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Background Alcoholics have been reported to have reduced levels of magnesium in both their extracellular and intracellular compartments. Calcium-dependent potassium channels (BK) are known to be one of ethanol (EtOH)'s better known molecular targets. Methods Using outside-out patches from hippocampal neuronal cultures, we examined the consequences of altered intracellular Mg2+ on the effects that EtOH has on BK channels. Results We find that the effect of EtOH is bimodally influenced by the Mg2+ concentration on the cytoplasmic side. More specifically, when internal Mg2+ concentrations are ≤200 μM, EtOH decreases BK activity, whereas it increases activity when Mg2+ is at 1 mM. Similar results are obtained when using patches from HEK cells expressing only the α-subunit of BK. When patches are made with the actin destabilizer cytochalasin D present on the cytoplasmic side, the potentiation caused by EtOH becomes independent of the Mg2+ concentration. Furthermore, in the presence of the actin stabilizer phalloidin, EtOH causes inhibition even at Mg2+ concentrations of 1 mM. Conclusions Internal Mg2+ can modulate the EtOH effects on BK channels only when there is an intact, internal actin interaction with the channel, as is found at synapses. We propose that the EtOH-induced decrease in cytoplasmic Mg2+ observed in frequent/chronic drinkers would decrease EtOH's actions on synaptic (e.g., actin-bound) BK channels, producing a form of molecular tolerance. [ABSTRACT FROM AUTHOR]

Background The large conductance Ca2+- 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 Et OH affects the expression and subcellular localization of endogenous BK channels over time. Results Exposure to Et OH 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 Et OH on perimembrane expression of BK. First, there was an increase in BK perimembrane expression after 10 minutes of Et OH exposure that remained evident after 3 hours, although not correlated to increases in functional channel expression. In contrast, after 6 hours of Et OH exposure, we observed a significant decrease in both BK perimembrane expression and functional channel expression. Furthermore, after 24 hours of Et OH exposure, perimembrane levels of BK had returned to baseline. Conclusions We report a complex time-dependent pattern in the effect of Et OH 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 Et OH 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. [ABSTRACT FROM AUTHOR]