Abstract
Recent studies suggest that Ca2+-dependent activation of the large-conductance voltage- and Ca2+-activated K+ (BK) channel is controlled by two RCK (Regulators of K+ Conductance) domains located in the C-terminus, encompassing high affinity Ca2+-sites D362/367 and M513 in RCK1, and a Ca2+-bowl (D894-898) in RCK2 (Schreiber, et al, 1999, Lingle et al. 2002, Bao et al. 2004). Previously, we characterized Ca2+-induced conformational changes and the role of the Ca2+ bowl in the hSloRCK2 domain using solution-based analysis (Yusifov et al., PNAS 2008).Using a similar approach, we are now investigating the Ca2+-dependent properties of the WT and D362/367A mutations of a purified protein corresponding to the amino acid sequence (322IIE¼DPL667) that forms the human BK channel's RCK1 domain.The calcium-binding activity of purified RCK1 (10ug), loaded on a nitrocellulose membrane, was directly probed by dot blot analysis of 45Ca2+-binding. Albumin and Troponin were used as negative and positive controls, respectively. RCK1 showed remarkable calcium-binding ability when compared to Albumin.Circular Dichroism (CD) analysis of the WT-RCK1 revealed a calcium-dependent spectral change, corresponding to an increased β-strand content of ∼9% as the free [Ca2+] was increased from 0.015 to 31.2μM. This change was paralleled by a similar decrease in α-helix content, while the turns and unordered fractions remained practically unchanged.On the other hand, the CD spectra of RCK1-D362/367A mutant displays ∼7-8% increased beta content, similar to the calcium-bound form of WT-RCK1. The Far-UV CD spectra obtained of hSloRCK1-D362/367A mutant in increasing free Ca2+ displayed no changes, suggesting a lack of substantial Ca2+-dependent structural changes. Based on these findings, we propose that the D362/367A mutation in hSloRCK1 may lead to a conformational state of hSloRCK1 that is unable to translate Ca2+-binding to channel gating.
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