The β2 subunit modulation of BK channels is determined by membrane-spanning and cytoplasmic domains In Slo1

Abstract

Ca2+ and voltage activated BK channels are composed of pore forming Slo1 subunits. These channels are modulated by various tissue-specific accessory β subunits, which render BK channels the phenotypes necessary for different physiological functions. Here we study Ca2+ sensitivity increase in BK channel activation by the β2 subunit, and elucidate the structural domains in Slo1 that determine this modulation. We found that β2ND (β2 with NH2-terminus deleted to remove inactivation) (Wallner et al., PNAS 96(7):4137-42, 1999) increased Ca2+ sensitivity in mouse Slo1 (mSlo1) but not in drosophila Slo1 (dSlo1). Taking advantage of these differential effects, chimeras of mSlo1 and dSlo1 were studied. When chimeras in the mSlo1 background contained the S0 transmembrane segment and the N-terminal region of RCK1 (regulator of K+ conductance) termed the AC region (Krishnamoorthy et al., JGP 126(3): 227-41, 2005) from dSlo1, β2ND failed to increase Ca2+ sensitivity. When these same regions from mSlo1 were in dSlo1, the channels showed increased Ca2+ sensitivity in association with β2ND. Thus, the mouse AC region and S0 segment are necessary and sufficient for the β2 subunit to increase Ca2+ sensitivity. Previous studies suggested that each Slo1 subunit contains two different Ca2+ binding sites (Xia et al., Nature 418(6900): 880-4, 2002). To further investigate the β2 subunit modulation, we studied the effect of β2ND with mutations of the binding sites. We found that the effect of β2ND was nearly intact when either site was ablated and was completely destroyed when both sites were mutated. These results suggest that the β2 subunit may affect an allosteric activation pathway that is common to both binding sites, and S0 or the AC region is part of such pathway.