RESUMEN
The transient potassium current (I(A)) plays an important role in shaping the firing properties of pyloric neurons in the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus. The shal gene encodes I(A) in pyloric neurons. However, when we over-expressed the lobster Shal protein by shal RNA injection into the pyloric dilator (PD) neuron, the increased I(A) had somewhat different properties from the endogenous I(A). The recently cloned K-channel interacting proteins (KChIPs) can modify vertebrate Kv4 channels in cloned cell lines. When we co-expressed hKChIP1 with lobster shal in Xenopus oocytes or lobster PD neurons, they produced A-currents resembling the endogenous I(A) in PD neurons; compared with currents evoked by shal alone, their voltage for half inactivation was depolarized, their kinetics of inactivation were slowed, and their recovery from inactivation was accelerated. We also co-expressed shal in PD neurons with lobster frequenin, which encodes a protein belonging to the same EF-hand family of Ca(2+) sensing proteins as hKChIP. Frequenin also restored most of properties of the shal-evoked currents to those of the endogenous A-currents, but the time course of recovery from inactivation was not corrected. These results suggest that lobster shal proteins normally interact with proteins in the KChIP/frequenin family to produce the transient potassium current in pyloric neurons.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Neuronas/fisiología , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Proteínas de Xenopus , Animales , Proteínas de Unión al Calcio/genética , Ganglios de Invertebrados/citología , Ganglios de Invertebrados/fisiología , Expresión Génica/fisiología , Proteínas de Interacción con los Canales Kv , Potenciales de la Membrana/fisiología , Microinyecciones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Sensoras del Calcio Neuronal , Neuropéptidos , Oocitos/fisiología , Palinuridae , Técnicas de Placa-Clamp , Potasio/metabolismo , Canales de Potasio/genética , Píloro/inervación , ARN/farmacología , Canales de Potasio Shal , XenopusRESUMEN
The Kv4 subfamily of voltage-gated potassium channels is responsible for the transient A-type potassium current that operates at subthreshold membrane potentials to control membrane excitability. Arachidonic acid was shown recently to modulate both the peak amplitude and kinetics of the hippocampal A-current. However, in Xenopus oocytes, arachidonic acid only inhibited the peak amplitude of Kv4 current without modifying its kinetics. These results suggest the existence of Kv4 auxiliary subunit(s) in native cells. We report here a K-channel interacting protein (KChIP)-dependent kinetic modulation of Kv4.2 current in Chinese hamster ovary cells and Kv4.2 and Kv4.3 currents in Xenopus oocytes by arachidonic acid at physiological concentrations. This concentration-dependent effect of arachidonic acid resembled that observed in cerebellar granule neurons and was fully reversible. Other fatty acids, including a nonhydrolyzable inhibitor of both lipooxygenase and cyclooxygenase, 5,8,11,14-eicosatetraynoic acid (ETYA), also mimicked arachidonic acid in modulating Kv4.3 and Kv4.3/KChIP1 currents. Compared with another transient potassium current formed by Kv1.1/Kvbeta1, Kv4.3/KChIP1 current was much more sensitive to arachidonic acid. Association between KChIP1 and Kv4.2 or Kv4.3 was not altered in the presence of 10 microm ETYA as measured by immunoprecipitation and association-dependent growth in yeast. Our data suggest that the KChIP proteins represent a molecular entity for the observed difference between arachidonic acid effects on A-current kinetics in heterologous cells and in native cells and are consistent with the notion that KChIP proteins modulate the subthreshold A-current in neurons.
Asunto(s)
Ácido Araquidónico/farmacología , Proteínas de Unión al Calcio/metabolismo , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Ácido 5,8,11,14-Eicosatetrainoico/farmacología , Animales , Células CHO , Proteínas de Unión al Calcio/genética , Células Cultivadas , Cricetinae , Relación Dosis-Respuesta a Droga , Ácidos Grasos/farmacología , Humanos , Proteínas de Interacción con los Canales Kv , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oocitos/metabolismo , Técnicas de Placa-Clamp , Potasio/metabolismo , Canales de Potasio/genética , Unión Proteica/efectos de los fármacos , Subunidades de Proteína , Ratas , Ratas Sprague-Dawley , Canales de Potasio Shal , Transfección , Técnicas del Sistema de Dos Híbridos , Xenopus laevisRESUMEN
In the brain and heart, rapidly inactivating (A-type) voltage-gated potassium (Kv) currents operate at subthreshold membrane potentials to control the excitability of neurons and cardiac myocytes. Although pore-forming alpha-subunits of the Kv4, or Shal-related, channel family form A-type currents in heterologous cells, these differ significantly from native A-type currents. Here we describe three Kv channel-interacting proteins (KChIPs) that bind to the cytoplasmic amino termini of Kv4 alpha-subunits. We find that expression of KChIP and Kv4 together reconstitutes several features of native A-type currents by modulating the density, inactivation kinetics and rate of recovery from inactivation of Kv4 channels in heterologous cells. All three KChIPs co-localize and co-immunoprecipitate with brain Kv4 alpha-subunits, and are thus integral components of native Kv4 channel complexes. The KChIPs have four EF-hand-like domains and bind calcium ions. As the activity and density of neuronal A-type currents tightly control responses to excitatory synaptic inputs, these KChIPs may regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium.