T-type Ca+ channel are implicated in diverse physiological processes, including cardiac pacemaker activity and spontaneous neuronal bursting in the central nervous system. This channel is also prominent in cells that are not spontaneously active, such as in mouse sperm where it is activated by the egg's extracellular matrix (the zona pellucida) during fertilization. However, efforts to identify the molecular constituents of this channel have been unsuccessful. This application takes advantage of unique features of mouse sperm that make it a feasible model system for the characterization of the constituents of the T channel. The objective during this funding period is to identify the constituents of the sperm T channel by classical biochemical and molecular genetic methods.
Aim 1 purifies a T channel- antagonist binding protein from sperm membrane preparations.
Aim 2 described the molecular cloning of cDNA that encode this protein using a reverse-genetic strategy.
In Aim 3, full length cDNA that encode the T channel antagonist binding site are expressed in Xenopus oocytes and assayed for Ca2+ channel activity.
In Aim 4 site-directed antibodies are generated against synthetic peptides from the channel protein. These antibodies are used to demonstrate the presence of the T channel protein on mouse sperm in immunocytochemical experiments. Finally, in Aim 5 additional protein associated with the T channel antagonist- binding protein will be identified by co-immunoprecipitation strategies using site-directed antibodies. These experiments will permit the first identification of the protein components of a low voltage-activated Ca2+ channel. As such, this study will contribute to our understanding of the mechanisms of mammalian fertilization and to the rationale design of channel-based contraceptives. In addition, this is the first step towards a molecular understanding of the function and the regulation of this channel in germ and in somatic cells.
| Lopez-Gonzalez, I; De La Vega-Beltran, J L; Santi, C M et al. (2001) Calmodulin antagonists inhibit T-type Ca(2+) currents in mouse spermatogenic cells and the zona pellucida-induced sperm acrosome reaction. Dev Biol 236:210-9 |
| Gonzalez-Martinez, M T; Galindo, B E; de De La Torre, L et al. (2001) A sustained increase in intracellular Ca(2+) is required for the acrosome reaction in sea urchin sperm. Dev Biol 236:220-9 |
| O'Toole, C M; Arnoult, C; Darszon, A et al. (2000) Ca(2+) entry through store-operated channels in mouse sperm is initiated by egg ZP3 and drives the acrosome reaction. Mol Biol Cell 11:1571-84 |
