Ion Permeabilities in Mouse Sperm Reveal an External Trigger for SLO3-Dependent Hyperpolarization

Abstract

Unlike most cells of the body which function in an ionic environment controlled within narrow limits, spermatozoa must function in a less controlled external environment. In order to better understand how sperm control their membrane potential in different ionic conditions, we measured mouse sperm membrane potentials under a variety of conditions and at different external K+ concentrations, both before and after capacitation. Experiments were undertaken using both wild-type, and mutant mouse sperm from the knock-out strain of the sperm-specific, pH-sensitive, SLO3 K+ channel. Membrane voltage data were fit to the Goldman-Hodgkin-Katz equation. Our study revealed a significant membrane permeability to both K+ and Cl− before capacitation, as well as Na+. The permeability to both K+ and Cl− has the effect of preventing large changes in membrane potential when the extracellular concentration of either ion is changed. Such a mechanism may protect against undesired shifts in membrane potential in changing ionic environments. We found that a significant portion of resting membrane potassium permeability in wild-type sperm was contributed by SLO3 K+ channels. We also found that further activation of SLO3 channels was the essential mechanism producing membrane hyperpolarization under two separate conditions, 1) elevation of external pH prior to capacitation and 2) capacitating conditions. Both conditions produced a significant membrane hyperpolarization in wild-type which was absent in SLO3 mutant sperm. Hyperpolarization in both conditions may result from activation of SLO3 channels by raising intracellular pH; however, demonstrating that SLO3-dependent hyperpolarization is achieved by an alkaline environment alone shows that SLO3 channel activation might occur independently of other events associated with capacitation. For example sperm may undergo stages of membrane hyperpolarization when reaching alkaline regions of the female genital tract. Significantly, other events associated with sperm capacitation, occur in SLO3 mutant sperm and thus proceed independently of hyperpolarization.

SLO3 K+ Channels Control Calcium Entry through CATSPER Channels in Sperm

“…2C could be that Slo3 Ϫ/Ϫ sperm fail to undergo internal alkalization when subjected to capacitating conditions. However, we (9,11,17) and other laboratories (16,18) have shown that a primary role of SLO3 channels is to evoke sperm membrane hyperpolarization when exposed to capacitating conditions. Thus, membrane hyperpolarization could be a factor that itself substitutes for an increase in intracellular pH, or SLO3-dependent membrane hyperpolarization could be involved in the mechanism by which internal alkalization occurs during capacitation.…”

“…The events that lead to the activation of SLO3 channels and membrane hyperpolarization are complex and varied, but generally, external conditions favorable for capacitation promote internal alkalization (5,6). Indeed, we have found that sperm encountering an external alkaline environment alone is sufficient to activate SLO3 channels (9), probably by an increase in pH i .…”

“…Both of these channels are activated by intracellular alkalization, and both are voltage-activated and open upon depolarization (15,20). Although voltage-sensitive, sperm-specific SLO3 channels can contribute to the resting potential (9) SLO3 channels have relatively high conductance (15), and the opening of only a relatively small number of SLO3 channels at resting potential in response to intracellular alkalization is sufficient to hyperpolarize the sperm membrane (9). We and others have shown that activation of the SLO3 K ϩ channel is the principal mechanism whereby the sperm plasma membrane hyperpolarizes during capacitation (16 -18).…”

Src Kinase Is the Connecting Player between Protein Kinase A (PKA) Activation and Hyperpolarization through SLO3 Potassium Channel Regulation in Mouse Sperm

“…After incubation for the indicated period, the potential-sensitive dye DiSC 3 -(5), which has been successfully used in mammalian sperm, was added to a final concentration of 1 M, and sperm were incubated for 2 min. No mitochondrial uncouplers were used because their contribution to the resting potential has been determined to be insignificant (17). The sperm were transferred to a gently stirred cuvette at 37°C, and the fluorescence was monitored with a Varian Cary Eclipse fluorescence spectrophotometer at 620/670 nm excitation/emission wavelength.…”

“…Recordings were initiated when steady-state fluorescence was reached (approximately 1 min). Calibration was performed by adding 1 M valinomycin and sequential additions of KCl as described previously (17). The final sperm membrane potential was obtained by linearly interpolating the theoretical E m values against arbitrary fluorescence units of each trace.…”