Taleh N. Yusifov

The RCK2 domain of the human BKCa channel is a calcium sensor

Large conductance voltage and Ca2+-dependent K+ channels (BKCa) are activated by both membrane depolarization and intracellular Ca2+. Recent studies on bacterial channels have proposed that a Ca2+-induced conformational change within specialized regulators of K+ conductance (RCK) domains is responsible for channel gating. Each pore-forming α subunit of the homotetrameric BKCa channel is expected to contain two intracellular RCK domains. The first RCK domain in BKCa channels (RCK1) has been shown to contain residues critical for Ca2+ sensitivity, possibly participating in the formation of a Ca2+-binding site. The location and structure of the second RCK domain in the BKCa channel (RCK2) is still being examined, and the presence of a high-affinity Ca2+-binding site within this region is not yet established. Here, we present a structure-based alignment of the C terminus of BKCa and prokaryotic RCK domains that reveal the location of a second RCK domain in human BKCa channels (hSloRCK2). hSloRCK2 includes a high-affinity Ca2+-binding site (Ca bowl) and contains similar secondary structural elements as the bacterial RCK domains. Using CD spectroscopy, we provide evidence that hSloRCK2 undergoes a Ca2+-induced change in conformation, associated with an α-to-β structural transition. We also show that the Ca bowl is an essential element for the Ca2+-induced rearrangement of hSloRCK2. We speculate that the molecular rearrangements of RCK2 likely underlie the Ca2+-dependent gating mechanism of BKCa channels. A structural model of the heterodimeric complex of hSloRCK1 and hSloRCK2 domains is discussed.

Structural changes in human tear lipocalins associated with lipid binding

Structural and conformational changes in tear lipocalins were detected in association with ligand binding and release. Circular dichroism measurements demonstrated that ligand binding induces beta structure formation, aromatic side chain asymmetry, and a more rigid state in tear lipocalins (TL). The exposure of the tyrosyl component is less in apo-TL than in holo-TL. The sole tryptophan residue, Trp17, is buried in both holo- and apo-TL. The steady state exposure of Trp17 is the same in holo- and apo-TL, but the dynamic exposure is two-fold greater in apo-TL. Maneuvers to unfold the protein with urea or incubation in an acidic environment resulted in increased exposure of aromatic amino acids. Electron paramagnetic resonance studies verified that lipids are liberated from TL in an acidic environment. Acidic pH promotes conformational changes in TL involving aromatic residues, particularly the conserved residue Trp17. These changes are associated with lipid release. The liberation of lipid from the cavity of TL under acidic conditions involves a molten globule state of the protein. We postulate that TL, exposed to the steep surface pH gradient that exists at lipid-aqueous interfaces, would release lipid in association with a molten globule transition. The data suggest a plausible regulatory mechanism for lipid delivery from lipocalins at the tear film surface.

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

Large-conductance voltage- and Ca2+-activated K+ (BKCa) channels play a fundamental role in cellular function by integrating information from their voltage and Ca2+ sensors to control membrane potential and Ca2+ homeostasis. The molecular mechanism of Ca2+-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 BKCa RCK1 domain adopts an α/β fold, binds Ca2+, and assembles into an octameric superstructure similar to prokaryotic RCK domains. Results from steady-state and time-resolved spectroscopy reveal Ca2+-induced conformational changes in physiologically relevant [Ca2+]. The neutralization of residues known to be involved in high-affinity Ca2+ sensing (D362 and D367) prevented Ca2+-induced structural transitions in RCK1 but did not abolish Ca2+ binding. We provide evidence that the RCK1 domain is a high-affinity Ca2+ sensor that transduces Ca2+ binding into structural rearrangements, likely representing elementary steps in the Ca2+-dependent activation of human BKCa channels.

The Contribution of RCK Domains to Human BK Channel Allosteric Activation

Background: In BK channels, Ca2+ and voltage sensors are allosterically connected to the pore. Results: We optically resolved voltage sensor rearrangements, initiated by Ca2+ binding to the intracellular domains RCK1 and RCK2. Impairing the RCK2 abolished this allosteric effect. Conclusion: The RCK2 Ca2+ sensor is required for the allosteric facilitation of voltage sensor activation. Significance: RCK1 and RCK2 Ca2+ sensors are not functionally homologous. Large conductance voltage- and Ca2+-activated K+ (BK) channels are potent regulators of cellular processes including neuronal firing, synaptic transmission, cochlear hair cell tuning, insulin release, and smooth muscle tone. Their unique activation pathway relies on structurally distinct regulatory domains including one transmembrane voltage-sensing domain (VSD) and two intracellular high affinity Ca2+-sensing sites per subunit (located in the RCK1 and RCK2 domains). Four pairs of RCK1 and RCK2 domains form a Ca2+-sensing apparatus known as the “gating ring.” The allosteric interplay between voltage- and Ca2+-sensing apparati is a fundamental mechanism of BK channel function. Using voltage-clamp fluorometry and UV photolysis of intracellular caged Ca2+, we optically resolved VSD activation prompted by Ca2+ binding to the gating ring. The sudden increase of intracellular Ca2+ concentration ([Ca2+]i) induced a hyperpolarizing shift in the voltage dependence of both channel opening and VSD activation, reported by a fluorophore labeling position 202, located in the upper side of the S4 transmembrane segment. The neutralization of the Ca2+ sensor located in the RCK2 domain abolished the effect of [Ca2+]i increase on the VSD rearrangements. On the other hand, the mutation of RCK1 residues involved in Ca2+ sensing did not prevent the effect of Ca2+ release on the VSD, revealing a functionally distinct interaction between RCK1 and RCK2 and the VSD. A statistical-mechanical model quantifies the complex thermodynamics interplay between Ca2+ association in two distinct sites, voltage sensor activation, and BK channel opening.

Tear Lipocalin: Structure, Function and Molecular Mechanisms of Action

Human tear lipocalin (TL), or von Ebner’s gland protein, accounts for about 15–33% of the protein in tears.1–4 TL has been identified in lacrimal gland, von Ebner’s gland, prostate, nasal and tracheal mucosa and skin.5–12 TL was recognized as a member of the lipocalin family when its primary sequence was determined.7,13,14 Lipocalins form a functionally diverse group of proteins with extremely varied amino acid sequences, yet some similar structural properties (Fig. 1). Eight strands (A-H) are arranged in a β-barrel and are joined by loops between the β-strands.15–19 Many of the lipocalins, including TL, are believed to function as dimers.20,21 There are highly conserved regions that are important to ligand affinity and lipocalin stability. Most lipocalins have 1–3 disulfides bonds and one in particular is conserved and may play a role in modulating ligand binding.15,22 A completely conserved tryptophan on the A strand is implicated in ligand binding and prevention of oxidation of retinol, as well as protein stability of various lipocalins.23,24

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

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.

Functional cavity dimensions of tear lipocalin

Purpose. We calibrated the cavity of tear lipocalin with a series of fluorescent labeled lipids of increasing chain length and varying diameter. Methods. Cavity length was assessed with competitive fluorescent assays in which DAUDA was displaced from apo-tear lipocalin with ligands of increasing carbon chain lengths from C12-C24. The concentrations of competitors that inhibit 50% of the binding of DAUDA (IC 50) were compared. Functional diameters of tear lipocalin and ß-lactoglobulin were estimated with fatty acids bearing fluorescent labels of various diameters. The cavity dimensions of other lipocalins were derived from their published crystal structure coordinates. Results. In tear lipocalin, the binding affinities of fatty acids increased up to a carbon chain length of 18 (22.5 Å) but remained constant from C18-C24. The cavity length of other lipocalins in crystal form were similar to tear lipocalin in solution. Tear lipocalin showed decreased binding affinities with progressively increasing ring dimensions of the ligand. In contrast to ß-lactoglobulin and retinol binding protein, tear lipocalin bound DAUDA and cholesterol in the calyx. Neither tear lipocalin nor ß-lactoglobulin bound P646 in their respective cavities. The calculated inter-sheet distances at the mouth of the crystallized lipocalins ranged from 16–22Å. Conclusions. Tear lipocalin is more promiscuous than ß-lactoglobulin or retinol binding protein because of a greater functional diameter. Differences in ligand specificity of the various lipocalins can not be explained simply by variation in cavity length or the intersheet distances at the calyx mouths as determined by crystal structure. Other factors may influence ligand specificity such as size and/or dynamic motion of loops between the ß strands.

Characterization of a lipophilin in rabbit tears.

A novel protein in human tears called lipophilin has homology to the uteroglobin superfamily.1–3 Putative functions of the members of the family include the binding of hydrophobic molecules, anti-inflammatory properties, anti-chemotactic properties, suppression of extracelluar matrix invasion by normal and cancer cells, and phospholipase A2 inhibition.4,5 Other proteins in the uteroglobin family include rat prostatem6 (a steroid binding protein), uteroglobin7,8 (a progesterone binding protein), Clara cell protein8 (a phosphatidylcholine and phosphatidylinositol binding protein), and mammaglobin9. The proteins share some structural features. In general, monomers are linked by disulfide bonds to form homo- (uteroglobin, Clara cell protein) or hetero-dimers (human lipophilin and prostatein). The crystal structure of uteroglobin reveals that the dimer is composed of two independent polypeptide chains of 70 residues linked by two disulfide bridges. Each monomer is folded into four alpha-helices. An oblong hydrophobic pocket composes the putative progesterone-binding site.10 The rat and human Clara cell protein also exists as 2 identical monomers joined together in an antiparallel manner to enclose a large internal hydrophobic cavity.8,11 The structural motif common to all members of the family is a hydrophobic tunnel created by the alpha helical monomers and bridged by multiple disulfide bonds. The disulfide bonds may be important in ligand binding and release. For uteroglobin, a reduction in the bonds induces a local unfolding of the N- and C-termini. The resulting separation of helices creates a channel to the binding site.12

Resolving near-ultraviolet circular dichroism spectra of single trp mutants in tear lipocalin.

Near-ultraviolet circular dichroism (near-UV CD) spectra of tryptophan residues in proteins are complicated because the line shapes are derived from the overlap of both the 1L(a) and the 1L(b) electronic bands that vary independently. Contributing to this complexity, tryptophan near-UV CD spectra differ in the relative amplitude of the 0-0 vibronic band compared to the rest of the 1L(b) spectrum, an inherent feature that may result in poor fitting. To resolve this problem, a computer program that incorporated the separation of the 0-0 transition of 1L(b) component from the rest of the 1L(b) was written in LabVIEW and its amplitude was allowed to vary independently. This method showed dramatically improved fitting of 1L(a) and 1L(b) components in the near-UV CD tryptophan spectra in tear lipocalin mutants featuring low intensity of the 0-0 1L(b) component. Side chain dynamic characteristics (mobility and accessibility to the solvent) identified from different spectroscopic techniques were related to differences in Trp near-UV CD spectra. This method is broadly applicable to different types of Trp near-UV CD spectra.

Vitamin E associated with the lipocalin fraction of human tears.

Ocular surface tissues are exposed to light and the accompanying photooxidative reactions that produce reactive oxygen species. Antioxidants are important to prevent free radical reactions with unsaturated fatty acids in cell membranes. Similarly, the reactive oxygen species may damage the lipids in the lipid layer of the tear film. Tears contain the water soluble antioxidants, ascorbic acid, uric acid, glutathione, and cysteine, that are capable of scavenging reactive oxygen metabolites.1–3 Recent data suggest that, together. ascorbic acid and uric acid account for about 44% of the total ferric reducing antioxidant activity in tears.4 However, α-tocopherol (vitamin E) is quite lipid soluble and a potential antioxidant for the lipid layer of tears.