Deletion of theSlo3gene 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.

Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells

“…Unexpectedly, sperm motility of Best1 −/− mice was not fully rescued by the hypertonic solution, in contrast to reports, for example, for spermatozoa from the Slo −/− mouse (43). As suggested by Voets et al (44), this finding may be explained by VRAC activation in Best1 −/− spermatozoa before ejaculation and could be triggered by reduced intracellular ionic strength.…”

Flagellar ion channels of sperm: similarities and differences between species

“…Slo3 was also sensitive to Ba 2+ , quinine and mibefradil that all reversibly inhibited murine Slo3 [29]. The identity and importance of the Slo3 channel for murine sperm physiology was confirmed by recordings from Slo3-deficient mice that also display severely reduced male fertility [25,31]. While Slo3 was also expected to form the potassium channel of human sperm, recent recording from ejaculated human spermatozoa indicates that in clear contrast to murine sperm, human sperm potassium current (KSper) is pH-independent and sensitive to [Ca 2+ ] i [20].…”

“…Noncapacitated epididymal murine spermatozoa are slightly depolarized at about −40 mV, however they hyperpolarize up to −60 mV upon capacitation [137]. This effect is attributed to potassium permeability and two members of the Slo family of potassium channels have been recently proposed to play a role in this process [21,25,26,29,31,33,[138][139][140][141]. Slo3 (Kcnu1), a pH-sensitive, calcium-independent and weakly voltage-sensitive channel, has been identified as the principal potassium channel in murine sperm [25,26,29,31,33,[141][142][143].…”

Cl− Channels and Transporters in Sperm Physiology

“…Motility is one of the most important functions carried out by the sperm because it is essential to achieve fertilization. Indeed, several sperm motility defects can cause male sterility (e.g., sAC, PKA, sNHE, GAPDHs, CatSper, PMCA4, SLO3) (Esposito et al 2004 ;Miki et al 2004 ;Nolan et al 2004 ;Okunade et al 2004 ;Quill et al 2001 ;Ren et al 2001 ;Santi et al 2010 ;Wang et al 2007 ;Zeng et al 2011 ). Sperm motility is driven by the fl agellum, an appendage with an ultrastructure very similar to that of cilia.…”

“…Achieving whole-cell patch-clamp recordings became more feasible when Kirichok et al ( 2006 ) were able to seal the cytoplasmic droplet of mouse epididymal spermatozoa and then mature human spermatozoa (Kirichok and Lishko 2011 ). This novel strategy is allowing the characterization of spermspecifi c channels such as CatSper (Kirichok et al 2006 ) and SLO3 (Navarro et al 2007 ;Santi et al 2010 ;Schreiber et al 1998 ;Zeng et al 2011 ), and of sperm anion channels that are present in other cell types Ferrera et al 2010 ). Additionally, a voltage-sensitive H + channel involved in the intracellular pH (pH i ) regulation in human sperm and less importantly in mouse sperm (Kirichok and Lishko 2011 ), and ATP-gated channels of the purinergic family, P2X2, in mouse epididymal sperm have been recorded (Navarro et al 2011 ).…”