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 Ca(2+) channels must open to initiate required, Ca(2+)-mediated events. However, the molecular identity and functional properties of sperm Ca(2+) 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 [Ca(2+)] evoked by approximately 10-s applications of an alkaline high [K(+)] medium directly reports activity of voltage-gated Ca(2+) channels. Little response occurs if external Ca(2+) is removed or if external or internal pH is elevated without depolarization. Responses are inhibited 30-40% by 30-100 micrometer Ni(2+) and more completely by 100-300 micrometer Cd(2+). They resist the dihydropyridines nitrendipine and PN200-110, but 1-10 micrometer mibefradil inhibits reversibly. They also resist the venom toxins calciseptine, omega-conotoxin MVIIC, and kurtoxin, but omega-conotoxin GVIA (5 micrometer) inhibits approximately 50%. GVIA also partially blocks transient, low voltage activated Ca(2+) currents of patch-clamped spermatids. Differential sensitivity of sperm responses to Ni(2+) and Cd(2+) and partial blockade by GVIA indicate that depolarization opens at least two types of voltage-gated Ca(2+) 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 Ca(2+) 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 micrometer mibefradil and an enhanced sensitivity of the GVIA-resistant component of response to Ni(2+) suggest participation of a Ca(V)2.3 (R-type) channel specified by previously found alpha(1E) subunits. Our examination of depolarization-evoked Ca(2+) entry indicates that mature sperm possess a larger palette of voltage-gated Ca(2+) channels than previously thought. Such diversity may permit specific responses to multiple cues encountered on the path to fertilization.
Wennemuth
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Departments of Physiology and Biophysics and Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195-7290, USA.
Westenbroek
R E
RE
Xu
T
T
Hille
B
B
Babcock
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DF
D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020864:Calcium Channels, N-Type / Q000145:classification / Q000235:genetics / Q000502:physiology*; D020908:Calcium Channels, R-Type / Q000145:classification / Q000235:genetics / Q000502:physiology*; D020013:Calcium Signaling / Q000502:physiology*; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D017275:Isradipine / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D020748:Mibefradil / Q000494:pharmacology; D051379:Mice; D009498:Neurotoxins / Q000494:pharmacology; D009532:Nickel / Q000494:pharmacology; D009568:Nitrendipine / Q000494:pharmacology; D012604:Scorpion Venoms / Q000494:pharmacology; D013094:Spermatozoa / Q000502:physiology*; D020866:omega-Conotoxin GVIA / Q000494:pharmacology; D020960:omega-Conotoxins / Q000494:pharmacology
D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D020908:Calcium Channels, R-Type; D009498:Neurotoxins; D012604:Scorpion Venoms; C118444:kurtoxin; D020960:omega-Conotoxins; D002104:Cadmium; C075851:omega-conotoxin-MVIIC; D020748:Mibefradil; D009532:Nickel; D020866:omega-Conotoxin GVIA; D009568:Nitrendipine; D002118:Calcium; D017275:Isradipine