Primary structure and functional expression of a cyclic nucleotidegated channel from rabbit aorta

Sequences specific for cyclic nucleotide-gated channels (CNG channels) have been amplified by PCR from cDNA of heart, aorta, sinoatrial node, cerebellum, C-cells and kidney. The complete amino acid sequence of a CNG channel from rabbit aorta has been deduced by cloning and sequence analysis of the cDNA. Synthetic RNA derived from this cDNA induces the formation of a functional CNG channel in Xenopus oocytes

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

Molecular architecture of a retinal cGMP-gated channel: the arrangement of the cytoplasmic domains

Cyclic nucleotide-gated (CNG) channels play a central role in the conversion of sensory information, such as light and scent, into primary electrical signals. We have purified the CNG channel from bovine retina and have studied it using electron microscopy and image processing. We present the structure of the channel to 35Å resolution. This three-dimensional reconstruction provides insight into the architecture of the protein, suggesting that the cyclic nucleotide-binding domains, which initiate the response to ligand, ‘hang’ below the pore-forming part of the channel, attached by narrow linkers. The structure also suggests that the four cyclic nucleotide-binding domains present in each channel form two distinct domains, lending structural weight to the suggestion that the four subunits of the CNG channels are arranged as a pair of dimers

Control of intracellular pH and bicarbonate by CO2 diffusion into human sperm

Abstract The reaction of CO2 with H2O to form bicarbonate (HCO3 −) and H+ controls sperm motility and fertilization via HCO3 −-stimulated cAMP synthesis. A complex network of signaling proteins participates in this reaction. Here, we identify key players that regulate intracellular pH (pHi) and HCO3 − in human sperm by quantitative mass spectrometry (MS) and kinetic patch-clamp fluorometry. The resting pHi is set by amiloride-sensitive Na+/H+ exchange. The sperm-specific putative Na+/H+ exchanger SLC9C1, unlike its sea urchin homologue, is not gated by voltage or cAMP. Transporters and channels implied in HCO3 − transport are not detected, and may be present at copy numbers < 10 molecules/sperm cell. Instead, HCO3 − is produced by diffusion of CO2 into cells and readjustment of the CO2/HCO3 −/H+ equilibrium. The proton channel Hv1 may serve as a unidirectional valve that blunts the acidification ensuing from HCO3 − synthesis. This work provides a new framework for the study of male infertility

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. In particular, the ion channels that are involved in Ca2+ influx and their mechanisms of gating are not known. Using rapid voltage-sensitive dyes and kinetic techniques, we record the voltage response that is evoked by the chemoattractant in sperm from the sea urchin Arbacia punctulata. We show that the chemoattractant evokes a brief hyperpolarization followed by a sustained depolarization. The hyperpolarization is caused by the opening of K+-selective cyclic-nucleotide-gated (CNG) channels in the flagellum. Ca2+ influx commences at the onset of recovery from hyperpolarization. The voltage threshold of Ca2+ entry indicates the involvement of low-voltage-activated Ca(v) channels. These results establish a model of chemosensory transduction in sperm whereby a cGMP-induced hyperpolarization opens Ca(v) channels by a 'recovery-from-inactivation' mechanism and unveil an evolutionary kinship between transduction mechanisms in sperm and photoreceptors