ISOLATION AND CHARACTERIZATION OF THE PRIMARY STRUCTURE OF TESTIS-SPECIFIC L-TYPE CALCIUM-CHANNEL - IMPLICATIONS FOR CONTRACEPTION

Therapeutic administration of calcium channel-blocking medications has been correlated with reduced mannose receptor expression and iatrogen ic human male infertility. In this report, we investigate whether the pharmacological activity of dihydropyridines, which block calcium infl ux through voltage-dependent calcium channels, contributes to the prod uction of an infertile state. An influx of extracellular calcium is an absolute requirement for the initiation of a progesterone-stimulated acrosome reaction by human spermatozoa. To determine whether dihydropy ridines could inhibit progesterone-induced acrosome loss, we have stud ied a protein expressed in rat and human spermatozoa which is related both antigenically and by cDNA sequence to the al subunit of the rat c ardiac muscle voltage-dependent calcium channel, which forms the pore of the channel. Using reverse transcription-polymerase chain reaction, we have isolated a 2169 base clone from rat testis mRNA whose sequenc e was largely identical to that of the al subunit of the rat cardiac m uscle calcium channel, but had an 84 base change, attributable to spli cing and alternate exon usage. This change inserts a peptide cassette encoding an amphipathic membrane-spanning helix that constitutes part of the ionic pore of the skeletal muscle calcium channel regulating th e kinetics of activation of the calcium channel and may serve as an in tramembrane dihydropyridine binding site. In parallel, human spermatoz oa from fertile donors were exposed to nifedipine in vitro. Nifedipine inhibited progesterone-stimulated calcium influx and subsequent acros ome reactions in human spermatozoa at concentrations effective in exci table cells, but required a prolonged time to do so. In contrast, prog esterone ligand binding was unaffected by nifedipine treatment, These data demonstrate that human spermatozoa express an L-type calcium chan nel which is responsive to nifedipine. Assuming sperm calcium transpor t pathways are highly conserved, the slow kinetics by which the blocka de of the human sperm channel was obtained can be correlated with alte rations in channel activation and conductance associated with isoform diversity generated by alternate splicing as observed in the rat. Thes e data provide unequivocal evidence for the presence of functional L-t ype voltage-dependent calcium channels in rat and human spermatozoa. T he data also define an altered binding site for calcium entry antagoni sts in this channel and offer a unique target for the design of new ma le contraceptive agents.