Ethanol Effect on BK Channels is Modulated by Magnesium

Abstract

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 Mg(2+) on the effects that EtOH has on BK channels.

Results: We find that the effect of EtOH is bimodally influenced by the Mg(2+) concentration on the cytoplasmic side. More specifically, when internal Mg(2+) concentrations are ≤200 μM, EtOH decreases BK activity, whereas it increases activity when Mg(2+) 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 Mg(2+) concentration. Furthermore, in the presence of the actin stabilizer phalloidin, EtOH causes inhibition even at Mg(2+) concentrations of 1 mM.

Conclusions: Internal Mg(2+) 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 Mg(2+) observed in frequent/chronic drinkers would decrease EtOH's actions on synaptic (e.g., actin-bound) BK channels, producing a form of molecular tolerance.

Keywords: BK Channel; Ethanol; Magnesium.

Figure 1. Magnesium-dependent ethanol influence on BK channels from hippocampal neurons

Shown are EtOH-induced changes from initial control NPo’s near 0.5-to-0.6. Significant EtOH-induced NPo reduction is observed at ≤200 μM free Mg²⁺ suggesting that the transition from potentiation to depression might be mostly dependent on the Mg²⁺ concentration. These results show that the 200 μM magnesium cases are a reasonable and consistent choice (see methods) for comparison with the 1 mM Mg²⁺ cases in the more rigorous tests that followed. Data shown is for internal 30 μM free Ca²⁺. Similar effects seen using 5 μM free Ca²⁺ (data not shown). White-filled bars are control and gray-filled are + EtOH. Error bars are SE. The brackets under the horizontal axis indicate significant difference (t-test’s p < 0.016) between a paired-case with the same free Mg²⁺ (bottom row), with n indicated below each control/+EtOH pair (row above magnesium concentrations). The control data was targeted to NPo values between 0.5 and 0.6. Since voltages for controls were different throughout the experiments, the +EtOH NPo is reported as that obtained at the same voltage as that of the corresponding control. Following ANOVA analysis of all data (+/− EtOH vs [Mg²⁺], p < 0.0001), Tukey tests indicated a significant difference limit at 200μM Mg²⁺ when +EtOH is compared to its control (at specific [Mg²⁺] for Control vs +EtOH; 100μM:p < 0.0145, 200μM:p <0.0485, 400μM: p = 1, 600μM:p > 0.9997, 1mM:p< 0.003).

Figure 2. Example of ethanol-induced potentiation of BK channels obtained from hippocampal neurons

Internal solution contained 30 μM free Ca²⁺ and 1 mM free Mg²⁺. (A) Addition of 20–25 mM EtOH causes an increase in activity. All-points histograms (sample of B = control & C = +EtOH) were used to obtain plots of NPo vs applied voltage (D). In D, fits made of these plots (dashed black = control, dashed gray = +EtOH) indicate that EtOH causes a left-ward voltage shift. The examples given in A (and for B&C) were from records taken at a voltage of −5 mV. In B and C, raw histograms are shown as black traces, Gaussian fits as gray traces and composite of Gaussian fits as dashed gray traces. Similar effects seen using 5 μM free Ca²⁺ (data not shown).

Figure 3. Example of ethanol-induced depression of BK channels obtained from hippocampal neurons

Internal solution contained 30 μM free Ca²⁺ and 200 μM free Mg²⁺. (A) Addition of EtOH causes a decrease in activity. All-points histograms (sample of B= control & C =+EtOH) were used to obtain plots of NPo-vs-applied voltage (D). In D, fits made for these plots (dashed black = control, dashed gray = +EtOH) indicate that EtOH causes a right-ward voltage-shift. The examples given in A (and for B & C) were from records taken at a voltage of −30 mV. All other plot details are as explained in figure 2.

Figure 4. Examples of ethanol-induced changes on BK channel activity from HEK cells expressing only the BK-α subunit

With 1 mM internal (cytoplasmic) free Mg²⁺, addition of EtOH causes an increase in activity (A). This activity increase is seen in the plots of NPo-vs-applied voltage (B) as a left-ward shift in V_1/2 (dashed black = control, dashed gray = +EtOH). There were 4 trials (n=4) made for this case, giving an average V_1/2 shift of (mV ±SE) −12.14 ±2.37. The example given in A was from records taken at −20 mV (contained in B). With 200 μM internal free Mg²⁺, addition of EtOH causes a decrease in activity (C), also seen in the plots of NPo-vs-applied voltage (D) as a right-ward shift in V_1/2 (dashed black = control, dashed gray = +EtOH). There were 4 trials (n=4) made for this case, giving an average V_1/2 shifts of (mV ±SE) 11.27 ±0.77, significantly different from the V_1/2 shift obtained with 1 mM free Mg²⁺ (t-test p < 0.0002). The example given in C was from records taken at −60 mV (contained in D). Internal solution for all tests had 30 μM free Ca²⁺.

Figure 5. Summary of ethanol-induced V_1/2 shifts from hippocampal neurons

Potentiation corresponds to negative V_1/2 shifts (bars below horizontal axis), while depression corresponds to positive V_1/2 shifts (bars above horizontal axis). DMSO was at 0.1%, also present at this concentration when phalloidin (Phal; 10 μM) or cytochalasin D (CD: 10 μM) were used. The n’s are given in the lowest row. Ethanol-induced potentiation is present with 1 mM free Mg²⁺, while depression is induced with 200 μM free Mg²⁺. The presence of cytochalasin D causes a persistent EtOH-induced potentiation, while phalloidin causes a persistent depression. Following ANOVA analysis of all data (treatment vs [Mg²⁺], p < 0.0001), Tukey tests indicated that within the 200μM Mg²⁺, the results with cytochalasin D were significantly different from the other three (largest p<0.0110), whereas at 1mM Mg²⁺ it was Phaloidin which was significantly different from the other three (largest p< 0.030). The “ * ” indicates the case which was significantly different from the others with the same Mg²⁺ concentration. Black capped lines are standard errors (SE).

Figure 6. Model of the mechanism of action of ethanol-induced changes in BK channel activity

An inherent mobility of the c-terminus (C-T) part of the channel is assumed and associated with BK activity. The basis for modulation of activity would be the c-terminus’ interaction with the α trans-membrane sections (and bilayer). Thus, one main feature of the model is the proposition that anything that alters the probability of the c-terminus being closer to the bilayer would also increase the probability of channel openings (depicted by thickness of the white arrows inside the trans-membrane section “α”, at top). The high/low Mg²⁺ (left/right of vertical dashed line) would correspondingly increase/decrease the open probability. Actin filaments would stabilize either state, thus determining the modulation of the system by EtOH. Thus, if the c-terminus was stabilized close to the bilayer (by actin, bottom drawing at left of vertical dashed line) addition of EtOH will further stabilize (or bring closer) the c-terminus. On the other hand, if the c-terminus were actin-stabilized away from the bilayer, addition of EtOH would further stabilize it away from the bilayer (bottom drawing at right of vertical dashed line). See discussion for more details.