西亚试剂：Metal-driven Operation of the Human Large-conductance Volta

Metal-driven Operation of the Human Large-conductance Voltage- and Ca2+-dependent Potassium Channel (BK) Gating Ring Apparatus

Anoosh D. Javaherian, Taleh Yusifov, Antonios Pantazis, Sarah Franklin, Chris S. Gandhi and Riccardo cese

Large-conductance voltage- and Ca2+-dependent K+ (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca2+ 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 Ca2+ 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 Ca2+- and Mg2+-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba2+-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca2+-binding site (the “calcium bowl”) reduced the Ca2+ and abolished the Mg2+ dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca2+-binding site and that Mg2+ can bind to the calcium bowl with less affinity than Ca2+. Dynamic light scattering analysis revealed a reversible Ca2+-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.