Human Slo1 Research Articles

Transmembrane determinants of voltage-gating differences between BK (Slo1) and Slo3 channels

Voltage-gated potassium channels are critical in modulating cellular excitability, with Slo (slowpoke) channels forming a unique family characterized by their large conductance and dual regulation by electrical signals and intracellular messengers. Despite their structural and evolutionary similarities, Slo1 and Slo3 channels exhibit significant differences in their voltage-gating properties. This study investigates the molecular determinants that differentiate the voltage-gating properties of human Slo1 and mouse Slo3 channels. Utilizing Slo1/Slo3 chimeras, we pinpointed the selectivity filter region as a key factor in the Slo3 channel’s reduced conductance at negative voltages. The S6 transmembrane (TM) segment was identified as pivotal for the Slo3 channel’s biphasic deactivation kinetics at these voltages. Additionally, the S4 and S6 TM segments were found to be responsible for the gradual slope in the Slo3 channel’s conductance-voltage relationship, while multiple TM regions appear to be involved in the Slo3 channel’s requirement of strong depolarization for activation. Mutations in the Slo1’s S5 and S6 TM segments revealed three residues (I233, L302, and M304) that likely play a crucial role in the allosteric coupling between the voltage sensors and the pore gate.

The sperm-specific K+ channel Slo3 is inhibited by albumin and steroids contained in reproductive fluids.

To locate and fertilize the egg, sperm probe the varying microenvironment prevailing at different stages during their journey across the female genital tract. To this end, they are equipped with a unique repertoire of mostly sperm-specific proteins. In particular, the flagellar Ca2+ channel CatSper has come into focus as a polymodal sensor used by human sperm to register ligands released into the female genital tract. Here, we provide the first comprehensive study on the pharmacology of the sperm-specific human Slo3 channel, shedding light on its modulation by reproductive fluids and their constituents. We show that seminal fluid and contained prostaglandins and Zn2+ do not affect the channel, whereas human Slo3 is inhibited in a non-genomic fashion by diverse steroids as well as by albumin, which are released into the oviduct along with the egg. This indicates that not only CatSper but also Slo3 harbours promiscuous ligand-binding sites that can accommodate structurally diverse molecules, suggesting that Slo3 is involved in chemosensory signalling in human sperm.

E-cigarette aerosols of propylene glycol impair BK channel activity and parameters of mucociliary function.

Propylene glycol (PG) is a common delivery vehicle for nicotine and flavorings in e-cigarette (e-cig) liquids and is largely considered safe for ingestion. However, little is known about its effects as an e-cig aerosol on the airway. Here, we investigated whether pure PG e-cig aerosols in realistic daily amounts impact parameters of mucociliary function and airway inflammation in a large animal model (sheep) in vivo and primary human bronchial epithelial cells (HBECs) in vitro. Five-day exposure of sheep to e-cig aerosols of 100% PG increased mucus concentrations (% mucus solids) of tracheal secretions. PG e-cig aerosols further increased the activity of matrix metalloproteinase-9 (MMP-9) in tracheal secretions. In vitro exposure of HBECs to e-cig aerosols of 100% PG decreased ciliary beating and increased mucus concentrations. PG e-cig aerosols further reduced the activity of large conductance, Ca2+-activated, and voltage-dependent K+ (BK) channels. We show here for the first time that PG can be metabolized to methylglyoxal (MGO) in airway epithelia. PG e-cig aerosols increased levels of MGO and MGO alone reduced BK activity. Patch-clamp experiments suggest that MGO can disrupt the interaction between the major pore-forming BK subunit human Slo1 (hSlo1) and the gamma regulatory subunit LRRC26. PG exposures also caused a significant increase in mRNA expression levels of MMP9 and interleukin 1 beta (IL1B). Taken together, these data show that PG e-cig aerosols cause mucus hyperconcentration in sheep in vivo and HBECs in vitro, likely by disrupting the function of BK channels important for airway hydration.

Homozygous mutation in SLO3 leads to severe asthenoteratozoospermia due to acrosome hypoplasia and mitochondrial sheath malformations

BackgroundPotassium channels are important for the structure and function of the spermatozoa. As a potassium transporter, the mSlo3 is essential for male fertility as Slo3 knockout male mice were infertile with the series of functional defects in sperm cells. However, no pathogenic variant has been detected in human SLO3 to date. Here we reported a human case with homozygous SLO3 mutation. The function of SLO3 in human sperm and the corresponding assisted reproductive strategy are also investigated.MethodsWe performed whole-exome sequencing analysis from a large cohort of 105 patients with asthenoteratozoospermia. The effects of the variant were investigated by quantitative RT-PCR, western blotting, and immunofluorescence assays using the patient spermatozoa. Sperm morphological and ultrastructural studies were conducted using haematoxylin and eosin staining, scanning and transmission electron microscopy.ResultsWe identified a homozygous missense variant (c.1237A > T: p.Ile413Phe) in the sperm-specific SLO3 in one Chinese patient with male infertility. This SLO3 variant was rare in human control populations and predicted to be deleterious by multiple bioinformatic tools. Sperm from the individual harbouring the homozygous SLO3 variant exhibited severe morphological abnormalities, such as acrosome hypoplasia, disruption of the mitochondrial sheath, coiled tails, and motility defects. The levels of SLO3 mRNA and protein in spermatozoa from the affected individual were reduced. Furthermore, the acrosome reaction, mitochondrial membrane potential, and membrane potential during capacitation were also afflicted. The levels of acrosome marker glycoproteins and PLCζ1 as well as the mitochondrial sheath protein HSP60 and SLO3 auxiliary subunit LRRC52, were significantly reduced in the spermatozoa from the affected individual. The affected man was sterile due to acrosome and mitochondrial dysfunction; however, intra-cytoplasmic sperm injection successfully rescued this infertile condition.ConclusionsSLO3 deficiency seriously impact acrosome formation, mitochondrial sheath assembly, and the function of K+ channels. Our findings provided clinical implications for the genetic and reproductive counselling of affected families.

Fe2+-Mediated Activation of BKCa Channels by Rapid Photolysis of CORM-S1 Releasing CO and Fe2.

Heme catabolism by heme oxygenase (HO) with a decrease in intracellular heme concentration and a concomitant local release of CO and Fe2+ has the potential to regulate BKCa channels. Here, we show that the iron-based photolabile CO-releasing molecule CORM-S1 [dicarbonyl-bis(cysteamine)iron(II)] coreleases CO and Fe2+, making it a suitable light-triggered source of these downstream products of HO activity. To investigate the impact of CO, iron, and cysteamine on BKCa channel activation, human Slo1 (hSlo1) was expressed in HEK293T cells and studied with electrophysiological methods. Whereas hSlo1 channels are activated by CO and even more strongly by Fe2+, Fe3+ and cysteamine possess only marginal activating potency. Investigation of hSlo1 mutants revealed that Fe2+ modulates the channels mainly through the Mg2+-dependent activation mechanism. Flash photolysis of CORM-S1 suits for rapid and precise delivery of Fe2+ and CO in biological settings.

Cloning and characterization of the rat Slo3 (KCa 5.1) channel: From biophysics to pharmacology.

The Slo3 potassium (KCa 5.1) channel, which is specifically expressed in the testis and sperm, is essential for mammalian male fertilization. The sequence divergence of the bovine, mouse and human Slo3 α-subunit revealed a rapid evolution rate across different species. The rat Slo3 (rSlo3) channel has not been cloned and characterized previously. We used molecular cloning, electrophysiology (inside-out patches and outside-out patches) and mutagenesis to investigate the biophysical properties and pharmacological characteristics of the rSlo3 channel. The rat Slo3 channel (rSlo3) is gated by voltage and cytosolic pH rather than intracellular calcium. The characteristics of voltage-dependent, pH-sensitivity and activation kinetics of the rSlo3 channel differ from the characteristics of other Slo3 orthologues. In terms of pharmacology, the 4-AP blockade of the rSlo3 channel also shows properties distinct from its blockade of the mSlo3 channel. Iberiotoxin and progesterone weakly inhibit the rSlo3 channel. Finally, we found that propofol, one of the widely used general anaesthetics, blocks the rSlo3 channel from both intracellular and extracellular sides, whereas ketamine only blocks the rSlo3 channel at the extracellular side. Our findings suggest that the rSlo3 channel possesses unique biophysical and pharmacological properties. Our results provide new insights into the diversities of the Slo3 family of channels, which are valuable for estimating the effects of the use of these drugs to improve sperm quality.

Molecular structures of the human Slo1 K+ channel in complex with β4.

Slo1 is a Ca2+- and voltage-activated K+ channel that underlies skeletal and smooth muscle contraction, audition, hormone secretion and neurotransmitter release. In mammals, Slo1 is regulated by auxiliary proteins that confer tissue-specific gating and pharmacological properties. This study presents cryo-EM structures of Slo1 in complex with the auxiliary protein, β4. Four β4, each containing two transmembrane helices, encircle Slo1, contacting it through helical interactions inside the membrane. On the extracellular side, β4 forms a tetrameric crown over the pore. Structures with high and low Ca2+ concentrations show that identical gating conformations occur in the absence and presence of β4, implying that β4 serves to modulate the relative stabilities of 'pre-existing' conformations rather than creating new ones. The effects of β4 on scorpion toxin inhibition kinetics are explained by the crown, which constrains access but does not prevent binding.

Large-conductance Ca2+- and voltage-gated K+ channels form and break interactions with membrane lipids during each gating cycle

Membrane depolarization and intracellular Ca2+ promote activation of the large-conductance Ca2+- and voltage-gated (Slo1) big potassium (BK) channel. We examined the physical interactions that stabilize the closed and open conformations of the ion conduction gate of the human Slo1 channel using electrophysiological and computational approaches. The results show that the closed conformation is stabilized by intersubunit ion-ion interactions involving negative residues (E321 and E324) and positive residues (329RKK331) at the cytoplasmic ends of the transmembrane S6 segments ("RKK ring"). When the channel gate is open, the RKK ring is broken and the positive residues instead make electrostatic interactions with nearby membrane lipid oxygen atoms. E321 and E324 are stabilized by water. When the 329RKK331 residues are mutated to hydrophobic amino acids, these residues form even stronger hydrophobic interactions with the lipid tails to promote the open conformation, shifting the voltage dependence of activation to the negative direction by up to 400 mV and stabilizing the selectivity filter region. Thus, the RKK segment forms electrostatic interactions with oxygen atoms from two sources, other amino acid residues (E321/E324), and membrane lipids, depending on the gate status. Each time the channel opens and closes, the aforementioned interactions are formed and broken. This lipid-dependent Slo1 gating may explain how amphipathic signaling molecules and pharmacologically active agents influence the channel activity, and a similar mechanism may be operative in other ion channels.

A Novel High-Throughput Screening Assay for State-Dependent and Subunit-Dependent BK Channel Modulators

BK potassium channels are attractive drug targets for a wide variety of human disorders affecting almost every organ system. However, the therapeutic potential of many BK channel modulators is limited by lack of tissue specificity, reflecting that the pore forming a-subunit (Slo1) is encoded by a single gene. Properties of modulators that can potentially enhance tissue specificity include (a) sensitivity to regulatory subunits that are expressed in a tissue-dependent manner and (b) state-dependent action, conferring sensitivity to tissue-specific differences in membrane voltage and Ca2+ that activate BK channels. We have developed a novel 384-well fluorescent thallium-flux kinetic (FLIPR) assay to identify modulators with these properties. The screen includes assays on a panel of BK channel variants composed of hyperactive human Slo1 mutants (F380Y or R275C) in the presence or absence of different regulatory subunits (β1, β2, β2αFIW, β4, γ1), expressed in U2OS cells with Bacmam virus. Hyperactive Slo1 mutants allow both inhibitors and activators to be reliably detected under resting cellular conditions. Comparison of R275C and F380Y identifies state-dependent modulators since the voltage-sensors of R275C are constitutively activated while F380Y are not. A test screen of small molecules and arachnid venoms revealed a surprising degree of variant-selectivity and identified novel modulators with activities that depend on voltage-sensor activation and/or regulatory subunit expression. Thus, the screen detects modulators that would fail to be detected by conventional screens while also providing information about mechanism of action that should facilitate SAR analysis and development of potent tissue-selective modulators.

Onion peel extract and its constituent, quercetin inhibits human Slo3 in a pH and calcium dependent manner.

Sperm function and male fertility are closely related to pH dependent K+ current (KSper) in human sperm, which is most likely composed of Slo3 and its auxiliary subunit leucine-rich repeat-containing protein 52 (LRRC52). Onion peel extract (OPE) and its major active ingredient quercetin are widely used as fertility enhancers; however, the effect of OPE and quercetin on Slo3 has not been elucidated. The purpose of this study is to investigate the effect of quercetin on human Slo3 channels. Human Slo3 and LRRC52 were co-transfected into HEK293 cells and pharmacological properties were studied with the whole cell patch clamp technique. We successfully expressed and measured pH sensitive and calcium insensitive Slo3 currents in HEK293 cells. We found that OPE and its key ingredient quercetin inhibit Slo3 currents. Inhibition by quercetin is dose dependent and this degree of inhibition decreases with elevating internal alkalization and internal free calcium concentrations. Functional moieties in the quercetin polyphenolic ring govern the degree of inhibition of Slo3 by quercetin, and the composition of such functional moieties are sensitive to the pH of the medium. These results suggest that quercetin inhibits Slo3 in a pH and calcium dependent manner. Therefore, we surmise that quercetin induced depolarization in spermatozoa may enhance the voltage gated proton channel (Hv1), and activate non-selective cation channels of sperm (CatSper) dependent calcium influx to trigger sperm capacitation and acrosome reaction.

A novel cross-species inhibitor to study the function of CatSper Ca2+ channels in sperm.

Sperm from many species share the sperm-specific Ca2+ channel CatSper that controls the intracellular Ca2+ concentration and, thereby, the swimming behaviour. A growing body of evidence suggests that the mechanisms controlling the activity of CatSper and its role during fertilization differ among species. A lack of suitable pharmacological tools has hampered the elucidation of the function of CatSper. Known inhibitors of CatSper exhibit considerable side effects and also inhibit Slo3, the principal K+ channel of mammalian sperm. The compound RU1968 was reported to suppress Ca2+ signaling in human sperm by an unknown mechanism. Here, we examined the action of RU1968 on CatSper in sperm from humans, mice, and sea urchins. We resynthesized RU1968 and studied its action on sperm from humans, mice, and the sea urchin Arbacia punctulata by Ca2+ fluorimetry, single-cell Ca2+ imaging, electrophysiology, opto-chemistry, and motility analysis. RU1968 inhibited CatSper in sperm from invertebrates and mammals. The compound lacked toxic side effects in human sperm, did not affect mouse Slo3, and inhibited human Slo3 with about 15-fold lower potency than CatSper. Moreover, in human sperm, RU1968 mimicked CatSper dysfunction and suppressed motility responses evoked by progesterone, an oviductal steroid known to activate CatSper. Finally, RU1968 abolished CatSper-mediated chemotactic navigation in sea urchin sperm. We propose RU1968 as a novel tool to elucidate the function of CatSper channels in sperm across species.

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 pH i However, in human sperm, the major K+ conductance is both Ca2+- and pH i -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.

Intracellular calcium-dependent regulation of the sperm-specific calcium-activated potassium channel, hSlo3, by the BKCa activator LDD175.

Plasma membrane hyperpolarization associated with activation of Ca2+-activated K+ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary γ2-subunit, LRRC52 (leucine-rich-repeat–containing 52), is known to mediate the pH-sensitive, sperm-specific K+ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical BKCa activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting γ2 subunit, hLRRC52. As previously reported, Slo3 K+ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 K+ current, and internal alkalinization and Ca2+ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 K+ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular Ca2+. In contrast, elevation of intracellular Ca2+ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate Ca2+-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.

Mechanistic insight into the heme-independent interplay between iron and carbon monoxide in CFTR and Slo1 BKCa channels.

Ion channels have been extensively reported as effectors of carbon monoxide (CO). However, the mechanisms of heme-independent CO action are still not known. Because most ion channels are heterologously expressed on human embryonic kidney cells that are cultured in Fe3+-containing media, CO may act as a small and strong iron chelator to disrupt a putative iron bridge in ion channels and thus to tune their activity. In this review CFTR and Slo1 BKCa channels are employed to discuss the possible heme-independent interplay between iron and CO. Our recent studies demonstrated a high-affinity Fe3+ site at the interface between the regulatory domain and intracellular loop 3 of CFTR. Because the binding of Fe3+ to CFTR prevents channel opening, the stimulatory effect of CO on the Cl- and HCO3- currents across the apical membrane of rat distal colon may be due to the release of inhibitive Fe3+ by CO. In contrast, CO repeatedly stimulates the human Slo1 BKCa channel opening, possibly by binding to an unknown iron site, because cyanide prohibits this heme-independent CO stimulation. Here, in silico research on recent structural data of the slo1 BKCa channels indicates two putative binuclear Fe2+-binding motifs in the gating ring in which CO may compete with protein residues to bind to either Fe2+ bowl to disrupt the Fe2+ bridge but not to release Fe2+ from the channel. Thus, these two new regulation models of CO, with iron releasing from and retaining in the ion channel, may have significant and extensive implications for other metalloproteins.

Pharmacology of hSlo3 channels and their contribution in the capacitation-associated hyperpolarization of human sperm

Slo3 channels (mSlo3) primarily mediate mouse sperm K+ currents and are essential for the capacitation-associated hyperpolarization (CAH). Whether Slo3 and/or Slo1, two Slo family K+ channels are functionally expressed in human sperm is controversial. Our recent pharmacological studies of the human sperm CAH suggested the participation of both. Lack of a detailed pharmacology of heterologously expressed human Slo3 (hSlo3) prevented precisely identifying the K+ channel(s) involved. In the present report, we compare the pharmacological profile of expressed hSlo3 in CHO cells with that of the CAH to advance this matter. Whole-cell patch-clamp recordings showed that hSlo3 currents are inhibited: significantly by progesterone, Ba2+ and quinidine; partially by Penitrem A and Charybdotoxin; and poorly by Iberiotoxin and Slotoxin. Surprisingly, hSlo3 currents were resistant to Clofilium and 60 mM TEA+ which inhibit mSlo3. Pharmacological comparison of the CAH and hSlo3 profiles indicates in addition to hSlo3, other K+ channels, possibly Slo1, may participate in CAH. The pharmacological profile of heterologously expressed hSlo3 channels differs from that of mSlo3 K+ channels, consistent with species-specific differences observed among other sperm ion channels. While the pharmacological correlation analysis of the hSlo3 currents and the CAH confirmed the participation of hSlo3 channels, it suggests that additional K+ channels may be involved, in particular Slo1 channels.

Two distinct effects of PIP2 underlie auxiliary subunit-dependent modulation of Slo1 BK channels.

Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a critical role in modulating the function of numerous ion channels, including large-conductance Ca(2+)- and voltage-dependent K(+) (BK, Slo1) channels. Slo1 BK channel complexes include four pore-forming Slo1 (α) subunits as well as various regulatory auxiliary subunits (β and γ) that are expressed in different tissues. We examined the molecular and biophysical mechanisms underlying the effects of brain-derived PIP2 on human Slo1 BK channel complexes with different subunit compositions that were heterologously expressed in human embryonic kidney cells. PIP2 inhibited macroscopic currents through Slo1 channels without auxiliary subunits and through Slo1 + γ1 complexes. In contrast, PIP2 markedly increased macroscopic currents through Slo1 + β1 and Slo1 + β4 channel complexes and failed to alter macroscopic currents through Slo1 + β2 and Slo1 + β2 Δ2-19 channel complexes. Results obtained at various membrane potentials and divalent cation concentrations suggest that PIP2 promotes opening of the ion conduction gate in all channel types, regardless of the specific subunit composition. However, in the absence of β subunits positioned near the voltage-sensor domains (VSDs), as in Slo1 and probably Slo1 + γ1, PIP2 augments the negative surface charge on the cytoplasmic side of the membrane, thereby shifting the voltage dependence of VSD-mediated activation in the positive direction. When β1 or β4 subunits occupy the space surrounding the VSDs, only the stimulatory effect of PIP2 is evident. The subunit compositions of native Slo1 BK channels differ in various cell types; thus, PIP2 may exert distinct tissue- and divalent cation-dependent modulatory influences.

Differential Effects of PIP2 on Slo1 BK Channels with Different Auxiliary Subunits

Phosphatidylinositol 4, 5-bisphosphate (PIP2) regulates numerous ion channels, including large-conductance Ca2+- and voltage-dependent K+ (Slo1 BK) channels (Vaithianathan et al. 2008, J Gen Physiol 132:13-28). We examined the molecular and biophysical mechanisms of the effects of PIP2 on human Slo1 BK channels with different auxiliary subunits heterologously expressed in HEK cells. In the absence of heterologously expressed auxiliary subunits, bovine brain PIP2 (10 µM) applied to the cytoplasmic side inhibits currents through Slo1 channels. This inhibition is associated with a shift in GV by 18 mV and deceleration of the activation kinetics at positive voltages. In contrast, PIP2 markedly increases currents through Slo1+β1 and hSlo1+β4 channels by shifting their GV curves by −46 mV and −34 mV, respectively. The stimulatory effect of PIP2 on Slo1+β1 channels does not require Ca2+-sensor activation or voltage-sensor activation. Currents through Slo1+β2 channels with a deletion in the N terminus of β2 to remove inactivation (Δ2-19) are not enhanced by PIP2 but neutralization of 3 negatively charged residues in the β2 Δ2-19 N terminus introduces a modest effect of PIP2. In Slo1+β1/β4, PIP2 accelerates the macroscopic activation kinetics at positive voltages and decelerates the macroscopic deactivation kinetics at negative voltages, but in Slo1+β2 Δ2-32 PIP2 has no effect on the deactivation kinetics. Measurements using chimeric β1-β2 subunits show that the second transmembrane domain and the C terminus of β1 is important for the large electrophysiological changes in Slo1+β1 channels caused by PIP2. Furthermore, Slo1 329RKK331, β1 R11, and β1 T14 are also critical to confer the effects of PIP2. Supported in part by the NIH, DFG HE 2993/8, and Shanghai Science and Technology Commission.

Membrane hyperpolarization during human sperm capacitation.

Sperm capacitation is a complex and indispensable physiological process that spermatozoa must undergo in order to acquire fertilization capability. Spermatozoa from several mammalian species, including mice, exhibit a capacitation-associated plasma membrane hyperpolarization, which is necessary for the acrosome reaction to occur. Despite its importance, this hyperpolarization event has not been adequately examined in human sperm. In this report we used flow cytometry to show that a subpopulation of human sperm indeed undergo a plasma membrane hyperpolarization upon in vitro capacitation. This hyperpolarization correlated with two other well-characterized capacitation parameters, namely an increase in intracellular pH and Ca(2+) concentration, measured also by flow cytometry. We found that sperm membrane hyperpolarization was completely abolished in the presence of a high external K(+) concentration (60 mM), indicating the participation of K(+) channels. In order to identify, which of the potential K(+) channels were involved in this hyperpolarization, we used different K(+) channel inhibitors including charybdotoxin, slotoxin and iberiotoxin (which target Slo1) and clofilium (a more specific blocker for Slo3). All these K(+) channel antagonists inhibited membrane hyperpolarization to a similar extent, suggesting that both members of the Slo family may potentially participate. Two very recent papers recorded K(+) currents in human sperm electrophysiologically, with some contradictory results. In the present work, we show through immunoblotting that Slo3 channels are present in the human sperm membrane. In addition, we found that human Slo3 channels expressed in CHO cells were sensitive to clofilium (50 μM). Considered altogether, our data indicate that Slo1 and Slo3 could share the preponderant role in the capacitation-associated hyperpolarization of human sperm in contrast to what has been previously reported for mouse sperm, where Slo3 channels are the main contributors to the hyperpolarization event.

The Ca2+-activated K+ current of human sperm is mediated by Slo3

Sperm are equipped with a unique set of ion channels that orchestrate fertilization. In mouse sperm, the principal K(+) current (IKSper) is carried by the Slo3 channel, which sets the membrane potential (Vm) in a strongly pHi-dependent manner. Here, we show that IKSper in human sperm is activated weakly by pHi and more strongly by Ca(2+). Correspondingly, Vm is strongly regulated by Ca(2+) and less so by pHi. We find that inhibitors of Slo3 suppress human IKSper, and we identify the Slo3 protein in the flagellum of human sperm. Moreover, heterologously expressed human Slo3, but not mouse Slo3, is activated by Ca(2+) rather than by alkaline pHi; current-voltage relations of human Slo3 and human IKSper are similar. We conclude that Slo3 represents the principal K(+) channel in human sperm that carries the Ca(2+)-activated IKSper current. We propose that, in human sperm, the progesterone-evoked Ca(2+) influx carried by voltage-gated CatSper channels is limited by Ca(2+)-controlled hyperpolarization via Slo3. DOI: http://dx.doi.org/10.7554/eLife.01438.001.

A Point Mutation that Impairs the Omega-3 Fatty Acid Sensitivity of Human Slo1 BK Channels

Docosahexaenoic acid (DHA), a long-chain omega-3 fatty acid with a 22 carbon chain, directly activates large-conductance calcium- and voltage-gated Slo1 BK channels with an EC50 = ∼500 nM and lowers blood pressure when injected into anesthetized mice (Hoshi et al. 2013 Proc Natl Acad Sci U S A. 110, 4816-21). The stimulatory action of DHA is observed in excised patches and reversible, suggesting that DHA directly interacts with the channel protein. Here we examined the molecular elements within Slo1 critical for the action of DHA. Unlike the clear stimulatory effect of DHA on human Slo1 (hSlo1) channels, we found that Drosophila Slo1 (dSlo1) channels expressed in mammalian cells were not stimulated by DHA, up to 10 μM. Prompted by this finding, we generated a series of chimeric dSlo1-hSlo1 channels and identified a single residue in S6 critical for the stimulatory effect of DHA on hSlo1. With the the human-to-Drosophila mutation Y318S, the current-enhancing effect of DHA on hSlo1, hSlo1+ β1 and hSlo1+β4 channels was severely impaired. The converse Drosophila-to-human mutation S332Y, however, failed to introduce sensitivity to DHA in dSlo1. Interestingly, the hSlo1 mutation Y318S altered the hSlo1+β1 channel sensitivity to 22- and 20-carbon omega-3 fatty acids but did not affect the response to alpha-linolenic acid with a 20-carbon chain or the omega-6 fatty acid arachidonic acid with a 20-carbon chain. Y318 in hSlo1 S6 is thus a critical determinant of the channel's sensitivity to select fatty acids. Supported in part through the NIH, DFG FOR 1738, and Shanghai Science and Technology Commission.