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 proteintubulin 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.

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.

Background: Large conductance voltage-and Ca 2ϩ -gated potassium channel (BK) ␤4 subunit profoundly influences BK acute ethanol tolerance with both physiological and behavioral consequences. Results: PKA, CaMKII, and phosphatases modulate BK, and influence its response to ethanol. The presence of ␤4 strongly regulates these responses. Conclusion:The control of BK ␤4 of kinase modulation is critical to ethanol response. Significance: The influence of ␤4 on kinase-mediated alcohol action provides insight into the molecular basis for alcohol tolerance.

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 EtOH. Methods Using primary cultures of hippocampal neurons, we combine total internal reflection fluorescence (TIRF) 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-min of EtOH exposure that remained evident after 3-hrs, though not correlated to increases in functional channel expression. In contrast, after 6-hrs of EtOH exposure we observed a significant decrease in both BK perimembrane expression and functional channel expression. Furthermore, after 24-hrs EtOH exposure, perimembrane levels of BK had returned to baseline. Conclusion 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-hrs of EtOH exposure. Possible mechanisms underlying this multiphasic trafficking are discussed. Since 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.

It has been suggested that drug tolerance represents a form of learning and memory, but this has not been experimentally established at the molecular level. We show that a component of alcohol molecular tolerance (channel internalization) from rat hippocampal neurons requires protein synthesis, in common with other forms of learning and memory. We identify ␤-catenin as a primary necessary protein.Alcohol increases ␤-catenin, and blocking accumulation of ␤-catenin blocks alcohol-induced internalization in these neurons. In transfected HEK293 cells, suppression of Wnt/␤-catenin signaling blocks ethanol-induced internalization. Conversely, activation of Wnt/␤-catenin reduces BK current density. A point mutation in a putative glycogen synthase kinase phosophorylation site within the S10 region of BK blocks internalization, suggesting that Wnt/␤-catenin directly regulates alcohol-induced BK internalization via glycogen synthase kinase phosphorylation. These findings establish de novo protein synthesis and Wnt/␤-catenin signaling as critical in mediating a persistent form of BK molecular alcohol tolerance establishing a commonality with other forms of long-term plasticity.