Real-time measurements of the interactions between fluorescent speract and its sperm receptor

Lytechinus 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 (k(on)) and dissociation (k(off)) rate constants. The high k(on) (2.4 x 10(7) M(-1 )s(-1)) and low k(off) (4.4 x 10(-6) s(-1) (95%) and 3.7 x 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 NH(4)Cl (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.