsis/metabolismoRESUMEN
A series of new tetracaine derivatives with substituents on the aromatic ring was prepared and evaluated for block of retinal rod cyclic nucleotide-gated (CNG) channels. Aromatic substitutions had little effect starting with the basic tetracaine scaffold, but electron-withdrawing substituents significantly improved the blocking potency of an octyl-tail derivative of tetracaine. In particular, halogen substitutions at either the 2- or 3-position on the ring resulted in compounds that were up to eight-fold more potent than the parent octyl-tail derivative and up to 50-fold more potent than tetracaine.
Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos/antagonistas & inhibidores , Halógenos/química , Tetracaína/químicaRESUMEN
To meet a pressing need for better cyclic nucleotide-gated (CNG) channel antagonists, we have increased the biological stability of tetracaine-based blockers by synthesizing amide and thioamide linkage substitutions of tetracaine (1) and a higher affinity octyl tail derivative (5). We report the apparent K(D) values, the mechanism of block, and the in vitro hydrolysis rates for these compounds. The ester linkage substitutions did not adversely affect CNG channel block; unexpectedly, thioamide substitution in 1 (compound 8) improved block significantly. Furthermore, the ester linkage substitutions did not appear to affect the mechanism of block in terms of the strong state preference for closed channels. All ester substituted compounds, especially the thioamide substitutions, were more resistant to hydrolysis by serum cholinesterase than their ester counterparts. These findings have implications for dissecting the physiological roles of CNG channels, treating certain forms of retinal degeneration, and possibly the current clinical uses of compound 1.
Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos/antagonistas & inhibidores , Tetracaína/análogos & derivados , Tetracaína/síntesis química , Animales , Butirilcolinesterasa/química , GMP Cíclico/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/química , Femenino , Humanos , Hidrólisis , Técnicas In Vitro , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Células Fotorreceptoras Retinianas Bastones/metabolismo , Relación Estructura-Actividad , Tetracaína/farmacología , XenopusRESUMEN
Transient, non-catastrophic brain ischaemia can induce either a protected state against subsequent episodes of ischaemia (ischaemic preconditioning) or delayed, selective neuronal death. Altered glutamatergic signalling and altered Ca(2+) homeostasis have been implicated in both processes. Here we use simultaneous patch-clamp recording and Ca(2+) imaging to monitor early changes in glutamate release and cytoplasmic [Ca(2+)] ([Ca(2+)](c)) in an in vitro slice model of hippocampal ischaemia. In slices loaded with the Ca(2+)-sensitive dye Fura-2, ischaemia leads to an early increase in [Ca(2+)](c) that precedes the severe ischaemic depolarization (ID) associated with pan necrosis. The early increase in [Ca(2+)](c) is mediated by influx through the plasma membrane and release from internal stores, and parallels an early increase in vesicular glutamate release that manifests as a fourfold increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs). However, the increase in mEPSC frequency is not prevented by blocking the increase in [Ca(2+)](c), and the early rise in [Ca(2+)](c) is not affected by blocking ionotropic and metabotropic glutamate receptors. Thus, the increase in [Ca(2+)](c) and the increase in glutamate release are independent of each other. Stabilizing actin filaments with jaspamide or phalloidin prevented vesicle release induced by ischaemia. Our results identify several early cellular cascades triggered by ischaemia: Ca(2+) influx, Ca(2+) release from intracellular stores, actin filament depolymerization, and vesicular release of glutamate that depends on actin dynamics but not [Ca(2+)](c). All of these processes precede the catastrophic ID by several minutes, and thus represent potential target mechanisms to influence the outcome of an ischaemic episode.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Isquemia Encefálica/fisiopatología , Región CA1 Hipocampal/fisiología , Calcio/fisiología , Glutamatos/fisiología , Vesículas Sinápticas/fisiología , Citoesqueleto de Actina/química , Animales , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/metabolismo , Quelantes/farmacología , Depsipéptidos/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Electrofisiología , Potenciales Postsinápticos Excitadores/fisiología , Colorantes Fluorescentes , Fura-2/análogos & derivados , Técnicas In Vitro , Técnicas de Placa-Clamp , Faloidina/farmacología , Polímeros , Terminales Presinápticos/fisiología , Células Piramidales/citología , Células Piramidales/metabolismo , Células Piramidales/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Despite lacking N-methyl-D-aspartate receptors, cerebellar Purkinje cells are highly vulnerable to ischaemic insults, which lead them to die necrotically in an -amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor-dependent manner. To investigate the electrical events leading to this cell death, we whole-cell clamped Purkinje cells in cerebellar slices. Simulated ischaemia evoked an initial hyperpolarization of Purkinje cells by 8.5 mV, followed by a regenerative 'anoxic depolarization' (AD) to -14 mV. The AD was prevented by glutamate receptor blockers. In voltage-clamp mode, we used the cells' glutamate receptors to sense the rise of extracellular glutamate concentration induced by ischaemia, with GABA(A) and GABA(B) receptors blocked and Cs+ as the main pipette cation. Ischaemia induced a small (<500 pA) slowly developing inward current in Purkinje cells, followed by a sudden large inward 'AD current' (approximately 6 nA) which was largely prevented by blocking AMPA receptors. Removing extracellular calcium reduced the large glutamate-mediated current by approximately 70% at early times (after 10 min ischaemia), but had no effect at later times (15 min). Blocking the operation of glutamate transporters, by preloading cells with the slowly transported glutamate analogue PDC (L-trans-pyrrolidine-2,4-dicarboxylate), reduced the current by approximately 88% at early and 83% at later times. In Purkinje cells in slices from mice lacking the glial glutamate transporters GLAST or GLT-1, the ischaemia-evoked AD current was indistinguishable from that in wild-type slices. These data suggest that, in cerebellar ischaemia, the dominant cause of the electrophysiological dysfunction of Purkinje cells is an activation of Purkinje cell AMPA receptors. The glutamate activating these receptors is released both by exocytosis (at early times) and by reversal of a glutamate transporter, apparently in neurons.