Cav2.2 and Cav2,3 (N- and R-type) Ca2+ channels in depolarization-evoked entry of Ca2+ into mouse sperm

As sperm prepare for fertilization, surface Ca2+ channels must open to initiate required, Ca2+-mediated events. However, the molecular identity and functional properties of sperm Ca2+ channels remain uncertain. Here, we use rapid local perfusion and single-cell photometry to examine the kinetics of calcium responses of mouse sperm to depolarizing stimuli. The linear rise of intracellular [Ca2+] evoked by similar to 10-s applications of an alkaline high [K+] medium directly reports activity of voltage-gated Ca2+ channels. Little response occurs if external Ca2+ is removed or if external or internal pH is elevated without depolarization. Responses are inhibited 30-40% by 30-100 mu M Ni2+ and more completely by 100-300 mu M Cd2+. They resist the dihydropyridines nitrendipine and PN200-110, but 1-10 mu M mibefradil inhibits reversibly. They also resist the venom toxins calciseptine, omega-conotoxin MVIIC, and kurtoxin, but omega-conotoxin GVIA (5 mu M) inhibits similar to 50%, GVIA also partially blocks transient, low voltage activated Ca2+ currents of patch-clamped spermatids, Differential sensitivity of sperm responses to Ni2+ and Cd2+ and partial blockade by GVIA indicate that depolarization opens at least two types of voltage-gated Ca2+ channels in epididymal sperm examined prior to capacitation. Involvement of a previously undetected Ca(V)2,2 (N-type) channel, suggested by the action of GVIA, is substantiated by immunodetection of Ca2+ channel alpha(1B) subunits in sperm and sperm extracts. Resistance to dihydropyridines, calciseptine, MVIIC, and kurtoxin indicates that Ca(V)1, Ca(V)2.1, and Ca(V)3 (L-, P/Q-, and T-type) channels contribute little to this evoked response. Partial sensitivity to 1 mu M mibefradil and an enhanced sensitivity of the GVIA-resistant component of response to Ni2+ suggest participation of a Ca(V)2.3 (R-type) channel specified by previously found alpha(1E) subunits. Our examination of depolarization-evoked Ca2+ entry indicates that mature sperm possess a larger palette of voltage-gated Ca2+ channels than previously thought. Such diversity may permit specific responses to multiple cues encountered on the path to fertilization.