Distinct regions of the slo subunit determine differential BKCa channel responses to ethanol

Abstract

Background: Ethanol at clinically relevant concentrations increases BKCa channel activity in dorsal root ganglia neurons, GH3 cells, and neurohypophysial terminals, leading to decreases in cell excitability and peptide release. In contrast, ethanol inhibits BKCa channels from aortic myocytes, which likely contributes to alcohol-induced aortic constriction. The mechanisms that determine differential BKCa channel responses to ethanol are unknown. We hypothesized that nonconserved regions in the BKCa channel-forming subunit (slo) are major contributors to the differential alcohol responses of different BKCa channel phenotypes.

Methods: We constructed chimeras by interchanging the core and the tail domains of two BKCa channel-forming subunits (mslo and bslo) that, after expression, differentially respond to ethanol (activation and inhibition, respectively), and studied ethanol action on these mbslo and bmslo chimeric channels using single-channel, patch-clamp techniques.

Results and conclusion: Data from cell-free membranes patches demonstrate that the activity of channels that share a mslo-type core-linker (wt mslo and the mbslo chimera) is consistently and significantly potentiated by acute exposure to ethanol. Thus, a mslo tail is not necessary for ethanol potentiation of slo channels. In contrast, the activity of channels that share a bslo-type core-linker (wt bslo and the bmslo chimera) display heterogenous responses to ethanol: inhibition (in the majority of cases), refractoriness, or activation. Overall, our data indicate that the slo core-linker is a critical region likely contributing to the differential responses of BKCa channels to ethanol.

Fig. 1

Depiction of the BK_Ca channel and construction of chimeric slo subunits. (A) BK_Ca channel heterodimer with its pore-forming α (slo) and modulatory β subunits. The slo pore region (P), RCK structure, Ca⁺⁺ bowl, and core and tail domains joined by the linker are indicated. Arrows point to regions with nonconserved residues between mslo and bslo. The C-terminus of slo is much longer in mslo than in bslo. The dotted line indicates the point at which mslo and bslo protein domains were swapped to construct chimeric channels; (B) Construction of chimeric slo by swapping the C terminal region of mslo and bslo. Thus, mbslo contains the mslo sequence from amino acid 1 to 676 (core and linker) and bslo from 677 to 1116 (tail), while bmslo contains the bslo sequence from amino acid 1 to 676 and mslo from 677 to 1169.

Fig. 2

The steady-state activity of both bmslo (top pair) and mbslo (bottom pair) channels increases with increases in internal [Ca⁺⁺]. Representative traces of activity showing increases in channel NP_o when free [Ca⁺⁺] at the intracellular side of I/O patches is raised from 100 nM to 1 μM (Vm = −20 mV). Each pair of traces was obtained from the same patch. Arrows indicate the baseline; channel openings are shown as downward deflections. For display, records were digitally filtered at 1 kHz. NP_o values were obtained from periods of at least 20 sec of continuous recording.

Fig. 3

As found with mslo, ethanol routinely increases mbslo channel NP_o. Representative single channel recordings obtained before and during the application of 100 mM EtOH to the cytosolic surface of the same I/O patch. Records were obtained >10 min after patch excision (Vm = 40 mV). Arrows indicate the baseline; channel openings are shown as upward deflections. For display, records were digitally filtered at 1 kHz. NP_o values were obtained from periods of at least 20 sec of continuous recording.

Fig. 4

Changes in the steady-state activity of wild type and chimeric channels in response to acute ethanol (100 mM). (A) Acute exposure of the cytosolic side of I/O patches to EtOH evoked varied responses in bslo and bmslo, the majority of channels being inhibited (see text), while routinely potentiating mslo and mbslo activity after expression in the same batches of oocytes. Ratios of NP_o values obtained in the presence and absence of EtOH (x100) are shown in a scatter graph, where each data point represents an individual patch/oocyte; (B) Average changes in channel NP_o in response to EtOH are shown as mean ± SEM, where n (number of patches/oocytes) is shown in parentheses at the bottom of each bar graph. In the presence of alcohol, NP_o reaches 67 ± 10 (bslo), 105 ± 22 (bmslo), 155 ± 15 (mbslo), and 196 ± 17% (mslo) of pre-EtOH values. *Significantly different when compared to bslo (p < 0.05); ^#Significantly different when compared to bslo (p < 0.001); ¶Significantly different when compared to bmslo (p < 0.01). Multicomparisons were performed with one-way ANOVA, followed by Tukey-Kramer's test. In both A and B, a dotted line highlights the point at which NP_o is unchanged by EtOH.