Juan Ferreira

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

Background: SLO3 and CATSPER are two sperm-specific ion channels. Results: SLO3 K+ channels control Ca2+ entry through CATSPER channels. Conclusion: SLO3 control of CATSPER channel activity involves an intermediary step in which SLO3-dependent hyperpolarization may elicit internal alkalization via a voltage-dependent mechanism. Significance: Understanding the control of Ca2+ entry in sperm is crucial to understanding fertility; this study also reveals an unusual role for a K+ channel. Here we show how a sperm-specific potassium channel (SLO3) controls Ca2+ entry into sperm through a sperm-specific Ca2+ channel, CATSPER, in a totally unanticipated manner. The genetic deletion of either of those channels confers male infertility in mice. During sperm capacitation SLO3 hyperpolarizes the sperm, whereas CATSPER allows Ca2+ entry. These two channels may be functionally connected, but it had not been demonstrated that SLO3-dependent hyperpolarization is required for Ca2+ entry through CATSPER channels, nor has a functional mechanism linking the two channels been shown. In this study we show that Ca2+ entry through CATSPER channels is deficient in Slo3 mutant sperm lacking hyperpolarization; we also present evidence supporting the hypothesis that SLO3 channels activate CATSPER channels indirectly by promoting a rise in intracellular pH through a voltage-dependent mechanism. This mechanism may work through a Na+/H+ exchanger (sNHE) and/or a bicarbonate transporter, which utilizes the inward driving force of the Na+ gradient, rendering it intrinsically voltage-dependent. In addition, the sperm-specific Na+/H+ exchanger (sNHE) possess a putative voltage sensor that might be activated by membrane hyperpolarization, thus increasing the voltage sensitivity of internal alkalization.

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

Background: Membrane potential (Em) hyperpolarization during sperm capacitation is necessary for acrosome reaction. Results: cSrc is activated downstream of PKA, regulating the SLO3 K+ channel and promoting membrane hyperpolarization. Conclusion: Acrosomal responsiveness of mouse sperm depends on cSrc activation of SLO3. Significance: This represents the first evidence of K+ channel regulation in mouse sperm by a tyrosine kinase, affecting Em. Plasma membrane hyperpolarization is crucial for mammalian sperm to acquire acrosomal responsiveness during capacitation. Among the signaling events leading to mammalian sperm capacitation, the immediate activation of protein kinase A plays a pivotal role, promoting the subsequent stimulation of protein tyrosine phosphorylation that associates with fertilizing capacity. We have shown previously that mice deficient in the tyrosine kinase cSrc are infertile and exhibit improper cauda epididymis development. It is therefore not clear whether lack of sperm functionality is due to problems in epididymal maturation or to the absence of cSrc in sperm. To further address this problem, we investigated the kinetics of cSrc activation using anti-Tyr(P)-416-cSrc antibodies that only recognize active cSrc. Our results provide evidence that cSrc is activated downstream of PKA and that inhibition of its activity blocks the capacitation-induced hyperpolarization of the sperm plasma membrane without blocking the increase in tyrosine phosphorylation that accompanies capacitation. In addition, we show that cSrc inhibition also blocks the agonist-induced acrosome reaction and that this inhibition is overcome by pharmacological hyperpolarization. Considering that capacitation-induced hyperpolarization is mediated by SLO3, we evaluated the action of cSrc inhibitors on the heterologously expressed SLO3 channel. Our results indicate that, similar to SLO1 K+ channels, cSrc blockers significantly decreased SLO3-mediated currents. Together, these results are consistent with findings showing that hyperpolarization of the sperm plasma membrane is necessary and sufficient to prepare the sperm for the acrosome reaction and suggest that changes in sperm membrane potential are mediated by cSrc activation.

A Genetic Variant of the Sperm-specific SLO3 K+ Channel has altered pH and Ca2+ sensitivities.

To fertilize an oocyte, sperm must first undergo capacitation in which the sperm plasma membrane becomes hyperpolarized via activation of potassium (K+) channels and resultant K+ efflux. Sperm-specific SLO3 K+ channels are responsible for these membrane potential changes critical for fertilization in mouse sperm, and they are only sensitive to pHi. However, in human sperm, the major K+ conductance is both Ca2+- and pHi-sensitive. It has been debated whether Ca2+-sensitive SLO1 channels substitute for human SLO3 (hSLO3) in human sperm or whether human SLO3 channels have acquired Ca2+ sensitivity. Here we show that hSLO3 is rapidly evolving and reveal a natural structural variant with enhanced apparent Ca2+ and pH sensitivities. This variant allele (C382R) alters an amino acid side chain at a principal interface between the intramembrane-gated pore and the cytoplasmic gating ring of the channel. Because the gating ring contains sensors to intracellular factors such as pH and Ca2+, the effectiveness of transduction between the gating ring and the pore domain appears to be enhanced. Our results suggest that sperm-specific genes can evolve rapidly and that natural genetic variation may have led to a SLO3 variant that differs from wild type in both pH and intracellular Ca2+ sensitivities. Whether this physiological variation confers differences in fertility among males remains to be established.

SLO2 Channels Are Inhibited by All Divalent Cations That Activate SLO1 K+ Channels

Two members of the family of high conductance K+ channels SLO1 and SLO2 are both activated by intracellular cations. However, SLO1 is activated by Ca2+ and other divalent cations, while SLO2 (Slack or SLO2.2 from rat) is activated by Na+. Curiously though, we found that SLO2.2 is inhibited by all divalent cations that activate SLO1, with Zn2+ being the most effective inhibitor with an IC50 of ∼8 μm in contrast to Mg2+, the least effective, with an IC50 of ∼ 1.5 mm. Our results suggest that divalent cations are not SLO2 pore blockers, but rather inhibit channel activity by an allosteric modification of channel gating. By site-directed mutagenesis we show that a histidine residue (His-347) downstream of S6 reduces inhibition by divalent cations. An analogous His residue present in some CNG channels is an inhibitory cation binding site. To investigate whether inhibition by divalent cations is conserved in an invertebrate SLO2 channel we cloned the SLO2 channel from Drosophila (dSLO2) and compared its properties to those of rat SLO2.2. We found that, like rat SLO2.2, dSLO2 was also activated by Na+ and inhibited by divalent cations. Inhibition of SLO2 channels in mammals and Drosophila by divalent cations that have second messenger functions may reflect the physiological regulation of these channels by one or more of these ions.