Limits...
Simultaneous knockout of Slo3 and CatSper1 abolishes all alkalization- and voltage-activated current in mouse spermatozoa.

Zeng XH, Navarro B, Xia XM, Clapham DE, Lingle CJ - J. Gen. Physiol. (2013)

Bottom Line: Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg.After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K(+) current hypothesized to reflect monovalent flux through CATSPER.Here, we address two questions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Life Science, Nanchang University, Nanchang, Jiangxi 330031, China.

ABSTRACT
During passage through the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that renders sperm competent to produce fertilization. Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg. In mouse sperm, the development of hyperactivated motility is dependent on cytosolic alkalization that then results in an increase in cytosolic Ca(2+). The elevation of Ca(2+) is thought to be primarily driven by the concerted interplay of two alkalization-activated currents, a K(+) current (KSPER) composed of pore-forming subunits encoded by the Kcnu1 gene (also termed Slo3) and a Ca(2+) current arising from a family of CATSPER subunits. After deletion of any of four CATSPER subunit genes (CATSPER1-4), the major remaining current in mouse sperm is alkalization-activated KSPER current. After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K(+) current hypothesized to reflect monovalent flux through CATSPER. Here, we address two questions. First, does the residual outward K(+) current present in the Slo3 (-/-) sperm arise from CATSPER? Second, can any additional membrane K(+) currents be detected in mouse sperm by patch-clamp methods other than CATSPER and KSPER? Here, using mice bred to lack both SLO3 and CATSPER1 subunits, we show conclusively that the voltage-activated outward current present in Slo3 (-/-) sperm is abolished when CATSPER is also deleted. Any leak currents that may play a role in setting the resting membrane potential in noncapacitated sperm are likely smaller than the pipette leak current and thus cannot be resolved within the limitation of the patch-clamp technique. Together, KSPER and CATSPER appear to be the sole ion channels present in mouse sperm that regulate membrane potential and Ca(2+) influx in response to alkalization.

Show MeSH

Related in: MedlinePlus

Residual current in dKO sperm is insensitive to 100 µM quinidine. (A) Currents were activated in a WT sperm with the indicated voltage protocol with symmetrical K+ gradients and pHi of 8.0. (left) Control saline. (right) 100 µM quinidine. Red traces correspond to 100-mV step. (B) In another WT sperm, the ±100-mV voltage ramp was used to activate currents before, during, and after application of 100 µM quinidine (red), highlighting effects of quinidine at more negative voltages. (C) Currents were activated in a dKO sperm as in A without (left) and with (right) 100 µM quinidine. Red traces correspond to 100 mV. (D) Ramp-activated currents in a dKO spermatozoa were monitored before, during (red), and after 100 µM quinidine. (A–D) The dashed lines represent 0 current level. (E) Mean conductances were calculated at the indicated voltages for six dKO sperm without (black) and with (red) 100 µM quinidine. There were no significant differences between groups. Error bars indicate SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC3753610&req=5

fig2: Residual current in dKO sperm is insensitive to 100 µM quinidine. (A) Currents were activated in a WT sperm with the indicated voltage protocol with symmetrical K+ gradients and pHi of 8.0. (left) Control saline. (right) 100 µM quinidine. Red traces correspond to 100-mV step. (B) In another WT sperm, the ±100-mV voltage ramp was used to activate currents before, during, and after application of 100 µM quinidine (red), highlighting effects of quinidine at more negative voltages. (C) Currents were activated in a dKO sperm as in A without (left) and with (right) 100 µM quinidine. Red traces correspond to 100 mV. (D) Ramp-activated currents in a dKO spermatozoa were monitored before, during (red), and after 100 µM quinidine. (A–D) The dashed lines represent 0 current level. (E) Mean conductances were calculated at the indicated voltages for six dKO sperm without (black) and with (red) 100 µM quinidine. There were no significant differences between groups. Error bars indicate SEM.

Mentions: The small residual conductance in the dKO sperm is certainly dominated by GL but might also include some small contribution of membrane ion channels, which might be important in intact sperm when KSPER and CATSPER are not activated. As an additional test of the nature of any conductance that might still be present in dKO sperm with pipette pH 8.0, we used quinidine, a rather general ion channel blocker which has been shown to block SLO3 currents (>90% inhibition at 100 µM quinidine at >100 mV; Tang et al., 2010; Zeng et al., 2011) as well as CATSPER (Zeng et al., 2011). In addition, quinidine also inhibits two pore leak channels (e.g., TASK2: 65% inhibition at 100 µM [Reyes et al., 1998]; and TASK3: 37% block by 100 µM [Kim et al., 2000]) that have also been proposed as possible sperm ion channels (Barfield et al., 2005), in addition to Kv channels (Fedida, 1997; Wang et al., 2003). Thus, because of the general effectiveness of quinidine on a host of cation channels, any sensitivity of the residual currents in the dKO sperm to quinidine might suggest the presence of additional types of channels. In WT sperm, 100 µM quinidine substantially inhibits both outward and inward currents recorded in symmetric K+ solutions with pipette pH of 8.0 (Fig. 2, A and B). This reflects inhibition of both KSPER and CATSPER currents (Zeng et al., 2011). Note that the residual conductance in WT sperm at negative potentials after application of 100 µM quinidine approaches that of the dKO sperm (Fig. 2, B and D). When 100 µM quinidine was applied to dKO sperm at pHi 8.0 (Figs. 1 D and 2, C and D), the residual current was little affected. The effects of 100 µM quinidine on conductances measured from −100 to 100 mV were determined for six dKO sperm (Fig. 2 E). Individual sperm in some cases exhibited small decreases in current during application of quinidine (e.g., Fig. 2 C). Although the differences measured over the set of cells was not significant, the mean conductance in the presence of quinidine (Fig. 2 C) corresponds to a decrease of 12.7 ± 4.9% compared with control levels. We conclude that quinidine-sensitive ion channels make minor contributions, if any, to the residual conductance of dKO sperm.


Simultaneous knockout of Slo3 and CatSper1 abolishes all alkalization- and voltage-activated current in mouse spermatozoa.

Zeng XH, Navarro B, Xia XM, Clapham DE, Lingle CJ - J. Gen. Physiol. (2013)

Residual current in dKO sperm is insensitive to 100 µM quinidine. (A) Currents were activated in a WT sperm with the indicated voltage protocol with symmetrical K+ gradients and pHi of 8.0. (left) Control saline. (right) 100 µM quinidine. Red traces correspond to 100-mV step. (B) In another WT sperm, the ±100-mV voltage ramp was used to activate currents before, during, and after application of 100 µM quinidine (red), highlighting effects of quinidine at more negative voltages. (C) Currents were activated in a dKO sperm as in A without (left) and with (right) 100 µM quinidine. Red traces correspond to 100 mV. (D) Ramp-activated currents in a dKO spermatozoa were monitored before, during (red), and after 100 µM quinidine. (A–D) The dashed lines represent 0 current level. (E) Mean conductances were calculated at the indicated voltages for six dKO sperm without (black) and with (red) 100 µM quinidine. There were no significant differences between groups. Error bars indicate SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3753610&req=5

fig2: Residual current in dKO sperm is insensitive to 100 µM quinidine. (A) Currents were activated in a WT sperm with the indicated voltage protocol with symmetrical K+ gradients and pHi of 8.0. (left) Control saline. (right) 100 µM quinidine. Red traces correspond to 100-mV step. (B) In another WT sperm, the ±100-mV voltage ramp was used to activate currents before, during, and after application of 100 µM quinidine (red), highlighting effects of quinidine at more negative voltages. (C) Currents were activated in a dKO sperm as in A without (left) and with (right) 100 µM quinidine. Red traces correspond to 100 mV. (D) Ramp-activated currents in a dKO spermatozoa were monitored before, during (red), and after 100 µM quinidine. (A–D) The dashed lines represent 0 current level. (E) Mean conductances were calculated at the indicated voltages for six dKO sperm without (black) and with (red) 100 µM quinidine. There were no significant differences between groups. Error bars indicate SEM.
Mentions: The small residual conductance in the dKO sperm is certainly dominated by GL but might also include some small contribution of membrane ion channels, which might be important in intact sperm when KSPER and CATSPER are not activated. As an additional test of the nature of any conductance that might still be present in dKO sperm with pipette pH 8.0, we used quinidine, a rather general ion channel blocker which has been shown to block SLO3 currents (>90% inhibition at 100 µM quinidine at >100 mV; Tang et al., 2010; Zeng et al., 2011) as well as CATSPER (Zeng et al., 2011). In addition, quinidine also inhibits two pore leak channels (e.g., TASK2: 65% inhibition at 100 µM [Reyes et al., 1998]; and TASK3: 37% block by 100 µM [Kim et al., 2000]) that have also been proposed as possible sperm ion channels (Barfield et al., 2005), in addition to Kv channels (Fedida, 1997; Wang et al., 2003). Thus, because of the general effectiveness of quinidine on a host of cation channels, any sensitivity of the residual currents in the dKO sperm to quinidine might suggest the presence of additional types of channels. In WT sperm, 100 µM quinidine substantially inhibits both outward and inward currents recorded in symmetric K+ solutions with pipette pH of 8.0 (Fig. 2, A and B). This reflects inhibition of both KSPER and CATSPER currents (Zeng et al., 2011). Note that the residual conductance in WT sperm at negative potentials after application of 100 µM quinidine approaches that of the dKO sperm (Fig. 2, B and D). When 100 µM quinidine was applied to dKO sperm at pHi 8.0 (Figs. 1 D and 2, C and D), the residual current was little affected. The effects of 100 µM quinidine on conductances measured from −100 to 100 mV were determined for six dKO sperm (Fig. 2 E). Individual sperm in some cases exhibited small decreases in current during application of quinidine (e.g., Fig. 2 C). Although the differences measured over the set of cells was not significant, the mean conductance in the presence of quinidine (Fig. 2 C) corresponds to a decrease of 12.7 ± 4.9% compared with control levels. We conclude that quinidine-sensitive ion channels make minor contributions, if any, to the residual conductance of dKO sperm.

Bottom Line: Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg.After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K(+) current hypothesized to reflect monovalent flux through CATSPER.Here, we address two questions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Life Science, Nanchang University, Nanchang, Jiangxi 330031, China.

ABSTRACT
During passage through the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that renders sperm competent to produce fertilization. Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg. In mouse sperm, the development of hyperactivated motility is dependent on cytosolic alkalization that then results in an increase in cytosolic Ca(2+). The elevation of Ca(2+) is thought to be primarily driven by the concerted interplay of two alkalization-activated currents, a K(+) current (KSPER) composed of pore-forming subunits encoded by the Kcnu1 gene (also termed Slo3) and a Ca(2+) current arising from a family of CATSPER subunits. After deletion of any of four CATSPER subunit genes (CATSPER1-4), the major remaining current in mouse sperm is alkalization-activated KSPER current. After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K(+) current hypothesized to reflect monovalent flux through CATSPER. Here, we address two questions. First, does the residual outward K(+) current present in the Slo3 (-/-) sperm arise from CATSPER? Second, can any additional membrane K(+) currents be detected in mouse sperm by patch-clamp methods other than CATSPER and KSPER? Here, using mice bred to lack both SLO3 and CATSPER1 subunits, we show conclusively that the voltage-activated outward current present in Slo3 (-/-) sperm is abolished when CATSPER is also deleted. Any leak currents that may play a role in setting the resting membrane potential in noncapacitated sperm are likely smaller than the pipette leak current and thus cannot be resolved within the limitation of the patch-clamp technique. Together, KSPER and CATSPER appear to be the sole ion channels present in mouse sperm that regulate membrane potential and Ca(2+) influx in response to alkalization.

Show MeSH
Related in: MedlinePlus