BK channels are dually regulated by voltage and Ca(2+), providing a cellular mechanism to couple electrical and chemical signalling. Intracellular Ca(2+) concentration is sensed by a large cytoplasmic region in the channel known as "gating ring", which is formed by four tandems of regulator of conductance for K(+) (RCK1 and RCK2) domains. The recent crystal structure of the full-length BK channel from Aplysia californica has provided new information about the residues involved in Ca(2+) coordination at the high-affinity binding sites located in the RCK1 and RCK2 domains, as well as their cooperativity. Some of these residues have not been previously studied in the human BK channel. In this work we have investigated, through site directed mutagenesis and electrophysiology, the effects of these residues on channel activation by voltage and Ca(2+). Our results demonstrate that the side chains of two non-conserved residues proposed to coordinate Ca(2+) in the A. californica structure (G523 and E591) have no apparent functional role in the human BK Ca(2+) sensing mechanism. Consistent with the crystal structure, our data indicate that in the human channel the conserved residue R514 participates in Ca(2+) coordination in the RCK1 binding site. Additionally, this study provides functional evidence indicating that R514 also interacts with residues E902 and Y904 connected to the Ca(2+) binding site in RCK2. Interestingly, it has been proposed that this interaction may constitute a structural correlate underlying the cooperative interactions between the two high-affinity Ca(2+) binding sites regulating the Ca(2+) dependent gating of the BK channel. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.