RESUMO
Recently, Conorfamide-Sr3 (CNF-Sr3) was isolated from the venom of Conus spurius and was demonstrated to have an inhibitory concentration-dependent effect on the Shaker K+ channel. The voltage-gated potassium channels play critical functions on cellular signaling, from the regeneration of action potentials in neurons to the regulation of insulin secretion in pancreatic cells, among others. In mammals, there are at least 40 genes encoding voltage-gated K+ channels and the process of expression of some of them may include alternative splicing. Given the enormous variety of these channels and the proven use of conotoxins as tools to distinguish different ligand- and voltage-gated ion channels, in this work, we explored the possible effect of CNF-Sr3 on four human voltage-gated K+ channel subtypes homologous to the Shaker channel. CNF-Sr3 showed a 10 times higher affinity for the Kv1.6 subtype with respect to Kv1.3 (IC50 = 2.7 and 24 µM, respectively) and no significant effect on Kv1.4 and Kv1.5 at 10 µM. Thus, CNF-Sr3 might become a novel molecular probe to study diverse aspects of human Kv1.3 and Kv1.6 channels.
Assuntos
Venenos de Moluscos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Animais , Caramujo Conus , Ativação do Canal Iônico , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Canal de Potássio Kv1.4/antagonistas & inibidores , Canal de Potássio Kv1.4/genética , Canal de Potássio Kv1.4/metabolismo , Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/genética , Canal de Potássio Kv1.5/metabolismo , Canal de Potássio Kv1.6/antagonistas & inibidores , Canal de Potássio Kv1.6/genética , Canal de Potássio Kv1.6/metabolismo , Potenciais da Membrana , Oócitos , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Xenopus laevisRESUMO
Postsynatptic density protein (PSD-95) is a 95 kDa scaffolding protein that assembles signaling complexes at synapses. Over-expression of PSD-95 in primary hippocampal neurons selectively increases synaptic localization of AMPA receptors; however, mice lacking PSD-95 display grossly normal glutamatergic transmission in hippocampus. To further study the scaffolding role of PSD-95 at excitatory synapses, we generated a recombinant PSD-95-4c containing a tetracysteine motif, which specifically binds a fluorescein derivative and allows for acute and permanent inactivation of PSD-95. Interestingly, acute inactivation of PSD-95 in rat hippocampal cultures rapidly reduced surface AMPA receptor immunostaining, but did not affected NMDA or transferrin receptor localization. Acute photoinactivation of PSD-95 in dissociated neurons causes â¼80% decrease in GluR2 surface staining observed by live-cell microscopy within 15 minutes of PSD-95-4c ablation. These results confirm that PSD-95 stabilizes AMPA receptors at postsynaptic sites and provides insight into the dynamic interplay between PSD-95 and AMPA receptors in live neurons.
Assuntos
Hipocampo/citologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Animais , Células COS , Chlorocebus aethiops , Proteína 4 Homóloga a Disks-Large , Hipocampo/metabolismo , Hipocampo/efeitos da radiação , Humanos , Canal de Potássio Kv1.4/metabolismo , Luz , Imagem Molecular , Neurônios/citologia , Neurônios/metabolismo , Neurônios/efeitos da radiação , Estabilidade Proteica/efeitos da radiação , Transporte Proteico/efeitos da radiação , Células Piramidais/citologia , Células Piramidais/metabolismo , Células Piramidais/efeitos da radiação , Ratos , Sinapses/efeitos da radiaçãoRESUMO
After removal of the fast N-type inactivation gate, voltage-sensitive Shaker (Shaker IR) K channels are still able to inactivate, albeit slowly, upon sustained depolarization. The classical mechanism proposed for the slow inactivation observed in cell-free membrane patches--the so called C inactivation--is a constriction of the external mouth of the channel pore that prevents K(+) ion conduction. This constriction is antagonized by the external application of the pore blocker tetraethylammonium (TEA). In contrast to C inactivation, here we show that, when recorded in whole Xenopus oocytes, slow inactivation kinetics in Shaker IR K channels is poorly dependent on external TEA but severely delayed by internal TEA. Based on the antagonism with internally or externally added TEA, we used a two-pulse protocol to show that half of the channels inactivate by way of a gate sensitive to internal TEA. Such gate had a recovery time course in the tens of milliseconds range when the interpulse voltage was -90 mV, whereas C-inactivated channels took several seconds to recover. Internal TEA also reduced gating charge conversion associated to slow inactivation, suggesting that the closing of the internal TEA-sensitive inactivation gate could be associated with a significant amount of charge exchange of this type. We interpreted our data assuming that binding of internal TEA antagonized with U-type inactivation (Klemic, K.G., G.E. Kirsch, and S.W. Jones. 2001. Biophys. J. 81:814-826). Our results are consistent with a direct steric interference of internal TEA with an internally located slow inactivation gate as a "foot in the door" mechanism, implying a significant functional overlap between the gate of the internal TEA-sensitive slow inactivation and the primary activation gate. But, because U-type inactivation is reduced by channel opening, trapping the channel in the open conformation by TEA would also yield to an allosteric delay of slow inactivation. These results provide a framework to explain why constitutively C-inactivated channels exhibit gating charge conversion, and why mutations at the internal exit of the pore, such as those associated to episodic ataxia type I in hKv1.1, cause severe changes in inactivation kinetics.