Metal-driven operation of the human large-conductance voltage- and Ca2+-dependent potassium channel (BK) gating ring apparatus.

Large-conductance voltage- and Ca(2+)-dependent K(+) (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca(2+) homeostasis. Their activation results from the complex synergy between the transmembrane voltage sensors and a large (>300 kDa) C-terminal, cytoplasmic complex (the "gating ring"), which confers sensitivity to intracellular Ca(2+) and other ligands. However, the molecular and biophysical operation of the gating ring remains unclear. We have used spectroscopic and particle-scale optical approaches to probe the metal-sensing properties of the human BK gating ring under physiologically relevant conditions. This functional molecular sensor undergoes Ca(2+)- and Mg(2+)-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba(2+)-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca(2+)-binding site (the "calcium bowl") reduced the Ca(2+) and abolished the Mg(2+) dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca(2+)-binding site and that Mg(2+) can bind to the calcium bowl with less affinity than Ca(2+). Dynamic light scattering analysis revealed a reversible Ca(2+)-dependent decrease of the hydrodynamic radius of the gating ring, consistent with a more compact overall shape. These structural changes, resolved under physiologically relevant conditions, likely represent the molecular transitions that initiate the ligand-induced activation of the human BK channel.

The RCK1 domain of the human BKCa channel transduces Ca2+ binding into structural rearrangements.

Large-conductance voltage- and Ca(2+)-activated K(+) (BK(Ca)) channels play a fundamental role in cellular function by integrating information from their voltage and Ca(2+) sensors to control membrane potential and Ca(2+) homeostasis. The molecular mechanism of Ca(2+)-dependent regulation of BK(Ca) channels is unknown, but likely relies on the operation of two cytosolic domains, regulator of K(+) conductance (RCK)1 and RCK2. Using solution-based investigations, we demonstrate that the purified BK(Ca) RCK1 domain adopts an alpha/beta fold, binds Ca(2+), and assembles into an octameric superstructure similar to prokaryotic RCK domains. Results from steady-state and time-resolved spectroscopy reveal Ca(2+)-induced conformational changes in physiologically relevant [Ca(2+)]. The neutralization of residues known to be involved in high-affinity Ca(2+) sensing (D362 and D367) prevented Ca(2+)-induced structural transitions in RCK1 but did not abolish Ca(2+) binding. We provide evidence that the RCK1 domain is a high-affinity Ca(2+) sensor that transduces Ca(2+) binding into structural rearrangements, likely representing elementary steps in the Ca(2+)-dependent activation of human BK(Ca) channels.