KSper, a pH-sensitive K+ current that controls sperm membrane potential

Abstract

Mature mammalian spermatozoa are quiescent in the male reproductive tract. Upon ejaculation and during their transit through the female reproductive tract, they undergo changes that enable them to fertilize the egg. During this process of capacitation, they acquire progressive motility, develop hyperactivated motility, and are readied for the acrosome reaction. All of these processes are regulated by intracellular pH. In the female reproductive tract, the spermatozoan cytoplasm alkalinizes, which in turn activates a Ca2+-selective current (I(CatSper)) required for hyperactivated motility. Here, we show that alkalinization also has a dramatic effect on membrane potential, producing a rapid hyperpolarization. This hyperpolarization is primarily mediated by a weakly outwardly rectifying K+ current (I(KSper)) originating from the principal piece of the sperm flagellum. Alkalinization activates the pH(i)-sensitive I(KSper), setting the membrane potential to negative potentials where Ca2+ entry via I(CatSper) is maximized. I(KSper) is one of two dominant ion currents of capacitated sperm cells.

Fig. 1.

pH_i sets the murine spermatozoan resting membrane potential. (a) Spermatozoan in whole-cell patch-clamp configuration. The pipette contains 1 mM Lucifer yellow, which fills the cytoplasmic space (Lower). Differential interference contrast image (Upper). (Scale bar, 10 μm.) (b) WT spermatozoan resting membrane potential (pH_i 6.0) before and after induction of intracellular alkalinization by bath perfusion with 5 mM NH₄Cl. Reduction of extracellular Cl⁻ ions, or the addition of 25 μM amiloride (ENaC channel antagonist), did not block the alkalinization-induced hyperpolarization. Representative of three similar recordings is shown. (c) WT I_KSper at pH_i 6.0 before (red) and after (black) addition of 5 mM NH₄Cl in normal saline (HS) or low Cl⁻ solution (green). The dashed line indicates 0 current level. Also shown is the voltage-clamp ramp protocol from −100 to +100 mV [1 sec; holding potential (HP), 0 mV]. (d) Mean membrane potential in varying pH_i. Alkalinization dramatically shifts the spermatozoan's resting membrane potential (averaged data from experiments similar to a).

Fig. 2.

Endogenous sperm cell K⁺ current (I_KSper). (a) Weakly outwardly rectifying K⁺ current from a WT spermatozoan measured in symmetrical 160 mM [K⁺] (pH_i 8.0) in response to a 1-sec ramp protocol from −100 to +100 mV [holding potential (HP), 0 mV]. (b) I_KSper of CatSper1^−/− spermatozoa in response to voltage steps. Whole-cell currents elicited by 1-sec steps from HP of 0 mV to test potentials between −100 and +100 mV (Δ = +20 mV; 5-sec intervals; pH_i 8.0). (c) Reversal potentials of a CatSper1^−/− spermatozoan were measured in response to changing [K⁺]_o (pH_i 8.0) from 160 mM (black trace; E_rev = 1.2 ± 0.34 mV; n = 30) to 5 mM (red trace; E_rev = −72.5 ± 0.71 mV; n = 21; predicted E_K = −87 mV). Green trace, 0 mM [K⁺]_o. (d) Head plus midpiece sperm fragment loaded with Lucifer yellow dye (Lower). Differential interference contrast (DIC) image (Upper). (Scale bar, 10 μm.) (e) Midpiece + principal piece sperm fragment loaded with Lucifer yellow dye (Lower). DIC image (Upper). (Scale bar, 10 μm.) (f) Current in CatSper1^−/− spermatozoa in response to a 1-sec voltage-clamp ramp from −100 to +100 mV from the head + midpiece (red) and from the midpiece + principal piece (black; pH_i 8.0).

Fig. 3.

Intracellular alkalinization strongly potentiates I_KSper. (a) Steady-state whole-cell currents of CatSper1^−/− spermatozoa at pH_i 6.0 (red), 7.0 (blue), and 8.0 (black). (b) Initial current (red) of CatSper1^−/− spermatozoa in symmetrical 160 mM [K⁺] at pH_i 6.0. Addition of 5 mM NH₄Cl to the bath alkalinized the cytoplasm and induced I_KSper (black). Alkalinization-induced I_KSper in 5 mM (purple) or 80 mM (green) [K⁺]_o. (c) Average I_KSper amplitudes at −100 and +100 mV at varying pH_i. HP, holding potential.

Fig. 4.

I_KSper antagonists and their effects on sperm membrane potential. (a) I_KSper antagonist effect on CatSper1^−/− spermatozoa (symmetrical 160 mM [K⁺] at pH_i 7.5, +100mV). (b) Spermatozoan membrane potential (WT; pH_i 7.0) was reversibly depolarized by 500 μM quinine but irreversibly depolarized by 50 μM clofilium (washout, 15 min). Addition of 5 mM NH₄Cl had a small effect on membrane potential. A representative trace from four independent experiments is shown. (c) Bath perfusion with 2 mM BaCl₂, 5 μM mibefradil, and 50 μM EIPA reversibly depolarized the WT sperm cell membrane potential (pH_i 7.0). A representative trace from four independent experiments is shown. (d) EIPA (50 μM) blocked WT sperm hyperpolarization induced by 1 mM NH₄Cl or 4 mM 4-AP (pH_i 6.0). Amiloride (25 μM) had no effect on V_m. A representative trace from three independent experiments is shown.