Ca(v)2.2 and Ca(v)2,3 (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 init iate required, Ca2+-mediated events. However, the molecular identity and fu nctional properties of sperm Ca2+ channels remain uncertain. Here, we use r apid local perfusion and single-cell photometry to examine the kinetics of calcium responses of mouse sperm to depolarizing stimuli. The linear rise o f intracellular [Ca2+] evoked by similar to 10-s applications of an alkalin e high [K+] medium directly reports activity of voltage-gated Ca2+ channels . Little response occurs if external Ca2+ is removed or if external or inte rnal 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 t he dihydropyridines nitrendipine and PN200-110, but 1-10 mu M mibefradil in hibits reversibly. They also resist the venom toxins calciseptine, omega-co notoxin MVIIC, and kurtoxin, but omega-conotoxin GVIA (5 mu M) inhibits sim ilar to 50%, GVIA also partially blocks transient, low voltage activated Ca 2+ currents of patch-clamped spermatids, Differential sensitivity of sperm responses to Ni2+ and Cd2+ and partial blockade by GVIA indicate that depol arization opens at least two types of voltage-gated Ca2+ channels in epidid ymal sperm examined prior to capacitation. Involvement of a previously unde tected Ca(V)2,2 (N-type) channel, suggested by the action of GVIA, is subst antiated by immunodetection of Ca2+ channel alpha(1B) subunits in sperm and sperm extracts. Resistance to dihydropyridines, calciseptine, MVIIC, and k urtoxin 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 compone nt 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 depol arization-evoked Ca2+ entry indicates that mature sperm possess a larger pa lette of voltage-gated Ca2+ channels than previously thought. Such diversit y may permit specific responses to multiple cues encountered on the path to fertilization.