Cholesterol Alters the Phase Separation in Model Membranes Containing hBest1.

Human retinal pigment epithelial (RPE) cells express the transmembrane Ca2+-dependent Cl- channel bestrophin-1 (hBest1) of the plasma membrane. Mutations in the hBest1 protein are associated with the development of distinct pathological conditions known as bestrophinopathies. The interactions between hBest1 and plasma membrane lipids (cholesterol (Chol), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and sphingomyelin (SM)) determine its lateral organization and surface dynamics, i.e., their miscibility or phase separation. Using the surface pressure/mean molecular area (π/A) isotherms, hysteresis and compressibility moduli (Cs-1) of hBest1/POPC/Chol and hBest1/SM/Chol composite Langmuir monolayers, we established that the films are in an LE (liquid-expanded) or LE-LC (liquid-condensed) state, the components are well-mixed and the Ca2+ ions have a condensing effect on the surface molecular organization. Cholesterol causes a decrease in the elasticity of both films and a decrease in the ΔGmixπ values (reduction of phase separation) of hBest1/POPC/Chol films. For the hBest1/SM/Chol monolayers, the negative values of ΔGmixπ are retained and equalized with the values of ΔGmixπ in the hBest1/POPC/Chol films. Shifts in phase separation/miscibility by cholesterol can lead to changes in the structure and localization of hBest1 in the lipid rafts and its channel functions.

Self-organization and surface properties of hBest1 in models of biological membranes.

The transmembrane Ca2+ - activated Cl- channel - human bestrophin-1 (hBest1) is expressed in retinal pigment epithelium and mutations of BEST1 gene cause ocular degenerative diseases colectivelly referred to as "bestrophinopathies". A large number of genetical, biochemical, biophysical and molecular biological studies have been performed to understand the relationship between structure and function of the hBest1 protein and its pathophysiological significance. Here, we review the current understanding of hBest1 surface organization, interactions with membrane lipids in model membranes, and its association with microdomains of cellular membranes. These highlights are significant for modulation of channel activity in cells.

Condensing Effect of Cholesterol on hBest1/POPC and hBest1/SM Langmuir Monolayers.

Human bestrophin-1 protein (hBest1) is a transmembrane channel associated with the calcium-dependent transport of chloride ions in the retinal pigment epithelium as well as with the transport of glutamate and GABA in nerve cells. Interactions between hBest1, sphingomyelins, phosphatidylcholines and cholesterol are crucial for hBest1 association with cell membrane domains and its biological functions. As cholesterol plays a key role in the formation of lipid rafts, motional ordering of lipids and modeling/remodeling of the lateral membrane structure, we examined the effect of different cholesterol concentrations on the surface tension of hBest1/POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and hBest1/SM Langmuir monolayers in the presence/absence of Ca2+ ions using surface pressure measurements and Brewster angle microscopy studies. Here, we report that cholesterol: (1) has negligible condensing effect on pure hBest1 monolayers detected mainly in the presence of Ca2+ ions, and; (2) induces a condensing effect on composite hBest1/POPC and hBest1/SM monolayers. These results offer evidence for the significance of intermolecular protein-lipid interactions for the conformational dynamics of hBest1 and its biological functions as multimeric ion channel.

Miscibility of hBest1 and sphingomyelin in surface films - A prerequisite for interaction with membrane domains.

Human bestrophin-1 (hBest1) is a transmembrane Ca2+- dependent anion channel, associated with the transport of Cl-, HCO3- ions, γ-aminobutiric acid (GABA), glutamate (Glu), and regulation of retinal homeostasis. Its mutant forms cause retinal degenerative diseases, defined as Bestrophinopathies. Using both physicochemical - surface pressure/mean molecular area (π/A) isotherms, hysteresis, compressibility moduli of hBest1/sphingomyelin (SM) monolayers, Brewster angle microscopy (BAM) studies, and biological approaches - detergent membrane fractionation, Laurdan (6-dodecanoyl-N,N-dimethyl-2-naphthylamine) and immunofluorescence staining of stably transfected MDCK-hBest1 and MDCK II cells, we report: 1) Ca2+, Glu and GABA interact with binary hBest1/SM monolayers at 35 °C, resulting in changes in hBest1 surface conformation, structure, self-organization and surface dynamics. The process of mixing in hBest1/SM monolayers is spontaneous and the effect of protein on binary films was defined as "fluidizing", hindering the phase-transition of monolayer from liquid-expanded to intermediate (LE-M) state; 2) in stably transfected MDCK-hBest1 cells, bestrophin-1 was distributed between detergent resistant (DRM) and detergent-soluble membranes (DSM) - up to 30 % and 70 %, respectively; in alive cells, hBest1 was visualized in both liquid-ordered (Lo) and liquid-disordered (Ld) fractions, quantifying protein association up to 35 % and 65 % with Lo and Ld. Our results indicate that the spontaneous miscibility of hBest1 and SM is a prerequisite to diverse protein interactions with membrane domains, different structural conformations and biological functions.

Effects of Ca2+, Glu and GABA on hBest1 and composite hBest1/POPC surface films.

Bestrophinopathies are ocular diseases caused by mutations in the human bestrophin-1 (hBest1) - transmembrane Ca2+-activated chloride channel protein, mainly expressed in the retinal pigment epithelium (RPE) cells. hBest1 is also an important transporter for neurotransmitters such as glutamate (Glu) and γ-aminobutyric acid (GABA) in the nervous system. Recently, a new biological role of hBest1, related to its possible involvement in the pathology of brain diseases (Alzheimer's, Parkinson's disease) has been proposed. Here, we report the effects of Ca2+, Glu and GABA on hBest1 and composite hBest1/POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) Langmuir and Langmuir-Blodgett monolayers based on surface dynamics (π/A isotherms, hysteresis and compressibility), morphology (Brewster angle microscopy, BAM) and visualization of protein molecular organization (Atomic force microscopy, AFM). Ca2+ ions and neurotransmitters Glu and GABA affect hBest1 topology at the air/water interface altering its surface activity, size, orientation and organization. In contrast, no significant changes were detected on π/A isotherms and hysteresis of the composite hBest1/POPC films but their effects on structure, aggregation state and orientation hBest1 established by BAM and AFM differentiate. We found that the binary films of hBest1 and POPC are phase separated at the air/water interface, suggesting stronger lipid-lipid and protein-protein interactions than lipid-protein interactions that can significantly alter the molecular organization and activity of hBest1 in cell membranes. Our data shed light on structure, surface behavior and organization of hBest1 that define relationship structure-functional activity of hBest1 as transport channel.

Effects of Ca2+ ions on bestrophin-1 surface films.

Human bestrophin-1 (hBest1) is a transmembrane calcium-activated chloride channel protein - member of the bestrophin family of anion channels, predominantly expressed in the membrane of retinal pigment epithelium (RPE) cells. Mutations in the protein cause ocular diseases, named Bestrophinopathies. Here, we present the first Fourier transform infrared (FTIR) study of the secondary structure elements of hBest1, π/A isotherms and hysteresis, Brewster angle microscopy (BAM) and atomic force microscopy (AFM) visualization of the aggregation state of protein molecules dispersed as Langmuir and Langmuir-Blodgett films. The secondary structure of hBest1 consists predominantly of 310-helices (27.2%), α-helixes (16.3%), ß-turns and loops (32.2%). AFM images of hBest1 suggest approximate lateral dimensions of 100×160Å and 75Å height. Binding of calcium ions (Ca2+) induces conformational changes in the protein secondary structure leading to assembly of protein molecules and changes in molecular and macro-organization of hBest1 in monolayers. These data provide basic information needed in pursuit of molecular mechanisms underlying retinal and other pathologies linked to this protein.

Interaction of Bestrophin-1 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in surface films.

Human bestrophin-1 (hBest1) is a transmembrane channel protein, predominantly expressed in the membrane of retinal pigment epithelium (RPE) cells. Although it is clear that hBest1's interactions with lipids are crucial for its function such studies were not performed as the protein was not purified. Here we describe an effective purification of hBest1 from Madin-Darby Canine Kidney (MDCK) cells via simple gel-filtration and affinity chromatographic steps, which makes possible to probe the protein interplay with lipids. The interaction of the purified hBest1 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was studied in Langmuir monolayers. The surface pressure (π)-area (A) isotherms and compression/expansion isocycles of POPC monolayer were recorded in absence and presence of hBest1 in the subphase. The π(A) isotherms were analyzed in terms of surface compressional modulus and via two-dimensional virial equation of state. The dilatational rheological properties of the surface films and their surface potential were also measured. The morphology of the films was observed by Brewster angle microscopy. The inclusion of the protein in the film subphase does not lead to in-depth penetration of hBest1 but interaction takes place in the headgroup region of the monolayer. The hBest1/POPC interaction resulted in formation of more condensed films, which rheological properties and lateral structure differed significantly from the pure POPC monolayers. Our study sheds light on the still unclear question how hBest1 gets in touch with biomembrane phospholipids of eukaryotic cells that might be of key importance for the proper structure and function of RPE biomembranes.