Revisiting the role of H+ in chemotactic signaling of sperm

© 2004 Solzin et al. This article is distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License. The definitive version was published in Journal of General Physiology 124 (2004): 115-124, doi:10.1085/jgp.200409030.Chemotaxis of sperm is an important step toward fertilization. During chemotaxis, sperm change their swimming behavior in a gradient of the chemoattractant that is released by the eggs, and finally sperm accumulate near the eggs. A well established model to study chemotaxis is the sea urchin Arbacia punctulata. Resact, the chemoattractant of Arbacia, is a peptide that binds to a receptor guanylyl cyclase. The signaling pathway underlying chemotaxis is still poorly understood. Stimulation of sperm with resact induces a variety of cellular events, including a rise in intracellular pH (pHi) and an influx of Ca2+; the Ca2+ entry is essential for the chemotactic behavior. Previous studies proposed that the influx of Ca2+ is initiated by the rise in pHi. According to this proposal, a cGMP-induced hyperpolarization activates a voltage-dependent Na+/H+ exchanger that expels H+ from the cell. Because some aspects of the proposed signaling pathway are inconsistent with recent results (Kaupp, U.B., J. Solzin, J.E. Brown, A. Helbig, V. Hagen, M. Beyermann, E. Hildebrand, and I. Weyand. 2003. Nat. Cell Biol. 5:109–117), we reexamined the role of protons in chemotaxis of sperm using kinetic measurements of the changes in pHi and intracellular Ca2+ concentration. We show that for physiological concentrations of resact (<25 pM), the influx of Ca2+ precedes the rise in pHi. Moreover, buffering of pHi completely abolishes the resact-induced pHi signal, but leaves the Ca2+ signal and the chemotactic motor response unaffected. We conclude that an elevation of pHi is required neither to open Ca2+-permeable channels nor to control the chemotactic behavior. Intracellular release of cGMP from a caged compound does not cause an increase in pHi, indicating that the rise in pHi is induced by cellular events unrelated to cGMP itself, but probably triggered by the consumption and subsequent replenishment of GTP. These results show that the resact-induced rise in pHi is not an obligatory step in sperm chemotactic signaling. A rise in pHi is also not required for peptide-induced Ca2+ entry into sperm of the sea urchin Strongylocentrotus purpuratus. Speract, a peptide of S. purpuratus may act as a chemoattractant as well or may serve functions other than chemotaxis.This work was supported by a grant from the Deutsche Forschungsgemeinschaft

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

AbstractPeptides 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 Ca2+ concentration. In contrast, cAMP levels did not change significantly and the Ca2+ 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

Experimente zur Phosphorylierung und Mikroheterogenität von Arrestin (48-kDa Protein) aus Rinderretina

1 . Arrestin (48-kDa Protein, S-Antigen) aus Rinderretina wurde säulenchromatographisch roh-gereinigt. Nahezu reines Arrestin wurde durch anschließende FPLC-Anionenaustauschchromatographie erhalten. Roh- und FPLC-gereinigtes Arrestin zeigen eine ladungsbedingte Heterogenität, die durch isoelektrische Fokussierung und Immunoblotting mit einem monoklonalen anti-S-Antigen-Antikörper (S6H8) nachgewiesen wurde. Das IEF-Muster von gereinigtem Arrestin besteht aus 2-4 Hauptbanden und mindestens 5-10 Nebenbanden ; die isoelektrischen Punkte liegen zwischen pH 5.5 - 6.1. Arrestin-Subspezies wurden durch FPLC-Anionenaustauschchromatographie teilweise voneinander getrennt; die basischeren Hauptbanden 2 und 3 (pH 6.0 - 6.1) werden in einem sog. Vorpeak eluiert, wohingegen im Hauptpeak Banden 0- und 1-Arrestin (pH 5.8 - 5.9) sowie die anodaleren Nebenbanden (pH < 5.75) vorzufinden sind. Eine fast vollständige Trennung einzelner Arrestin-Subspezies voneinander habe ich durch FPLC-Chromatofokussierung erreicht. 2. Um einen postulierten lichtabhängigen ATP/ADP-Austausch am Arrestin zu überprüfen, habe ich Nukleotidaustausch- und Bindungsexperimente im rekonstituierten System, d. h. gereinigtes Arrestin + ROS-Membranen $\pm$ Licht, durchgeführt. Ich konnte keine lichtabhängige ATP-Bindungskapazität am Arrestin nachweisen, fand aber eine lichtabhängige Phosphorylierung am Arrestin. 3. Ich untersuchte, ob Arrestin von einer ROS-Protein-Kinase stöchiometrisch phosphoryliert wird und inwieweit die ladungsbedingte Heterogenität von Arrestin durch Phosphorylierung erklärt werden kann. [...

Experimente zur Phosphorylierung und Mikroheterogenität von Arrestin (48-kDa-Protein) aus Rinderretina

1 . Arrestin (48-kDa Protein, S-Antigen) aus Rinderretina wurde säulenchromatographisch roh-gereinigt. Nahezu reines Arrestin wurde durch anschließende FPLC-Anionenaustauschchromatographie erhalten. Roh- und FPLC-gereinigtes Arrestin zeigen eine ladungsbedingte Heterogenität, die durch isoelektrische Fokussierung und Immunoblotting mit einem monoklonalen anti-S-Antigen-Antikörper (S6H8) nachgewiesen wurde. Das IEF-Muster von gereinigtem Arrestin besteht aus 2-4 Hauptbanden und mindestens 5-10 Nebenbanden ; die isoelektrischen Punkte liegen zwischen pH 5.5 - 6.1. Arrestin-Subspezies wurden durch FPLC-Anionenaustauschchromatographie teilweise voneinander getrennt; die basischeren Hauptbanden 2 und 3 (pH 6.0 - 6.1) werden in einem sog. Vorpeak eluiert, wohingegen im Hauptpeak Banden 0- und 1-Arrestin (pH 5.8 - 5.9) sowie die anodaleren Nebenbanden (pH < 5.75) vorzufinden sind. Eine fast vollständige Trennung einzelner Arrestin-Subspezies voneinander habe ich durch FPLC-Chromatofokussierung erreicht. 2. Um einen postulierten lichtabhängigen ATP/ADP-Austausch am Arrestin zu überprüfen, habe ich Nukleotidaustausch- und Bindungsexperimente im rekonstituierten System, d. h. gereinigtes Arrestin + ROS-Membranen $\pm$ Licht, durchgeführt. Ich konnte keine lichtabhängige ATP-Bindungskapazität am Arrestin nachweisen, fand aber eine lichtabhängige Phosphorylierung am Arrestin. 3. Ich untersuchte, ob Arrestin von einer ROS-Protein-Kinase stöchiometrisch phosphoryliert wird und inwieweit die ladungsbedingte Heterogenität von Arrestin durch Phosphorylierung erklärt werden kann. [...

Molecular basis of coupled transport and anion conduction in excitatory amino acid transporters

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. After its release from presynaptic nerve terminals, glutamate is quickly removed from the synaptic cleft by excitatory amino acid transporters (EAATs) 1–5, a subfamily of glutamate transporters. The five proteins utilize a complex transport stoichiometry that couples glutamate transport to the symport of three Na(+) ions and one H(+) in exchange with one K(+) to accumulate glutamate against up to 10(6)-fold concentration gradients. They are also anion-selective channels that open and close during transitions along the glutamate transport cycle. EAATs belong to a larger family of secondary-active transporters, the SLC1 family, which also includes purely Na(+)- or H(+)-coupled prokaryotic transporters and Na(+)-dependent neutral amino acid exchangers. In recent years, molecular cloning, heterologous expression, cellular electrophysiology, fluorescence spectroscopy, structural approaches, and molecular simulations have uncovered the molecular mechanisms of coupled transport, substrate selectivity, and anion conduction in EAAT glutamate transporters. Here we review recent findings on EAAT transport mechanisms, with special emphasis on the highly conserved hairpin 2 gate, which has emerged as the central processing unit in many of these functions

CRIS—A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending

The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca(2+) regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca(2+)-regulated proteins that--in mature sperm--are involved in flagellar bending