Deletion of the Slo3 gene abolishes alkalization-activated K+ current in mouse spermatozoa

Abstract

Mouse spermatozoa express a pH-dependent K(+) current (KSper) thought to be composed of subunits encoded by the Slo3 gene. However, the equivalence of KSper and Slo3-dependent current remains uncertain, because heterologous expression of Slo3 results in currents that are less effectively activated by alkalization than are native KSper currents. Here, we show that genetic deletion of Slo3 abolishes all pH-dependent K(+) current at physiological membrane potentials in corpus epididymal sperm. A residual pH-dependent outward current (I(Kres)) is observed in Slo3(-/-) sperm at potentials of >0 mV. Differential inhibition of KSper/Slo3 and I(Kres) by clofilium reveals that the amplitude of I(Kres) is similar in both wild-type (wt) and Slo3(-/-) sperm. The properties of I(Kres) suggest that it likely represents outward monovalent cation flux through CatSper channels. Thus, KSper/Slo3 may account for essentially all mouse sperm K(+) current and is the sole pH-dependent K(+) conductance in these sperm. With physiological ionic gradients, alkalization depolarizes Slo3(-/-) spermatozoa, presumably from CatSper activation, in contrast to Slo3/KSper-mediated hyperpolarization in wt sperm. Slo3(-/-) male mice are infertile, but Slo3(-/-) sperm exhibit some fertility within in vitro fertilization assays. Slo3(-/-) sperm exhibit a higher incidence of morphological abnormalities accentuated by hypotonic challenge and also exhibit deficits in motility in the absence of bicarbonate, revealing a role of KSper under unstimulated conditions. Together, these results show that KSper/Slo3 is the primary spermatozoan K(+) current, that KSper may play a critical role in acquisition of normal morphology and sperm motility when faced with hyperosmotic challenges, and that Slo3 is critical for fertility.

Fig. 1.

KO of Slo3 results in removal of most K⁺ current in corpus epididymal sperm. (A) A 1-s voltage ramp was used to activate current from wt, Slo3^−/− (red), Slo3^+/− (blue), and Slo3–eGFP^+/+ (green) spermatozoa. Each example approximates the average behavior for that particular genotype. Pipette pH was 8.0; intracellular and extracellular salines each contained 160 mM K·Mes, with 2 mM extracellular Ca²⁺ to reduce monovalent flux through CatSper channels. (B) Mean current estimates at +100 mV with a cytosolic pH of 8.0 are plotted for four genotypes (wt: n = 25 cells, 17 mice; Slo3^+/−: 10 cells, 4 mice; Slo3^−/−: 13 cells, 8 mice; Slo3–eGFP: 6 cells, 4 mice). (C) Mean current measured at +100 mV is plotted for wt and Slo3^−/− sperm at pH 6.0 (wt: n = 8, 2 mice; KO: n = 10, 4 mice), 7.0 (wt: 9 cells, 2 mice; KO: 6 cells, 2 mice), and 8.0 (wt: 25 cells, 17 mice; KO:13 cells, 8 mice). (D) Currents were activated in a Slo3^−/− sperm with the ±100-mV voltage ramp with a pH 6.0 pipette solution. Current during application of 10 mM NH₄Cl is in red. (E) Mean current was measured at +100 mV in Slo3^−/− sperm before and during application of NH₄Cl with a pipette solution at either pH 6.0 (n = 5, two mice) or pH 8.0 (n = 3, one mouse).

Fig. 2.

Distinct voltage dependence and kinetic properties of current present following Slo3 deletion. (A) Traces show current activated in a wt sperm from a holding potential of −100 mV with steps up to +200 mV. Red trace highlights current activated at +100 mV. (Pipette pH 8.0.) (B) Activation of current from traces in A after subtraction of uncompensated capacity currents (using the step to 0 mV for digital subtraction) reveals both instantaneous and time-dependent current activation. (C) Traces show currents from a Slo3^−/− sperm. (Pipette pH 8.0.) (D) After subtraction of uncompensated capacity transients, currents in Slo3^−/− sperm are only activated at potentials positive to 0 mV and exhibit prominent voltage-dependent increases in current activation. (E) Steady-state currents for wt (n = 11) and Slo3^−/− (n = 9) sperm were converted to conductances assuming a 0-mV reversal potential. At potentials negative to 0 mV, essentially all residual K⁺ conductance is absent in Slo3^−/− sperm.

Fig. 3.

Clofilium distinguishes between I_KSper and I_Kres, revealing that I_Kres is identical in both wt and Slo3^−/− sperm. (A) Top traces show currents activated in a wt sperm with cytosolic pH 8.0 with voltage steps up to +200 mV (red, +100 mV). Capacity transients were digitally subtracted. Application of 50 μM clofilium (middle traces) blocks most rapidly activating current and most current at +100 mV. Most current at +100 mV remains inhibited following washout of clofilium (bottom traces). (B) The time course of onset and recovery from block of wt currents by 50 μM clofilium (at +200 mV) is plotted. Washout from clofilium shows both rapid and essentially irreversible components of recovery. (C) Clofilium inhibits outward current in Slo3^−/− sperm, but clofilium inhibition is quickly reversed upon washout. (D) Time course of clofilium block and recovery in Slo3^−/− sperm. (E) Average net conductance from wt sperm before (open circles), during block by 50 μM clofilium (red circles), and after ~180 s washout of clofilium (diamonds). (n = 5 cells.) (F) Average net conductance from Slo3^−/− sperm for control (open circles), 50 μM clofilium (red circles), and after wash (diamonds). (n = 4 cells.) (G) The component of net conductance irreversibly blocked by clofilium for wt and Slo3^−/− sperm is plotted.

Fig. 4.

Spermatozoa from Slo3^−/− mice lack alkalization-mediated hyperpolarization. (A) Membrane potential was recorded in a wt mouse spermatozoan bathed in HS medium, and 10 mM NH₄Cl was applied as indicated. (Pipette saline pH 6.0.) (B) Membrane potential in a Slo3^−/− spermatozoan was monitored, and NH₄Cl was applied as indicated. (C) Mean values for resting potential for either Slo3^+/+, Slo3^+/−, or Slo3^−/− sperm are plotted for both HS and for HS supplemented with 10 mM NH₄Cl, all with pH 6.0 intracellular saline. (D) Mean values for membrane potential for Slo3^+/+, Slo3^+/−, and Slo3^−/− sperm are plotted. (Pipette saline pH 8.0.)

Fig. 5.

Slo3^−/− sperm have osmoregulatory deficits. (A) Caudal epididymal sperm were allowed to swim out into a 430 mmol/kg HS medium. At t = 0, aliquots were diluted into solutions of different osmolality with added 25 mM NaHCO₃. Moving sperm were categorized as either normal or abnormal (bent or hairpin) morphology. The occurrence of abnormal morphology is increased in wt sperm with reductions in osmolality, and this effect is increased in Slo3^−/− sperm. Each red point represents the estimate from over 100 motile sperm from a single mouse, with error bars indicating SEM. (B) Osmolality-related deficits in morphology also occur in the absence of NaHCO₃. Each red dot corresponds to the estimate from sperm from a single mouse (n = 3–5 for each condition).