Structural Studies of the Apo and Ca^(2+)-Bound States of the Human BK (SLO1) Channel Gating Ring in Solution

The gating ring (GR) regulates the activity of large-conductance voltage- and Ca^(2+)-activated K^+ channels (BK) by interacting with intracellular signaling molecules. To understand the operation of this biological sensor under physiological conditions, we performed Small-Angle X-ray Scattering (SAXS) analysis, at beamline 4-2 at the Stanford Synchrotron Radiation laboratory. SAXS measurements of the purified GR were performed in the absence or in the presence of 35 μM free Ca^(2+), found to be a saturating concentration in previous work. The quality of the circularly-averaged scattering data was evaluated with Guinier analysis, while the ATSAS software suite was used to derive structural information. The radius of gyration (R_g) and maximum interparticle distance (D_(max)) of the apo GR were 48.65±1.372 Å and 185 Å, respectively. These values are comparable to data obtained from crystal structure of GR (3NAF), where the envelope R_g, calculated with CRYSOL, is 45.55 Å, and its diameter 155.6 Å. Ca^(2+)-bound GR shows a decrease in R_g to 42.77±1.058 Å and D_(max) to 160 Å, demonstrating the structural response of GR to Ca^(2+). Low-resolution structural models of the GR were generated from the experimental scattering pattern using DAMMIN. The Ca^(2+)-bound GR revealed notable changes in both flexible and assembly interfaces of the superstructure's constituent RCK1 (Regulator of Conductance for K^+) and RCK2 domains. Since the structural changes are resolved under physiologically-relevant conditions, we speculate that they represent the molecular transitions that initiate the Ca^(2+)-induced activation of human BK channels

Tear lipocalin: evidence for a scavenging function to remove lipids from the human corneal

PURPOSE. Lipid contamination of the cornea may create an unwettable surface and result in desiccation of the corneal epithelium. Tear lipocalin (TL), also known as lipocalin-1, is the principal lipid-binding protein in tears. TL has been shown to scavenge lipids from hydrophobic surfaces. The hypothesis that TL can remove contaminating fatty acids and phospholipids from the human corneal surface was tested. METHODS. TL was purified from pooled human tear samples by size exclusion and ion exchange chromatographies. Tears depleted of TL were reconstituted from fractions eluted by size exclusion chromatography that did not contain TL. Fresh and formalin-fixed human corneas were obtained from exenteration specimens. Fluorescent analogs of either palmitic acid or phosphatidylcholine were applied to the corneal epithelial surface. Tears, TL, or tears depleted of TL were applied over the corneas, and spectrofluorometry and fluorescent stereomicroscopy were used to monitor the removal of fluorescent lipids. Tears used in the experiments were then fractionated by size exclusion chromatography to determine the component of tears associated with fluorescent lipids. RESULTS. Significant enhancement of fluorescence for 16AP and NBD C 6 -HPC was evident in solutions incubated with whole tears and purified TL but not with tears depleted of TL for fixed and unfixed corneas. After the experiment, size exclusion fractions of tears showed that the fluorescence component coeluted with TL. CONCLUSIONS. TL scavenges lipids from the human corneal surface and delivers them into the aqueous phase of tears. TL may have an important role in removing lipids from the corneal surface to maintain the wettability and integrity of the ocular surface. (Invest Ophthalmol Vis Sci. 2005;46:3589 -3596

The RCK1 domain of the human BK_(Ca) channel transduces Ca^(2+) 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 Ca2+ sensors to control membrane potential and Ca^(2+) homeostasis. The molecular mechanism of Ca^(2+)-dependent regulation of BKCa 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 α/β 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

Metal-driven Operation of the Human Large-conductance Voltage- and Ca^(2+)-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