Incorporation of membrane proteins into large single bilayer vesicles. Application to rhodopsin.

A general procedure to incorporate membrane proteins in a native state into large single bilayer vesicles is described. The results obtained with rhodopsin from vertebrate and invertebrate retinas are presented. The technique involves: (a) the direct transfer of rhodopsin-lipid complexes from native membranes into ether or pentane, and (b) the sonication of the complex in apolar solvent with aqueous buffer followed by solvent evaporation under reduced pressure. The spectral properties of rhodopsin in the large vesicles are similar to those of rhodopsin in photoreceptors; furthermore, bleached bovine rhodopsin is chemically regenerable with 9-cis retinal. These results establish the presence of photochemically functional rhodopsin in the large vesicles. Freeze-fracture replicas of the vesicles reveal that both internal and external leaflets contain numerous particles approximately 80 A in diameter, indicating that rhodopsin is symmetrically distributed within the bilayer. More than 75% of the membrane area is incorporated into vesicles larger than 0.5 micron and approximately 40% into vesicles larger than 1 micron

Real-Time Measurements of the Interactions between Fluorescent Speract and Its Sperm Receptor

AbstractLytechinus pictus sea urchin sperm express receptors for speract, a sperm-activating peptide derived from the homologous egg jelly coat. We found that the fluorescence of fluorophore-labeled, active, speract analogs is quenched upon receptor binding. This property allowed us to perform real-time measurements of speract–receptor interactions using intact sperm and to determine, for the first time, their association (kon) and dissociation (koff) rate constants. The high kon (2.4 × 107 M−1 s−1) and low koff (4.4 × 10−6 s−1 (95%) and 3.7 × 10−4 s−1 (5%)) can account for the sperm response to picomolar concentrations of speract. We also examined the influence of extracellular ions on speract–receptor interactions using the fluorescence quenching method described in this study. The association rate of speract to the receptor is dramatically reduced in Na+-free seawater (NaFSW), divalent cation-free seawater (DCFSW), and high-K+ seawater (HKSW). In seawater speract induces an increase in intracellular pH (pHi), while it is unable to do so in either NaFSW or HKSW. To test if the lack of this pHi change causes the reduction in the speract association rate, pHi was increased with NH4Cl (10 mM) at the time labeled speract was added. Interestingly, this procedure completely (in HKSW) or partially (in NaFSW and DCFSW) restored the speract association rate to its receptor. These findings indicate that an increase in sperm pHi positively affects the receptor binding activity for this peptide and may partially explain the positive binding cooperativity displayed by the speract receptor

Functional reassembly of membrane proteins in planar lipid bilayers

Recent progress in membrane biology has brought us to a stage where it is possible to associate complex biological processes to identifiable membrane proteins. Technical advances in the biochemical characterization and purification of membrane proteins have contributed a wealth of structural information. The reconstitution approach has proved to be valuable in our efforts to understand the molecular mechanisms of membrane transport and energy transductio

Modular analysis of the control of flagellar Ca2+-spike trains produced by CatSper and CaV channels in sea urchin sperm

Intracellular calcium ([Ca2+]i) is a basic and ubiquitous cellular signal controlling a wide variety of biological processes. A remarkable example is the steering of sea urchin spermatozoa towards the conspecific egg by a spatially and temporally orchestrated series of [Ca2+]i spikes. Although this process has been an experimental paradigm for reproduction and sperm chemotaxis studies, the composition and regulation of the signalling network underlying the cytosolic calcium fluctuations are hitherto not fully understood. Here, we used a differential equations model of the signalling network to assess which set of channels can explain the characteristic envelope and temporal organisation of the [Ca2+]i-spike trains. The signalling network comprises an initial membrane hyperpolarisation produced by an Upstream module triggered by the egg-released chemoattractant peptide, via receptor activation, cGMP synthesis and decay. Followed by downstream modules leading to intraflagellar pH (pHi), voltage and [Ca2+]i fluctuations. The Upstream module outputs were fitted to kinetic data on cGMP activity and early membrane potential changes measured in bulk cell populations. Two candidate modules featuring voltage-dependent Ca2+-channels link these outputs to the downstream dynamics and can independently explain the typical decaying envelope and the progressive spacing of the spikes. In the first module, [Ca2+]i-spike trains require the concerted action of a classical CaV-like channel and a potassium channel, BK (Slo1), whereas the second module relies on pHi-dependent CatSper dynamics articulated with voltage-dependent neutral sodium-proton exchanger (NHE). We analysed the dynamics of these two modules alone and in mixed scenarios. We show that the [Ca2+]i dynamics observed experimentally after sustained alkalinisation can be reproduced by a model featuring the CatSper and NHE module but not by those including the pH-independent CaV and BK module or proportionate mixed scenarios. We conclude in favour of the module containing CatSper and NHE and highlight experimentally testable predictions that would corroborate this conclusion

Speract induces calcium oscillations in the sperm tail

Sea urchin sperm motility is modulated by sperm-activating peptides. One such peptide, speract, induces changes in intracellular free calcium concentration ([Ca2+]i). High resolution imaging of single sperm reveals that speract-induced changes in [Ca2+]i have a complex spatiotemporal structure. [Ca2+]i increases arise in the tail as periodic oscillations; [Ca2+]i increases in the sperm head lag those in the tail and appear to result from the summation of the tail signal transduction events. The period depends on speract concentration. Infrequent spontaneous [Ca2+]i transients were also seen in the tail of unstimulated sperm, again with the head lagging the tail. Speract-induced fluctuations were sensitive to membrane potential and calcium channel blockers, and were potentiated by niflumic acid, an anion channel blocker. 3-isobutyl-1-methylxanthine, which potentiates the cGMP/cAMP-signaling pathways, abolished the [Ca2+]i fluctuations in the tail, leading to a very delayed and sustained [Ca2+]i increase in the head. These data point to a model in which a messenger generated periodically in the tail diffuses to the head. Sperm are highly polarized cells. Our results indicate that a clear understanding of the link between [Ca2+]i and sperm motility will only be gained by analysis of [Ca2+]i signals at the level of the single sperm

ATPase activity of thylakoid membranes in CTAB-hexanol-octane low water system

AbstractThylakoid membranes transferred into a low water system composed of n-octane, the cationic surfactant cetyltrimethylammonium bromide (CTAB), and 1-hexanol as cosurfactant, displayed protein- and substrate-dependent ATPase activities for more than 60 min. This activity was enhanced 7–10-fold and 3–4-fold with 28%-vol. of methanol and 21%-vol of tert-butanol present in the polar phase, respectively, in a fashion reminiscent of what occurs in aqueous media. Approximately 25% and 10% of control and methanol-enhanced ATPase activities found in buffer were detected in the low water system, respectively, and both activities showed a pronounced dependency on the amount of water present (between 2.5 and 15% of water (v/v)). 1H-Nuclear Magnetic Resonance (1H-NMR) studies revealed that the bound/free water ratio (a) increased with decreasing concentration of water in the reverse micellar phase and (b) slightly increased in the presence of methanol. The results altogether suggest that the amount and physical state of water significantly contribute to determine the ATPase activity in the low water system

Mouse sperm membrane potential: changes induced by Ca2+

AbstractMouse sperm resting membrane potential (Er) (−42±8.8 mV), determined with a potential sensitive dye, depended on extracellular K+ and, in the absence of extracellular Ca2+ ([Ca2+]e), on external Na+ ([Na+]e). Ca2+ addition (>5 μM) to sperm in Ca-free media induced a transient hyperpolarization (Ca-ith) which strongly depended on [Na+]e and less on external Cl− ([Cl−]e). Cd2+ and Mn2+ (μM) mimicked the Ca2+ effect, but not Ba2+. The Ca-ith was partially inhibited by ouabain (74%, IC50 = 5.8 μM) and niflumic acid (38%, IC50 = 240 μM), indicating the participation of the Na-K ATPase and Cl− channels. In Ca-free low-Na+ media, Ca2+ addition caused a depolarization sensitive to: nimodipine (25 μM), trifluoperazine (12.5 μM) and Mg2+ (1.2 mM), suggesting the participation of Ca2+ channels. Since some inhibitors of the sperm Ca-ith block the acrosome reaction (AR), both processes may share transport systems

Mitochondrial inhibitors activate influx of external Ca2+ in sea urchin sperm

AbstractSea urchin sperm have a single mitochondrion which, aside from its main ATP generating function, may regulate motility, intracellular Ca2+ concentration ([Ca2+]i) and possibly the acrosome reaction (AR). We have found that acute application of agents that inhibit mitochondrial function via differing mechanisms (CCCP, a proton gradient uncoupler, antimycin, a respiratory chain inhibitor, oligomycin, a mitochondrial ATPase inhibitor and CGP37157, a Na+/Ca2+ exchange inhibitor) increases [Ca2+]i with at least two differing profiles. These increases depend on the presence of extracellular Ca2+, which indicates they involve Ca2+ uptake and not only mitochondrial Ca2+ release. The plasma membrane permeation pathways activated by the mitochondrial inhibitors are permeable to Mn2+. Store-operated Ca2+ channel (SOC) blockers (Ni2+, SKF96365 and Gd2+) and internal-store ATPase inhibitors (thapsigargin and bisphenol) antagonize Ca2+ influx induced by the mitochondrial inhibitors. The results indicate that the functional status of the sea urchin sperm mitochondrion regulates Ca2+ entry through SOCs. As neither CCCP nor dicycloexyl carbodiimide (DCCD), another mitochondrial ATPase inhibitor, eliminate the oligomycin induced increase in [Ca2+]i, apparently oligomycin also has an extra mitochondrial target