On the Properties of the RCK1 Domain of the Human BK (SLO1) Channel

Abstract

In BK channels, four RCK1 and four RCK2 domains assemble into a cytosolic ligand-sensing superstructure known as the gating ring. While electrophysiological data suggest that both RCK1 and RCK2 contain high affinity Ca2+-sensing sites, recent crystallographic data (Wu et al., 2010) has revealed Ca2+ binding only within the RCK2 domain. Does the RCK1 domain bind Ca2+? What is the role of the high-affinity Ca2+ sensing residues D362/D367 (Xia, et al 2002) located in RCK1 domain? To address these questions, we expressed and purified the region of the human BK channel C-terminus corresponding to the amino acid sequence 322IIE¼HDP667. We have probed the structure and Ca2+ sensing properties of the RCK1 domain in solution, under physiologically relevant conditions. In addition to the α/β fold shared with its bacterial counterparts and human BK RCK2 domain, the RCK1 domain preferentially self-assembles into a homo-octameric structure. We recently reported that RCK1 undergoes Ca2+-dependent conformational changes similarly to the RCK2 domain (Yusifov et al., 2008 and 2010). The neutralization of residues D362/D367 altered the secondary and quaternary structure of the RCK1 domain and prevented Ca2+-induced structural transitions in RCK1. 45Ca2+ overlay assay suggested that the neutralization of D362/367 did not abolish the Ca2+-binding activity of RCK1 in the range of 2.1-115 µM free Ca2+. While it cannot be excluded that D362/D367 are elements of a Ca2+ binding site with multiple coordinating residues, our results favor the view that D362 /D367 have a predominantly structural role. Possibly, these mutations set RCK1 domains in a conformational state that hampers the propagation of structural changes caused by ligand binding.