RESUMEN
BACKGROUND: The large conductance Ca(2+) - and voltage-activated K(+) channel (BK) is an important player in molecular and behavioral alcohol tolerance. Trafficking and surface expression of ion channels contribute to the development of addictive behaviors. We have previously reported that internalization of the BK channel is a component of molecular tolerance to ethanol (EtOH). METHODS: Using primary cultures of hippocampal neurons, we combine total internal reflection fluorescence microscopy, electrophysiology, and biochemical techniques to explore how exposure to EtOH affects the expression and subcellular localization of endogenous BK channels over time. RESULTS: Exposure to EtOH changed the expression of endogenous BK channels in a time-dependent manner at the perimembrane area (plasma membrane and/or the area adjacent to it), while total protein levels of BK remain unchanged. These results suggest a redistribution of the channel within the neurons rather than changes in synthesis or degradation rates. Our results showed a temporally nonlinear effect of EtOH on perimembrane expression of BK. First, there was an increase in BK perimembrane expression after 10 minutes of EtOH exposure that remained evident after 3 hours, although not correlated to increases in functional channel expression. In contrast, after 6 hours of EtOH exposure, we observed a significant decrease in both BK perimembrane expression and functional channel expression. Furthermore, after 24 hours of EtOH exposure, perimembrane levels of BK had returned to baseline. CONCLUSIONS: We report a complex time-dependent pattern in the effect of EtOH on BK channel trafficking, including successive increases and decreases in perimembrane expression and a reduction in active BK channels after 3 and 6 hours of EtOH exposure. Possible mechanisms underlying this multiphasic trafficking are discussed. As molecular tolerance necessarily underlies behavioral tolerance, the time-dependent alterations we see at the level of the channel may be relevant to the influence of drinking patterns on the development of behavioral tolerance.
Asunto(s)
Etanol/metabolismo , Etanol/farmacología , Hipocampo/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Neuronas/metabolismo , Animales , Células Cultivadas , Femenino , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Embarazo , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Ratas , Factores de TiempoRESUMEN
Tolerance is a well described component of alcohol abuse and addiction. The large conductance voltage- and Ca(2+)-gated potassium channel (BK) has been very useful for studying molecular tolerance. The influence of association with the ß4 subunit can be observed at the level of individual channels, action potentials in brain slices, and finally, drinking behavior in the mouse. Previously, we showed that 50 mm alcohol increases both α and αß4 BK channel open probability, but only α BK develops acute tolerance to this effect. Currently, we explore the possibility that the influence of the ß4 subunit on tolerance may result from a striking effect of ß4 on kinase modulation of the BK channel. We examine the influence of the ß4 subunit on PKA, CaMKII, and phosphatase modulation of channel activity, and on molecular tolerance to alcohol. We record from human BK channels heterologously expressed in HEK 293 cells composed of its core subunit, α alone (Insertless), or co-expressed with the ß4 BK auxiliary subunit, as well as, acutely dissociated nucleus accumbens neurons using the cell-attached patch clamp configuration. Our results indicate that BK channels are strongly modulated by activation of specific kinases (PKA and CaMKII) and phosphatases. The presence of the ß4 subunit greatly influences this modulation, allowing a variety of outcomes for BK channel activity in response to acute alcohol.
Asunto(s)
Etanol/química , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Caribdotoxina/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Electrofisiología , Células HEK293 , Humanos , Neuronas/metabolismo , Núcleo Accumbens/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Potasio/metabolismo , Factores de TiempoRESUMEN
Motor proteins are involved in a wide range of cellular and subcellular movements. Recent studies have implicated two motor proteins in particular, myosin II and cytoplasmic dynein, in diverse aspects of cell migration. This review focuses on emerging roles for these proteins in the nervous system, with particular emphasis on migrating neurons and neuronal growth cones. The former cells exhibit unusual features of centrosome and nuclear movement, whereas growth cones offer an opportunity to evaluate motor protein function in a region of cytoplasm free of these organelles.
Asunto(s)
Movimiento Celular , Dineínas/metabolismo , Miosina Tipo II/metabolismo , Neuronas/citología , Animales , Citoplasma/metabolismo , Humanos , Neuronas/metabolismoRESUMEN
Recent evidence has implicated dynein and its regulatory factors dynactin and LIS1 in neuronal and non-neuronal cell migration. In the current study we sought to test whether effects on neuronal cell motility might reflect, in part, a role for these proteins in the growth cone. In chick sensory neurons subjected to acute laminin treatment dynein, dynactin, and LIS1 were mobilized strikingly and rapidly to the leading edge of the growth cone, where they were seen to be associated with microtubules converging into the laminin-induced axonal outgrowths. To interfere acutely with LIS1 and dynein function and to minimize secondary phenotypic effects, we injected antibodies to these proteins just before axon initiation. Antibody to both proteins produced an almost complete block of laminin-induced growth cone remodeling and the underlying reorganization of microtubules. Penetration of microtubules into the peripheral zone of differentiating axonal growth cones was decreased dramatically by antibody injection, as judged by live analysis of enhanced green fluorescent protein-tubulin and the microtubule tip-associated EB3 (end-binding protein 3). Dynein and LIS1 inhibition had no detectable effect on microtubule assembly but reduced the ability of microtubules to resist retrograde actin flow. In hippocampal neurons dynein, dynactin, and LIS1 were enriched in axonal growth cones at stage 3, and both growth cone organization and axon elongation were altered by LIS1 RNA interference. Together, our data indicate that dynein and LIS1 play a surprisingly prominent role in microtubule advance during growth cone remodeling associated with axonogenesis. These data may explain, in part, the role of these proteins in brain developmental disease and support an important role in diverse aspects of neuronal differentiation and nervous system development.