A K+-selective CNG channel orchestrates Ca2+ signalling in zebrafish sperm
- Center of Advanced European Studies and Research, Germany
- Ruhr-Universität Bochum, Germany
- Forschungszentrum Jülich, Germany
- University of Massachusetts Boston, United States
- Helmholtz-Zentrum München, Germany
Figures
Figure 1 with 3 supplements
Identification of DrCNGK channel homologues and of a K+ channel in D. rerio sperm.
(A) Phylogenetic tree (Page, 1996) of various ion channel families. The CNGK channel family exists in protozoa (dark blue), marine invertebrates and fish (medium blue), and freshwater fish (light …
Figure 1—figure supplement 1
Amino-acid sequence of the DrCNGK channel.
Different colors indicate the transmembrane segments (red shades, repeats 1–4), the four pore regions (green), the four CNBDs (gray), and the unusual insert in the C-linker of the third repeat …
Figure 1—figure supplement 2
Separation of heads and flagella from whole sperm.
Dark-field micrographs of whole sperm (left), purified heads (middle), and purified flagella (right). Bar represents 100 µm.
Figure 1—figure supplement 3
Electrophysiological characterization of currents recorded from zebrafish sperm.
(A) Whole-cell recordings at different intracellular Cl- concentrations (Cli).
Figure 2
Localization of the DrCNGK channel.
(A) Western blot of membrane proteins (15 µg) from CHOK1 cells transfected with cDNA encoding either DrCNGK with a C-terminal HA-tag alone (lane C) or with both, a C-terminal HA-tag and an …
-
Figure 2—source data 1
Indicators of merit for the mass spectrometric results of a testis preparation.
a m indicates oxidized methionine, b△M [ppm] relative mass error, c XCorr, △Score, and △Cn indicate results based on searches with the sequest algorithm in Proteome Discoverer; d PEP (Posterior Error Probability) describes the probability that the observed hit is a chance event.
- https://doi.org/10.7554/eLife.07624.008
-
Figure 2—source data 2
Indicators of merit for the mass spectrometric results of different sperm preparations: whole sperm, head and flagella.
a m indicates oxidized methionine, b△M [ppm] for all peptides is below ± 2.5, c XCorr indicates results based on searches with the sequest algorithm; Amanda indicates results based on searches with the MS Amanda algorithm, d PEP (Posterior Error Probability) describes the probability that the observed hit is a chance event; △Score and △Cn are not indicated separately since the scores for all peptides are 1 and 0, respectively.
- https://doi.org/10.7554/eLife.07624.009
Figure 3 with 5 supplements
Cyclic nucleotides do not activate K+ channels in sperm.
(A) Current amplitude of whole-cell recordings from zebrafish sperm at +25 mV in the absence or presence of 100 µM cAMP or cGMP in the pipette (control: 91 ± 49 pA (n = 23); cAMP: 73 ± 25 pA (n = 6);…
Figure 3—figure supplement 1
K+ dependence of heterologously expressed DrCNGK channels in oocytes and channel block by tetraethylammonium (TEA).
(A) Two-Electrode Voltage-Clamp recordings of heterologously expressed DrCNGK channels in the presence of different K+ concentrations (left panel: 7 mM, middle panel: 96 mM) and corresponding IV …
Figure 3—figure supplement 2
Sequence alignment of the individual CNBDs from the DrCNGK and ApCNGK channels.
The secondary structure elements of CNBDs are indicated above the sequences. A key Arg residue between β6 and β7 is indicated by an asterisk. An FGE motif important for interaction with cyclic …
Figure 3—figure supplement 3
Photo-release of cyclic nucleotides in HEK cells expressing ApCNGK channels and use of 8Br-analogs in ApCNGK-injected oocytes.
(A) Left and middle panel: Pooled IV curves of ApCNGK channels heterologously expressed in HEK cells before (-cGMP or -cAMP) and after the release of cGMP or cAMP. Cells were loaded with 100 µM …
Figure 3—figure supplement 4
Photo-release of cyclic nucleotides (A) or Ca2+ (B) in sperm.
(A) Mean velocity (averaged-path velocity, VAP) before (-) and after (+) release of cAMP or cGMP. Sperm were loaded with 30 µM DEACM-caged cAMP or DEACM-caged cGMP. Individual data (symbols) and …
Figure 3—figure supplement 5
Control of loading and release of DEACM-cAMP in zebrafish sperm.
(A) Dark-field micrograph (using red light) of sperm loaded with DEACM-caged cAMP (30 µM). (B) Fluorescence image after 15 s of continuous illumination with 365 nm UV light (1.75 mW power). (C) Time …
Figure 4
Comparison of sperm K+ current with current from heterologously expressed ApCNGK channels.
(A) Normalized IV relations of whole-cell recordings from zebrafish sperm and ApCNGK channels expressed in HEK293 cells. Pipette solution: standard IS. Bath solution: standard ES. Currents were …
Figure 5 with 3 supplements
pH regulation of the DrCNGK channel.
(A) Whole-cell recordings from zebrafish sperm after perfusion with NH4Cl or propionic acid. Voltage steps as shown in Figure 1C. Recordings at extracellular pH 7.4 and pipette pH 6.4 (left). NH4Cl …
Figure 5—figure supplement 1
pH dependence of heterologously expressed DrCNGK channels in oocytes.
Two-Electrode Voltage-Clamp recordings of DrCNGK-injected (A) and uninjected (B) oocytes. Cells were reversibly perfused with 96 mM K+ bicarbonate followed by 1 mM NH4Cl added to K+ gluconate (10 …
Figure 5—figure supplement 2
High intracellular Ca2+ does not suppress DrCNGK currents.
Pooled IV relations of whole-cell recordings from whole sperm and isolated heads under standard conditions (ES/IS, pH 7.4) (Figure 1G) and from whole sperm, when [Ca2+]i in the pipette solution was …
Figure 5—figure supplement 3
Hypoosmotic conditions do not stimulate or diminish DrCNGK currents in Xenopus oocytes.
DrCNGK-injected oocytes were recorded in the TEVC mode in ND96-7K and ND48-7K olutions (n = 4). No significant differences were observed.
Figure 6
Sperm swimming behaviour upon Ca2+ release.
(A), (B), and (C) representative swimming paths of three different DMSO loaded sperm before and after application of UV light. (D), (E), and (F) representative averaged swimming paths of three …
Figure 7
Models of signalling pathways in sea urchin and zebrafish sperm.
Sea urchin (upper panel): Binding of the chemoattractant resact to a receptor guanylyl cyclase (GC) activates cGMP synthesis. Cyclic GMP opens K+-selective CNG channels (CNGK), thereby, causing a …
Videos
Video 1
Behavioural response of zebrafish sperm to successive Ca2+ release.
Representative recording of zebrafish sperm loaded with NP-EGTA (40 µM). Upon release of Ca2+, the swimming path curvature increases and, eventually, sperm spin against the wall of the recording …
