Calmodulin antagonists inhibit T-type Ca(2+) currents in mouse spermatogenic cells and the zona pellucida-induced sperm acrosome reaction.
The sperm acrosome reaction (AR) is a regulated exocytotic process required for gamete fusion. It depends on an increase in [Ca(2+)](i) mediated by Ca(2+) channels. Although calmodulin (CaM) has been reported to regulate several events during the AR, it is not known whether it modulates sperm Ca(2+) channels. In the present study we analyzed the effects of CaM antagonists W7 and trifluoroperazine on voltage-dependent T-type Ca(2+) currents in mouse spermatogenic cells and on the zona pellucida-induced AR in sperm. We found that these CaM antagonists decreased T-currents in a concentration-dependent manner with IC(50) values of approximately 10 and approximately 12 microM, respectively. W7 altered the channels' voltage dependence of activation and slowed both activation and inactivation kinetics. It also induced inactivation at voltages at which T-channels are not activated, suggesting a promotion of inactivation from the closed state. Consistent with this, W7 inhibited the ZP-induced [Ca(2+)](i) transients in capacitated sperm. Likewise, W7 and TFP inhibited the AR with an IC(50) of approximately 10 microM. In contrast, inhibitors of CaM-dependent kinase II and protein kinase A, as well as a CaM-activated phosphatase, had no effect either on T-currents in spermatogenic cells or on the sperm AR. Together these results suggest a functional interaction between CaM and the sperm T-type Ca(2+) channel. They are also consistent with the involvement of T-channels in the AR.
López-González
I
I
Department of Genetics and Molecular Physiology, Institute of Biotechnology, UNAM, Mexico.
De La Vega-Beltrán
J L
JL
Santi
C M
CM
Florman
H M
HM
Felix
R
R
Darszon
A
A
D020101:Acrosome Reaction; D000818:Animals; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology*; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008297:Male; D051379:Mice; D018408:Patch-Clamp Techniques; D013094:Spermatozoa / Q000166:cytology* / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D013997:Time Factors; D014268:Trifluoperazine / Q000494:pharmacology; D015044:Zona Pellucida / Q000378:metabolism*
D015220:Calcium Channels; D002147:Calmodulin; D004791:Enzyme Inhibitors; D013449:Sulfonamides; D014268:Trifluoperazine; C017967:W 7; D002118:Calcium