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

Abstract

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.

Keywords: Ca2+-activated K+ channel; Slo3; human sperm; ion channels; patch-clamp recordings.

Figure 1.. Voltage- and alkaline-activated K⁺ currents in human sperm.

(A) Whole-cell currents before and after application of 10 mM NH₄Cl. Traces at 35 mV and 85 mV are depicted in blue and red, respectively. (B) Current-voltage relation of recordings from (A) and currents recorded in 5 mM [K⁺]_o. (C) Mean V_rev of currents at pH_i 7.3 in different extracellular solutions (n = 3–5). (D) Currents recorded at pH_i 6.2. (E) Current-voltage relation of recordings from (D). (F) Mean currents before and after application of NH₄Cl (10 mM) and with Cs⁺-based intracellular solution (180 mM Cs⁺) (n = 3–6). DOI: http://dx.doi.org/10.7554/eLife.01438.003

Figure 1—figure supplement 1.. Voltage-gated currents in human sperm are carried by K⁺ channels.

(A) Whole-cell currents from human sperm recorded in K⁺-based intracellular solution at pH_i 7.3 and in extracellular solutions containing (in mM): 5 K⁺/150 Cl⁻ or 5 K⁺/7 Cl⁻. Current traces at +35 mV and +85 mV are depicted in blue and red, respectively. (B) Current-voltage relation of recordings from part (A). (C) Whole-cell currents recorded from human sperm in Cs⁺-based (left) and K⁺-based (right) intracellular solutions at pH_i 7.3 in 5 mM extracellular K⁺. Current traces at 85 mV and 135 mV are depicted in red and green, respectively. (D) Mean current–voltage relation of recordings as in panel C. (E) Whole-cell currents recorded from human sperm in Cs⁺-based intracellular solutions at pH_i 7.3 in 5 mM extracellular K⁺ and Ca²⁺ as indicated. Current traces at +85 mV and +135 mV are depicted in red and green, respectively. (F) Mean current–voltage relation recorded from human sperm in Cs⁺-based intracellular solutions at pH_i 7.3 in HS (2 mM Ca²⁺, 2 mM Mg²⁺), HS 100 µM Ca²⁺ (2 mM Mg²⁺), HS 0 Ca²⁺ (2 mM Mg²⁺), and NaDVF (0 Ca²⁺, 0 Mg²⁺). DOI: http://dx.doi.org/10.7554/eLife.01438.004

Figure 2.. V_m of human sperm is controlled by [Ca²⁺]_i rather than pH_i.

(A) Current-clamp recording from human sperm (pH_i 6.2) in extracellular solutions containing different [K⁺] and [Cl⁻] (in mM). Intracellular alkalization was evoked by superfusion with 10 mM NH₄Cl. (B) Current-clamp recording at pH_i 7.3. (C) Left panel: mean V_m under conditions as described in panel A and B; right panel: mean V_m at indicated [Ca²⁺]_i, and at 1 mM [Ca²⁺]_i under conditions described in panel D (n = 3–4). (D) Current-clamp recording at pH_i 7.3 and 1 mM [Ca²⁺]_i. DOI: http://dx.doi.org/10.7554/eLife.01438.005

Figure 3.. Ca²⁺ enhances K⁺ currents in human sperm.

(A) Whole-cell currents recorded at pH_i 7.3 with 5 mM DMNP-EDTA, 4 mM Ca²⁺, and 10 µM Fluo-4 in 5 mM extracellular K⁺ solution before (control) and after photorelease of Ca²⁺; for simplification, only currents evoked by −105, −55, −5 (grey), 45, and 95 mV are depicted. (B) Current-voltage relation of recordings from A. (C) Whole-cell-currents at pH_i 7.3 at 0 (left), 40 µM (middle) and 1 mM [Ca²⁺]_i (right) in extracellular solutions containing 5 mM K⁺; for simplification, only currents evoked by −105, −55, −5 (grey), 45, and 95 mV are depicted. (D) Current-voltage relations of currents recorded at 2.5–1000 µM [Ca²⁺]_i; I–V curves were normalized to the amplitude at 115 mV. (E) Current-voltage relation of currents recorded at pH_i 7.3 and 1 mM [Ca²⁺]_i; extracellular solutions contained 150 mM K⁺ or 5 mM K⁺. (F) Mean V_rev of currents at pH_i 7.3, 1 mM [Ca²⁺]_i, and different [K⁺]_o and [Cl⁻]_o (in mM) (n = 3–4). (G) Whole-cell currents recorded at pH_i 6.2, 1 mM [Ca²⁺]_i, and 5 mM [K⁺]_o, before and after superfusion with 10 mM NH₄Cl. (H) Current-voltage relation of recordings from (G). DOI: http://dx.doi.org/10.7554/eLife.01438.006

Figure 3—figure supplement 1.. Ca²⁺-activated currents in human sperm are carried by K⁺ channels.

(A) Whole-cell currents from human sperm recorded in K⁺-based intracellular solution at pH_i 7.3 and 1 mM [Ca²⁺]_i in extracellular solutions containing (in mM): 5 K⁺/150 Cl⁻ or 5 K⁺/7 Cl⁻. (B) Current-voltage relation of recordings from panel (A). (C) Mean currents from human sperm recorded in K⁺-based intracellular solution at pH_i 7.3 and different [Ca²⁺]_i in 5 mM extracellular K⁺ (n = 3–9). DOI: http://dx.doi.org/10.7554/eLife.01438.007

Figure 4.. Ca²⁺-activated K⁺ currents in human sperm exhibit hallmarks of Slo3 channels.

(A–D, G) Current-voltage relation of whole-cell currents from human sperm recorded in K⁺-based intracellular solution at pH_i 7.3 and 1 mM [Ca²⁺]_i in 5 mM extracellular K⁺ before, during, and after application of inhibitors. (E) Mean outward currents at 65 mV in the presence of 500 µM quinidine, 10 mM TEA, 50 µM clofilium, or 100 nM IBTX. (F) Current-clamp recording from human sperm in intracellular solution (pH_i 7.3) containing 180 mM K⁺ and 1 mM Ca²⁺. Sperm were bathed in extracellular solution containing 5 mM K⁺ and 150 mM Cl⁻. Concentrations of drugs were as in (A–E, G). (H) Current trace recorded at pH_i 7.3 and 70 µM [Ca²⁺]_i at −60 mV in HS (top) and K-based HS (bottom) Filter: 2 kHz. (I) Segments indicated by the red bars in panel (H) shown on an extended time scale (5 kHz), revealing opening events of one (top) and two K⁺ channels (bottom). Red lines correspond to conductance levels of 0 (c), 65 (o), and 130 pS (o). (J) Histogram of current amplitudes recorded in 5 mM K⁺ and 150 mM K⁺, at the conditions described in (H). DOI: http://dx.doi.org/10.7554/eLife.01438.008

Figure 5.. Human sperm express Slo3 and its auxiliary subunit LRRC52.

(A) Predicted membrane topology of hSlo3 (yellow) and hLRRC52 (red) polypeptides. Proteotypic peptides identified in human sperm by targeted protein mass-spectrometry are indicated in grey. (B) Western blot of total proteins of human sperm, CHO cells (wt), and CHO cells transfected with HA-tagged hSlo3 (hSlo3). The Western blot was probed with an anti-hSlo3 and anti-HA antibody. The molecular masses (kDa) of the protein standard are indicated on the right. (C) Human sperm stained with an antibody directed against hSlo3 (red). The DNA in the head was stained with DAPI (blue). Scale bar: 30 µM. DOI: http://dx.doi.org/10.7554/eLife.01438.009

Figure 5—figure supplement 1.. Specificity of anti-hSlo3 antibody.

CHO cells heterologously expressing human Slo3 that was modified with a C-terminal hemagglutinin (HA) tag were stained with an anti-hSlo3 antibody (green) and an antibody directed against the HA-tag (red). DAPI (blue) was used to label the nucleus. Scale bar: 10 µM. DOI: http://dx.doi.org/10.7554/eLife.01438.010

Figure 6.. Activation of heterologous hSlo3 by intracellular Ca²⁺.

(A) Families of hSlo3 + hLRRC52 currents in oocytes at pH 7 and 8 with indicated [Ca²⁺]_i. Current trace at +200 mV is depicted in red. (B and C) Current-voltage relations of tail currents determined at −140 mV; amplitudes were normalized to the amplitude evoked by step to 200 mV, 0 [Ca²⁺]_i, and pH 8. (D) Tail current amplitudes (activated by 200 mV) as function of [Ca²⁺]_i for pH 7 and pH 8. Normalization as in (B). (E) Current-voltage relation of steady-state hSlo3 + hLRRC52 currents in CHO cells at pH_i 7.3 and 0, 70 µM, and 1 mM [Ca²⁺]_i. Currents were normalized to the amplitude evoked at 115 mV. (F) Current-voltage relation of hSlo3 + hLRRC52 currents in CHO cells and I_KSper recorded from human sperm at 0 and 1 mM [Ca²⁺]_i (pH_i 7.2). Normalization as in panel (D). DOI: http://dx.doi.org/10.7554/eLife.01438.011

Figure 6—figure supplement 1.. Ca²⁺ increases hSlo3 single-channel openings at −60 mV.

(A, C, E) hSlo3 + hLRRC52 openings at −60 mV. Filter: 2 kHz. (B, D, F) Segments highlighted with red bars in panel (A, C, E) at faster time base (5 kHz filtering). (G) Total amplitude histogram of a set of current records as in (B, D, F). (H) NP_o at 60 and 300 µM Ca²⁺ normalized to NP_o at 0 Ca²⁺ (n = 7). Red symbols: individual estimates. Black symbols: mean and SD. DOI: http://dx.doi.org/10.7554/eLife.01438.012

Figure 6—figure supplement 2.. Currents carried by hSlo3 co-expressed with hLRRC52 in CHO cells.

(A) Whole-cell hSlo3 + hLRRC52 currents at pH_i 7.3. Traces at +35 mV and +85 mV are depicted in blue and red, respectively. (B) Current-voltage relation of hSlo3 + hLRRC52 currents recorded in 140 mM and 5 mM [K⁺]_o. (C) Currents recorded at pH_i 6.2, before and after intracellular alkalization by NH₄Cl (10 mM). (D) Current-voltage relation of recordings from panel C. (E) Mean current amplitudes at pH_i 6.2 and pH_i 7.3 before and after application of NH₄Cl (10 mM) (n = 3). (F) Currents recorded at 1 mM [Ca²⁺]_i and 5 mM [K⁺]_o. (G) Current-voltage relation of recordings at 1 mM [Ca²⁺]_i in 140 mM and 5 mM [K⁺]_o. DOI: http://dx.doi.org/10.7554/eLife.01438.013

Figure 7.. Co-expression of mLRRC52 does not confer Ca²⁺ sensitivity on mSlo3.

(A) Currents were activated with 0 [Ca²⁺]_i pH 7, 0 [Ca²⁺]_i pH 8, 60 µM [Ca²⁺]_i pH 8, and 300 µM [Ca²⁺]_i pH 8. Red trace corresponds to step to 60 mV. (B) Larger gain display of tail currents from (A). (C) Normalized current–voltage relations of tail currents determined at −140 mV; currents were normalized to tail current amplitude following the step to 200 mV. (D) Normalized tail-current amplitude as a function of [Ca²⁺]_i. Tail currents were normalized to 0 [Ca²⁺]_i at pH 8. DOI: http://dx.doi.org/10.7554/eLife.01438.014

Figure 7—figure supplement 1.. Co-expression of hLRRC52 with mSlo3 does not confer Ca²⁺ sensitivity on mSlo3.

(A) Currents were activated by the indicated voltage-protocol with 0 Ca²⁺ pH 7, 0 Ca²⁺ pH 8, 60 µM Ca²⁺ pH 8, and 300 µM Ca²⁺ pH 8, from top to bottom. Red trace corresponds to step to +60 mV. (B) Larger gain display of tail currents from panel (A). (C) Comparison of currents activated at the indicated voltages at pH 8 with either 0 Ca²⁺ (black) or 60 µM Ca²⁺ (red). (D) Normalized conductances determined from tail currents following each activation voltage for mSlo3 + hLRRC52 channels for the indicated conditions. (E) Normalized conductance as a function of Ca²⁺ determined from tail currents following activation at either +100 or +200 mV at pH 8. DOI: http://dx.doi.org/10.7554/eLife.01438.015

Figure 7—figure supplement 2.. mSlo3 is more sensitive to voltage-dependent block by Ca²⁺ than hSlo3.

(A) Steady-state conductance-voltage (G-V) relationships for hSlo3 + hLRRC52 at different [Ca²⁺]_i. (B) Steady-state G-V relationships for mSlo3 + mLRRC52 at different [Ca²⁺]_i and pH_i. (C) Steady-state G-V relationships for mSlo3 + hLRRC52 at different [Ca²⁺]_i and pH_i. (D) Fractional inhibition of conductance at three voltages is plotted as a function of [Ca²⁺]_i; lines indicate the fit of I(Ca²⁺) = 1/(1+[Ca²⁺]/K_D). For each [Ca²⁺]_i, steady-state conductance reflects both the increase in conductance from Ca²⁺-dependent inactivation and voltage-dependent block. Values of steady-state conductance from a panel were, therefore, corrected to reflect the measured Ca²⁺-dependent increase of conductance determined from tail currents. K_D values were 128.0 ± 77.6 µM, 428.3 ± 165.1 µM, and 5.4 ± 2.8 mM for +240, +200 and +160 mV, respectively. (E) Fractional inhibition of mSlo3 + mLRRC52 as a function of Ca²⁺ is plotted along with the best fit curves. K_D values were 16.9 ± 8.2, 43.7 ± 12.9, and 350.2 ± 132.6 µM, for +240, +200, and +160 mV, respectively. (F) Fractional inhibition of mSlo3 + hLRRC52 as a function of Ca²⁺. K_D values were 20.8 ?13.9, 52.2 ?23.0, and 345.8 ± 215.3 µM for +240, +200, and +160 mV, respectively. (G) Voltage-dependence of the K_D for Ca²⁺ inhibition is plotted for mSlo3 + mLRRC52 along with the values for hSlo3 + hLRRC52 and mSlo3 + hLRRC52. DOI: http://dx.doi.org/10.7554/eLife.01438.016

Figure 8.. Progesterone-evoked Ca²⁺ responses in human sperm.

(A) Ca²⁺ signal evoked by progesterone (2 µM) and ionomycin (2 µM) in sperm loaded with different Ca²⁺ indicators. (B) Relative amplitude of the progesterone- vs ionomycin-induced Ca²⁺ signal in sperm loaded with indicators of various Ca²⁺ sensitivity. (C) Dynamic range of Ca²⁺ sensitivity for different indicators assuming 1:1 binding of Ca²⁺. DOI: http://dx.doi.org/10.7554/eLife.01438.017

Figure 9.. Progesterone inhibits hSlo3 but not hSlo1.

(A) Whole-cell hSlo3 + hLRRC52 currents recorded in CHO cells at pH_i 7.3 before and after perfusion with 30 µM progesterone. (B) hSlo1 currents recorded in outside-out patches excised from CHO cells at pH_i 7.3 and 70 µM [Ca²⁺]_i before and after perfusion with 30 µM progesterone. (C) Relative amplitude of hSlo1 and hSlo3 + hLRRC52 currents at 80 mV in CHO cells in the presence of progesterone. DOI: http://dx.doi.org/10.7554/eLife.01438.018

Figure 9—figure supplement 1.. Lack of homology among Slo3 sequences in the ligand-sensing cytosolic domain.

(A) Alignments of human Slo1 and Slo3 from various species are shown for the membrane-associated, pore-forming part of the channels, indicating the relatively high extent of conservation through this part of the Slo3 protein. The Slo1 N-terminus is omitted to minimize effects of S0–S1 linker gaps on the alignment. Slo1 numbering starts from amino acids MDAL. Tick marks below each segment of residues counts every 10 residues in human Slo3. (B) Alignments of human Slo1 and Slo3 from various species are shown for the cytosolic gating ring domain beginning with the conserved sequence at the beginning of the first RCK domain. Blue highlights residue differences between human Slo1 and human Slo3. Yellow highlights differences of Slo3 of various species to human Slo3. Alignments were generated by Clustal 1.2.0 and minor adjustments were made based on structural considerations (Leonetti et al., 2012). Above the residues, the correspondence of particular amino acid segments to structurally defined α-helical and β-strand segments is shown based on Leonetti et al. (2012). In red, residues or segments identified in Slo1 or Slo3 isoforms which are implicated in ligand-sensing or species-specific functional differences are highlighted. Although extensive information is available regarding loci important in ligand-sensing in Slo1, such information for Slo3 remains lacking. Numbers identify the following: 1, the sequence of residues termed the Ca²⁺ bowl (Schreiber and Salkoff, 1997), for which there is good correspondence of mutations affecting Ca2+-dependent function (Bao et al., 2004) and coordination of density in a crystal structure (Yuan et al., 2012); 2, the D367 residue implicated in the role of the RCK1 domain in Ca²⁺-dependent activation (Xia et al., 2002) which is clearly distinct from Ca²⁺-bowl dependent activation (Zeng et al., 2005); 3, the M513 residue, which also affects Ca²⁺⁻dependent activation involving the RCK1 domain (Bao et al., 2002), but probably is not involved in ligand coordination; 4, residues E374 and E399 which have been implicated in low affinity effects of divalent cations, specifically Mg²⁺ (Shi et al., 2002; Xia et al., 2002; Yang et al., 2006); 5, residue E535 which may also be involved in Ca²⁺ coordination in RCK1 (Zhang et al., 2010); 6, residues H365 and H394, which have been implicated in proton-dependent activation of Slo1 and also influence Ca²⁺-dependent activation when protonated (Hou et al., 2008); 7, H417 and segment 368–475, which influence pH-sensing in mouse Slo3 (Zhang et al., 2006); 8, segment 495–515 in bovine Slo3 which accounts for part of the different in functional properties between mouse Slo3 and bovine Slo3 (Santi et al., 2009). Illustrated sequences and accession numbers include: HsSlo1 (Homo sapiens), NP_001154824, Gene ID 3778; HsSlo3 (Homo sapiens), NP_001027006, Gene ID 157855; MmSlo3 (Mus musculus), NP_032458, Gene ID 16532; RnSlo3 (Rattus norvegicus), XP_006253398, Gene ID 680912; TcSlo3 (Tupaia chinensis, Chinese tree shrew), XP_006171561, Gene ID 102493286; CcSlo3 (Condylura cristata, star-nosed mole), XP_004682520, Gene ID 101620543; CfSlo3 (Canis lupus familiaris), XP_539971, Gene ID 482856; BtSlo3 (Bos taurus), NP_001156721, Gene ID 524144; OaSlo3 (Ovis aries, sheep), XP_004021821, Gene ID 10110209. DOI: http://dx.doi.org/10.7554/eLife.01438.019

Author response image 1.

Relative amplitude of hSlo1 (at 80 mV) before and after superfusion with IBTX (100 nM). Relative amplitude of hSlo3 + hLRRC52 currents (at 80 mV) before and after superfusion with IBTX (100 nM), CTX (1 μM), or Paxilline (100 nM). hSlo1 and hSlo3 + hLRRC52 were expressed in CHO cells.