Volker Hagen

The signal flow and motor response controling chemotaxis of sea urchin sperm

The signalling pathway and the behavioural strategy underlying chemotaxis of sperm are poorly understood. We have studied the cellular events and motor responses that mediate chemotaxis of sperm from the sea urchin Arbacia punctulata. Here we show that resact, a chemoattractant peptide, initiates a rapid and transient rise in the concentration of cyclic GMP, followed by a transient influx of Ca2+. The binding of a single resact molecule elicits a Ca2+ response, and 50–100 bound molecules saturate the response. The ability to register single molecules is reminiscent of the single-photon sensitivity of rod photoreceptors. Both resact and cyclic nucleotides cause a turn or brief tumbling in the swimming path of sperm. We conclude that a cGMP-mediated increase in the Ca2+ concentration induces the primary motor response of sperm to the chemoattractant.

Ca2+ permeation in cyclic nucleotide‐gated channels

Cyclic nucleotide‐gated (CNG) channels conduct Na+, K+ and Ca2+ currents under the control of cGMP and cAMP. Activation of CNG channels leads to depolarization of the membrane voltage and to a concomitant increase of the cytosolic Ca2+ concentration. Several polypeptides were identified that constitute principal and modulatory subunits of CNG channels in both neurons and non‐excitable cells, co‐assembling to form a variety of heteromeric proteins with distinct biophysical properties. Since the contribution of each channel type to Ca2+ signaling depends on its specific Ca2+ conductance, it is necessary to analyze Ca2+ permeation for each individual channel type. We have analyzed Ca2+ permeation in all principal subunits of vertebrates and for a principal subunit from Drosophila melanogaster. We measured the fractional Ca2+ current over the physiological range of Ca2+ concentrations and found that Ca2+ permeation is determined by subunit composition and modulated by membrane voltage and extracellular pH. Ca2+ permeation is controlled by the Ca2+‐binding affinity of the intrapore cation‐binding site, which varies profoundly between members of the CNG channel family, and gives rise to a surprising diversity in the ability to generate Ca2+ signals.

Ca2+ spikes in the flagellum control chemotactic behavior of sperm

The events that occur during chemotaxis of sperm are only partly known. As an essential step toward determining the underlying mechanism, we have recorded Ca2+ dynamics in swimming sperm of marine invertebrates. Stimulation of the sea urchin Arbacia punctulata by the chemoattractant or by intracellular cGMP evokes Ca2+ spikes in the flagellum. A Ca2+ spike elicits a turn in the trajectory followed by a period of straight swimming (‘turn‐and‐run’). The train of Ca2+ spikes gives rise to repetitive loop‐like movements. When sperm swim in a concentration gradient of the attractant, the Ca2+ spikes and the stimulus function are synchronized, suggesting that precise timing of Ca2+ spikes controls navigation. We identified the peptide asterosap as a chemotactic factor of the starfish Asterias amurensis. The Ca2+ spikes and swimming behavior of sperm from starfish and sea urchin are similar, implying that the signaling pathway of chemotaxis has been conserved for almost 500 million years.

Relating ligand binding to activation gating in CNGA2 channels

Cyclic nucleotide-gated (CNG) ion channels mediate sensory signal transduction in photoreceptors and olfactory cells. Structurally, CNG channels are heterotetramers composed of either two or three homologue subunits. Although it is well established that activation is a cooperative process of these subunits, it remains unknown whether the cooperativity is generated by the ligand binding, the gating, or both, and how the subunits interact. In this study, the action of homotetrameric olfactory-type CNGA2 channels was studied in inside-out membrane patches by simultaneously determining channel activation and ligand binding, using the fluorescent cGMP analogue 8-DY547-cGMP as the ligand. At concentrations of 8-DY547-cGMP < 1 μM, steady-state binding was larger than steady-state activation, whereas at higher concentrations it was smaller, generating a crossover of the steady-state relationships. Global analysis of these relationships together with multiple activation time courses following cGMP jumps showed that four ligands bind to the channels and that there is significant interaction between the binding sites. Among the binding steps, the second is most critical for channel opening: its association constant is three orders of magnitude smaller than the others and it triggers a switch from a mostly closed to a maximally open state. These results contribute to unravelling the role of the subunits in the cooperative mechanism of CNGA2 channel activation and could be of general relevance for the action of other ion channels and receptors.

Structural Proton Diffusion along Lipid Bilayers

For H(+) transport between protein pumps, lateral diffusion along membrane surfaces represents the most efficient pathway. Along lipid bilayers, we measured a diffusion coefficient of 5.8 x 10(-5) cm(2) s(-1). It is too large to be accounted for by vehicle diffusion, considering proton transport by acid carriers. Such a speed of migration is accomplished only by the Grotthuss mechanism involving the chemical exchange of hydrogen nuclei between hydrogen-bonded water molecules on the membrane surface, and the subsequent reorganization of the hydrogen-bonded network. Reconstitution of H(+)-binding sites on the membrane surface decreased the velocity of H(+) diffusion. In the absence of immobile buffers, structural (Grotthuss) diffusion occurred over a distance of 100 micro m as shown by microelectrode aided measurements of the spatial proton distribution in the immediate membrane vicinity and spatially resolved fluorescence measurements of interfacial pH. The efficiency of the anomalously fast lateral diffusion decreased gradually with an increase in mobile buffer concentration suggesting that structural diffusion is physiologically important for distances of approximately 10 nm.

A sperm-activating peptide controls a cGMP signaling pathway in starfish sperm

Peptides released from eggs of marine invertebrates play a central role in fertilization. About 80 different peptides from various phyla have been isolated, however, with one exception, their respective receptors on the sperm surface have not been unequivocally identified and the pertinent signaling pathways remain ill defined. Using rapid mixing techniques and novel membrane-permeable caged compounds of cyclic nucleotides, we show that the sperm-activating peptide asterosap evokes a fast and transient increase of the cGMP concentration in sperm of the starfish Asterias amurensis, followed by a transient cGMP-stimulated increase in the Ca(2+) concentration. In contrast, cAMP levels did not change significantly and the Ca(2+) response evoked by photolysis of caged cAMP was significantly smaller than that using caged cGMP. By cloning of cDNA and chemical crosslinking, we identified a receptor-type guanylyl cyclase in the sperm flagellum as the asterosap-binding protein. Sperm respond exquisitely sensitive to picomolar concentrations of asterosap, suggesting that the peptide serves a chemosensory function like resact, a peptide involved in chemotaxis of sperm of the sea urchin Arbacia punctulata. A unifying principle emerges that chemosensory transduction in sperm of marine invertebrates uses cGMP as the primary messenger, although there may be variations in the detail.

Cyclic AMP is sufficient for triggering the exocytic recruitment of aquaporin‐2 in renal epithelial cells

The initial response of renal epithelial cells to the antidiuretic hormone arginine vasopressin (AVP) is an increase in cyclic AMP. By applying immunofluorescence, cell membrane capacitance and transepithelial water flux measurements we show that cAMP alone is sufficient to elicit the antidiuretic cellular response in primary cultured epithelial cells from renal inner medulla, namely the transport of aquaporin‐2 (AQP2)‐bearing vesicles to, and their subsequent fusion with, the plasma membrane (AQP2 shuttle). The AQP2 shuttle is evoked neither by AVP‐independent Ca2+ increases nor by AVP‐induced Ca2+ increases. However, clamping cytosolic Ca2+ concentrations below resting levels at 25 nM inhibited exocytosis. Exocytosis was confined to a slow monophasic response, and readily releasable vesicles were missing. Analysis of endocytic capacitance steps revealed that cAMP does not decelerate the retrieval of AQP2 from the plasma membrane. Our data suggest that cAMP initiates an early step, namely the transport of AQP2‐bearing vesicles towards the plasma membrane, and do not support a regulatory function for Ca2+ in the AQP2 shuttle.

[7-(Dialkylamino)coumarin-4-yl]methyl-Caged Compounds as Ultrafast and Effective Long-Wavelength Phototriggers of 8Bromo-Substituted Cyclic Nucleotides

[7‐(Dimethylamino)coumarin‐4‐yl]methyl (DMACM) and [7‐(diethylamino)coumarin‐4‐yl]methyl (DEACM) esters of 8bromoadenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP) and 8bromoguanosine 3′,5′‐cyclic monophosphate (8‐Br‐cGMP) are described as novel caged compounds for 8bromo‐substituted cyclic nucleotides. Synthesis is accomplished by treatment of the free acids of the cyclic nucleotides with the corresponding 7(dialkylamino)‐substituted 4(diazomethyl)coumarins. Irradiation of the DMACM‐ and DEACM‐caged cyclic nucleotides with UV light stimulates the release of the cyclic nucleotides within roughly a nanosecond. The new caged compounds are resistant to hydrolysis in aqueous buffers and exhibit long‐wavelength absorption properties with maxima at 400 nm, high extinction coefficients, and high quantum yields (0.15–0.31). Their favorable properties render these compounds the most efficient and rapid phototriggers of 8bromo‐substituted cyclic nucleotides known. The usefulness of the compounds for physiological studies under nondamaging light conditions was examined in HEK293 cells expressing the α subunit of the cyclic‐nucleotide‐gated (CNG) channel of cone photoreceptors (CNGA3) and of olfactory neurons (CNGA2) by using confocal laser scanning microscopy and the patch clamp technique.

Wavelength-selective photoactivatable protecting groups for thiols.

We developed and characterized efficient, remarkably water-soluble photolabile protecting groups for thiols based on 2-nitrobenzyl and (coumarin-4-yl)methyl chromophores, among them two new ones. The protecting groups allow, due to their different absorption maxima, wavelength-selective photocleavage of binary mixtures of cysteine derivatives protected at the thiol function with various photolabile protecting groups by irradiation with light. The concept was also functional with the two different S-protected cysteine residues in derivatives of the model peptide resact. Selective photolysis could be achieved for the peptides Ac(0)-Cys(1)(BCMACMOC),Cys(8)(7,8BCMCMOC)-resact and Ac(0)-Cys(1)(C4MNB),Cys(8)(BCMACMOC)-resact by irradiation with light of > or = 402 nm or > or = 436 nm wavelength, respectively, followed by irradiation at lambda > or = 325 nm.

Protons migrate along interfacial water without significant contributions from jumps between ionizable groups on the membrane surface

Proton diffusion along membrane surfaces is thought to be essential for many cellular processes such as energy transduction. Commonly, it is treated as a succession of jumps between membrane-anchored proton-binding sites. Our experiments provide evidence for an alternative model. We released membrane-bound caged protons by UV flashes and monitored their arrival at distant sites by fluorescence measurements. The kinetics of the arrival is probed as a function of distance for different membranes and for different water isotopes. We found that proton diffusion along the membrane is fast even in the absence of ionizable groups in the membrane, and it decreases strongly in D2O as compared to H2O. We conclude that the fast proton transport along the membrane is dominated by diffusion via interfacial water, and not via ionizable lipid moieties.