Cd2+ sensitivity and permeability of a low voltage-activated Ca2+ channel with CatSper-like selectivity filter

• CatSper-like selectivity filter built in a CaV3.1 channel reduces sensitivity to Cd2+.
• The higher number of aspartates in the filter gives the higher permeability to Cd2+.
• Mn2+ influx is also enhanced in the CatSper-like selectivity filter.
• Progesterone, a potent CatSper activator, increases Cd2+ influx in human sperm.

CatSper is a sperm-specific Ca2+ channel that plays an essential role in the male fertility. However, its biophysical properties have been poorly characterized mainly due to its deficient heterologous expression. As other voltage-gated Ca2+ channels (CaVs), CatSper possesses a conserved Ca2+-selective filter motif ([T/S]x[D/E]xW) in the pore region. Interestingly, CatSper conserves four aspartic acids (DDDD) as the negatively charged residues in this motif while high voltage-activated CaVs have four glutamic acids (EEEE) and low voltage-activated CaVs possess two glutamic acids and two aspartic acids (EEDD). Previous studies based on site-directed mutagenesis of L- and T-type channels showed that the number of D seems to have a negative correlation with their cadmium (Cd2+) sensitivity. These results suggest that CatSper (DDDD) would have low sensitivity to Cd2+. To explore Cd2+-sensitivity and -permeability of CatSper, we performed two types of experiments: 1) Electrophysiological analysis of heterologously expressed human CaV3.1 channel and three pore mutants (DEDD, EDDD and DDDD), 2) Cd2+ imaging of human spermatozoa with FluoZin-1. Electrophysiological studies showed a significant increase in Cd2+ and manganese (Mn2+) currents through the CaV3.1 mutants as well as a reduction in the inhibitory effect of Cd2+ on the Ca2+ current. In fluorescence imaging with human sperm, we observed an increase in Cd2+ influx potentiated by progesterone, a potent activator of CatSper. These results support our hypothesis, namely that Cd2+-sensitivity and -permeability are related to the absolute number of D in the Ca2+-selective filter independently to the type of the Cav channels.

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