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-fold concentration gradients.

Allosteric gate modulation confers K coupling in glutamate transporters.

Excitatory amino acid transporters (EAATs) mediate glial and neuronal glutamate uptake to terminate synaptic transmission and to ensure low resting glutamate concentrations. Effective glutamate uptake is achieved by cotransport with 3 Na and 1 H , in exchange with 1 K . The underlying principles of this complex transport stoichiometry remain poorly understood.

High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor.

Guanylyl cyclases (GCs), which synthesize the messenger cyclic guanosine 3',5'-monophosphate, control several sensory functions, such as phototransduction, chemosensation, and thermosensation, in many species from worms to mammals. The GC chemoreceptor in sea urchin sperm can decode chemoattractant concentrations with single-molecule sensitivity. The molecular and cellular underpinnings of such ultrasensitivity are not known for any eukaryotic chemoreceptor.

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.

Induced fit substrate binding to an archeal glutamate transporter homologue.

Excitatory amino acid transporters (EAATs) are a class of glutamate transporters that terminate glutamatergic synaptic transmission in the mammalian CNS. GltPh, an archeal EAAT homolog from Pyrococcus horikoshii, is currently the only member with a known 3D structure. Here, we studied the kinetics of substrate binding of a single tryptophan mutant (L130W) GltPh in detergent micelles.

The CatSper channel: a polymodal chemosensor in human sperm.

The sperm-specific CatSper channel controls the intracellular Ca(2+) concentration ([Ca(2+)](i)) and, thereby, the swimming behaviour of sperm. In humans, CatSper is directly activated by progesterone and prostaglandins-female factors that stimulate Ca(2+) influx. Other factors including neurotransmitters, chemokines, and odorants also affect sperm function by changing [Ca(2+)](i).

The CatSper channel mediates progesterone-induced Ca2+ influx in human sperm.

In the oviduct, cumulus cells that surround the oocyte release progesterone. In human sperm, progesterone stimulates a Ca(2+) increase by a non-genomic mechanism. The Ca(2+) signal has been proposed to control chemotaxis, hyperactivation and acrosomal exocytosis of sperm.

Caged progesterone: a new tool for studying rapid nongenomic actions of progesterone.

Ketalization of the biomolecule progesterone with (6-bromo-7-hydroxycoumarin-4-yl)ethane-1,2-diol gives the photolabile progesterone derivatives 3 and 4. These compounds display dramatically reduced bioactivity and release progesterone upon irradiation with UV/vis or IR light. In particular, 4 can be used to perform concentration-jump experiments with high temporal and spatial resolution that allows one to study elegantly the mechanisms of rapid nongenomic cellular events evoked by progesterone.

Mechanisms of sperm chemotaxis.

Sperm are attracted by chemical factors that are released by the egg-a process called chemotaxis. Most of our knowledge on sperm chemotaxis originates from the study of marine invertebrates. In recent years, the main features of the chemotactic signaling pathway and the swimming behavior evoked by chemoattractants have been elucidated in sea urchins.

A K+-selective cGMP-gated ion channel controls chemosensation of sperm.

Eggs attract sperm by chemical factors, a process called chemotaxis. Sperm from marine invertebrates use cGMP signalling to transduce incident chemoattractants into changes in the Ca2+ concentration in the flagellum, which control the swimming behaviour during chemotaxis. The signalling pathway downstream of the synthesis of cGMP by a guanylyl cyclase is ill-defined.

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.

Revisiting the role of H+ in chemotactic signaling of sperm.

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.

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.

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+.