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

Acute exposure to clinically relevant concentrations of ethanol (EtOH) reversibly increases BKCa channel activity in neurohypophysial terminals, where drug action likely contributes to inhibition of vasopressin release and, thus, diuresis (Dopico et al., 1996). A similar potentiation of BKCa channels by acute EtOH was reported in GH3 cells (Jakab et al., 1997), and neurons in dorsal root ganglia, where alcohol action on BKCa channels effectively reduces neuronal excitability (Gruss et al., 2001). On the other hand, intoxicating [EtOH] usually decrease the activity of BKCa channels from aortic myocytes, which is thought to contribute to alcohol constriction of aortic smooth muscle (Dopico, 2003; Walters et al., 2000). While advance was made in linking EtOH action on BKCa channels to alcohol effects in the body, the molecular determinants of differential BKCa responses to EtOH are unknown. In supraoptic neurons, EtOH increases nerve ending BKCa channel activity but fails to modulate cell body BKCa channels. Data were obtained in cell-free, membrane patches, indicating that the continuous presence of cytosolic messengers is not required for EtOH to evoke differential responses from native BKCa channels (Dopico et al., 1999). Native BKCa channels consist of pore-forming (α, encoded by the slo1 or KCNMA1 gene), and accessory (β) subunits (Fig. 1A). Following expression in Xenopus oocytes, mslo (from mouse brain; Butler et al., 1993) channel activity is consistently potentiated by 10–200 mM EtOH (Dopico et al., 1998). In contrast, EtOH action on bslo channels (from bovine aortic smooth muscle; Moss et al., 1996) expressed in the same system and under similar conditions, primarily decreases activity. This distinct action of EtOH on bslo channels is not modified by the presence of modulatory β1-subunits (Dopico, 2003). Fig. 1 Depiction of the BKCa channel and construction of chimeric slo subunits. (A) BKCa 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 ... Since differential responses to EtOH were found with slo expressed in a similar proteolipid environment, we hypothesized that nonconserved regions between mslo and bslo primarily determine the differential channel responses to EtOH. The slo subunit has seven putative transmembrane segments (S0–6), a long C-terminus, which includes four hydrophobic regions (S7–10), and a “linker” between S8 and S9 (Fig. 1A) (Weiger et al. 2002). This linker connects two functional domains that determine distinct functional properties of slo channels (Wei et al., 1994). The “core” domain (S0–8) includes the voltage-sensor (in S4), the pore (between S5 and S6), and the RCK structure, which contributes to divalent sensitivity (Jiang et al., 2001). On the other hand, the “tail” domain (S9–10) includes the “Ca++-bowl,” a major determinant of the channel Ca++ sensitivity (Schreiber and Salkoff, 1997). Primary sequence alignment of mslo (mbr5) and bslo shows that nonconserved regions are scattered all around core, linker and tail (Fig. 1A). The least conserved region, however, is the C terminus, a modulatory region located in the tail: in mslo this region consists of 60 residues; the first 8 being substituted (4 are nonconserved) and the remaining absent in bslo, resulting in a shorter C-end (Butler et al., 1993; Moss et al., 1996). Thus, to identify which region of the subunit is the primary structural determinant of channel responses to EtOH, “bmslo” and “mbslo” chimeras were constructed by swapping the mslo core-linker with the bslo tail (Fig. 1B). The chimeras were expressed in oocytes and their basic functional properties as BKCa channel-forming proteins were determined. Then, we evaluated mbslo and bmslo, as well as wild-type (wt) mslo and bslo, channel responses to EtOH under identical conditions after expression in the same batches of oocytes.