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A K+-selective cGMP-gated ion channel controls chemosensation of sperm

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Eggs attract sperm by chemical factors, a process called chemotaxis. Sperm from marine invertebrates use cGMP signalling to transduce incident chemoattractants into changes in the Ca2+ concentration in the flagellum, which control the swimming
  LETTERS NATURE CELL BIOLOGY   VOLUME 8 | NUMBER 10 | OCTOBER 2006 1149 A K + -selective cGMP-gated ion channel controlschemosensation of sperm Timo Strünker 1,2 , Ingo Weyand 1,2 , Wolfgang Bönigk  1 , Qui Van 1 , Astrid Loogen 1 , Joel E. Brown 2 ,Nachiket Kashikar 1 , Volker Hagen 3 , Eberhard Krause 3 and U. Benjamin Kaupp 1,2,4 Eggs attract sperm by chemical factors, a process calledchemotaxis. Sperm from marine invertebrates use cGMPsignalling to transduce incident chemoattractants into changesin the Ca 2+ concentration in the flagellum, which control theswimming behaviour during chemotaxis 1–3 . The signallingpathway downstream of the synthesis of cGMP by a guanylylcyclase is ill-defined. In particular, the ion channels that areinvolved in Ca 2+ influx and their mechanisms of gating arenot known 4 . Using rapid voltage-sensitive dyes and kinetictechniques, we record the voltage response that is evoked bythe chemoattractant in sperm from the sea urchin Arbacia punctulata  . We show that the chemoattractant evokes a briefhyperpolarization followed by a sustained depolarization. Thehyperpolarization is caused by the opening of K + -selectivecyclic-nucleotide-gated (CNG) channels in the flagellum.Ca 2+ influx commences at the onset of recovery fromhyperpolarization. The voltage threshold of Ca 2+ entry indicatesthe involvement of low-voltage-activated Ca v channels. Theseresults establish a model of chemosensory transduction insperm whereby a cGMP-induced hyperpolarization opensCa v channels by a ‘recovery-from-inactivation’ mechanismand unveil an evolutionary kinship between transductionmechanisms in sperm and photoreceptors. Using fast mixing an flash photolysis techniques, we were able to recoroptically the voltage response from intact motile sperm of the sea urchin  Arbacia punctulata . We have chosen the fluorescent ye i-8-ANEPPS asa potentiometric probe ue to its rapi response time, its exclusive stain-ing of plasma membrane an its electrochromic mechanism of voltagesensing. Depolarization an hyperpolarization shift the emission spec-trum of i-8-ANEPPS by a few nanometres to lower an higher wave-lengths, respectively, by a mechanism known as electrochromism 5,6 (seeSupplementary Information, Fig. S1a). This electrochromic shift allowsfor ual-emission ratio measurements 7 ; thereby, fluorescence signals thatare unrelate to changes in membrane voltage are largely eliminate.A suitable pair of wavelengths for the ratiometric measurement wereerive from the ifference between i-8-ANEPPS emission spectra thatwere recore in sperm suspension uner normal conitions (artificialsea water, ASW) an epolarizing conitions (high potassium ASW,K-ASW). The maximum emission of the spectrum was λ max = 566 nm,an the ifference spectrum isplaye a positive an negative peak at theflanks of the emission maximum (~540 nm an ~595 nm, respectively)(see Supplementary Information, Fig. S1b).As expecte from the ifference spectrum, stimulation of ye-stainesperm with the chemoattractant peptie resact gave rise to oppositechanges in fluorescence F 535 an F 580 that were superpose on a persist-ent increase of fluorescence at both wavelengths (Fig. 1a). By taking theratio F 580 /F 535 (R or ∆R, correcte for the control), the steay componentof the fluorescence signal was eliminate. ∆R ecrease in a pulse-likefashion, inicating a rapi transient hyperpolarization, followe by aslower increase above baseline, reflecting a more persistent epolariza-tion (Fig. 1b). We sought inepenent evience that ΔR represents agenuine voltage signal. A istinct electrochromic ye 7,8 , RH414, reportea resact-inuce ΔR signal that was similar to that of i-8-ANEPPS (seeSupplementary Information, Fig. S1c).Voltage signals coul be recore for concentrations of resact thatwere as low as 0.25 pM, an the hyperpolarization signal saturate atroughly 25 nM (Fig. 1c, , g). The hyperpolarization graually increaseover five orers of magnitue of resact concentrations (Fig. 1). Theshape of the voltage signals change with concentration of resact: Withincreasing concentrations, the with of the hyperpolarizing pulse firstbecame smaller an then broaene again — that is, the recovery fromhyperpolarization became slower. Except for very low concentrationsof resact, the cell epolarize after the return from hyperpolarization(compare Fig. 1c,  an i). The epolarization was smaller than thehyperpolarization, isplaye a ifferent concentration epenence anpersiste for at least 10 s (Figs 1, 3e).The hyperpolarization inicates that resact opens a K + -selective chan-nel; alternatively, non-selective cation channels may close, as in verte-brate photoreceptors 9 , or Cl – channels may open. To etermine the ionic 1 Institut für Neurowissenschaft und Biophysik, Abteilung Zelluläre Signalverarbeitung, INB-1, Forschungszentrum Jülich, 52425 Jülich, Germany. 2 Marine BiologicalLaboratory, Woods Hole, MA 02543, USA. 3 Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Str., 13125 Berlin, Germany. 4 Correspondence should be addressed to U.B.K. (e-mail: 4 May 2006; accepted 5 July 2006; published online 10 September 2006; DOI: 10.1038/ncb1473 NaturePublishingGroup ©200 6  1150   NATURE CELL BIOLOGY   VOLUME 8 | NUMBER 10 | OCTOBER 2006 LETTERS selectivity of the resact-inuce conuctance, we measure the voltagesignals evoke by saturating concentrations of resact (25 nM) in thepresence of various external K + concentrations, [K + ] o . Resact was in thehigh K-ASW solution, thus stimulation an high K + conitions wereapplie simultaneously. With increasing [K + ] o , the hyperpolarizationbecame smaller an the rate of ecay from the peak ecrease signifi-cantly (Fig. 1e). The sign of the voltage response with respect to the rest-ing voltage (V rest ) reverse at [K + ] o = 62 mM (Fig. 1f), yieling a V rest of –48.1 mV (assuming a [K + ] i of 423 mM — that is, equal to the [Na + ] o ) .  When [K + ] o was lowere by mixing sperm with 0 K + -ASW, the hyperpo-larization was enhance. Substitution of Na + by  N  -methyl-d-glucamine orCl – by gluconate ha no effect on the voltage response (see Supplementary Information, Fig. S1). Together, these results inicate that the hyperpo-larization is cause by opening of K + -selective channels.The changes in fluorescence can be calibrate into millivolts, proviethat the K + channel represents the ominant conuctance uring therise time of the response. ΔR was linearly relate to log [K + ] o (Fig. 1f)— that is, the response isplaye an almost perfect Nernstian behav-iour. The slope of the graph is 6.6 ± 0.4%/57.7 mV or 1.14%/10 mV. Inneurons, in which absolute calibration is feasible by voltage-clamping,relative changes of ∆R between 0.8 an 1.1%/10 mV were etermine fori-8-ANEPPS 6 . Thus, the voltage sensitivity of i-8-ANEPPS in spermmembranes is similar to that in neurons.The calibration allowe us to estimate the maximal hyperpolari-zation (ΔV h ) an epolarization (ΔV  ) an V rest . Given that V m at thepeak of the hyperpolarization equals the K + equilibrium potential (E K ),then V rest = E K – ΔV h . With E K = –96 mV (assuming an intracellular[K + ] of 423 mM) an ΔV h = –52.7 ± 7.2 mV ( n = 6), we obtaine a 0 1 2 340.360.370.380.390.40250 pM25 pM6.25 pM ASWTime (s) Time (s)Time (s)Time (s) a c 0 1 2 34 − 0.06 − 0.04 − 1.0 − 0.06 − 1 2 3 4 − 0.04 −       ∆    R       ∆    R       ∆    R       ∆    R       ∆    R       ∆    R   n  o  r  m       ∆    R +0.0 0.5 1.0 − 0.04 − bd − 0.08 − 0.06 − 0.04 − 0.02012340. − 0.08 − 0.06 − 0.04 − e − 0.08 − 0.06 − 0.04 − f 012345670.0000.0020.0040.0060.0080.010Mean number of bound molecules h 01234 − 0.02 − (s)Time (s) g 01234 − 1.0 − i 10 − 13 10 − 12 10 − 11 10 − 10 10 − 9 10 − 8 10 − 7 10 − 6 [Resact] (M)    F    5   3   5   a  n   d   F    5   8   0    (  a  r   b   i   t  r  a  r  y  u  n   i   t  s   )       ∆    R   m  a  x 110100[K + ] o (mM) ∆ R/p[K + ] = 0.066 (± 0.004) Figure 1 Resact-induced changes in membrane voltage. ( a ) Resact-inducedchanges in the fluorescence of di-8-ANEPPS F 535 (red traces) and F 580 (blacktraces) after stimulation of sperm at t  = 0. ( b ) Resact-induced changes in theratio F 580  /F 535 (or ∆ R, see Methods); 6.25 pM of resact (black); 25 pM (grey);125 pM (green). Inset: signals shown on an extended time scale. ( c ) Changesin Δ R evoked by resact: 2.5 pM (black), 6.25 pM (red), 25 pM (green), 50 pM(dark blue), 125 pM (light blue), 250 pM (magenta), 500 pM (yellow); and25 nM (olive); traces represent the average of six recordings. Inset: Signalsshown on an extended time scale. ( d ) Dependence of the hyperpolarization(closed symbols) and the depolarization (open symbols) on the concentrationof resact. Bars indicate s.d. ( n = 3–4). ( e ) Resact-induced changes in Δ R atvarious [K + ] o (in mM): 4.5 (black), 9 (red), 19.5 (green), 24.5 (dark blue),29.5 (light blue), 49.5 (magenta); and 99.5 (yellow) — average of sixrecordings; and 25 nM resact. ( f ) Relationship between the changes in Δ R andthe logarithm of [K + ] o . Bars indicate s.d. ( n = 5). ( g ) Changes in Δ R evokedby 0.25 pM resact (black), 0.5 pM (red), 1.25 pM (green), 2.5 pM (blue) —average of nine recordings; and 6.25 pM (olive) — average of six recordings.( h ) Dependence of Δ R on the mean number of bound molecules m  . The sperm‘concentration’ ranged from 0.40 to 0.43 pM. Bars indicate s.d. ( n = 4).( i ) Time course of normalized Δ R responses evoked by 0.25 pM resact (grey),0.5 pM (red); and 1.25 pM (green). At 0.5 pM resact, the mean number ofbound molecules m  was 1.2. NaturePublishingGroup ©200 6  NATURE CELL BIOLOGY   VOLUME 8 | NUMBER 10 | OCTOBER 2006 1151 LETTERS V rest of –43.3 mV. The maximal epolarization was ΔV  = 19.1 ± 1.0 mV( n = 6). Thus, sperm maintain a V rest that is significantly less negativethan that of most other cells, except ro photoreceptors; uring stimula-tion, the voltage can maximally range from –24.2 mV to –96 mV.Sperm are highly sensitive; a single molecule of resact evokes a Ca 2+  signal 1 . To extract information about the unitary voltage response evokeby a single molecule of resact, we recore ΔR at low concentrations of resact when sperm cells receive, on average, a single or a few molecules 1  (Fig. 1g). Assuming a Poisson istribution of molecules among sperm,the signal amplitue becomes:  A = anP n . Σ n1 wherein a enotes the amplitue of the unitary response, n thenumber of boun molecules an P  n the Poisson probability that spermboun n molecules. The foot of the ose–response relationship waslinear up to a mean number of boun molecules m   ≅ 1 (Fig. 1h). Theinitial slope a yiele a quantal response of –2.4 mV. At m ≥ 1.5, therelationship starte to become sublinear (Fig. 1h). This fining isremarkable as it shows that unitary responses — evoke by two mol-ecules that might be asorbe at istant places on the flagellum — onot sum linearly.In the single-molecule regime, resact-inuce Ca 2+ signals scale– that is, their normalize time course becomes almost ientical 1 . The voltage signals for 0.25–1.25 pM of resact also scale, inicating that,in this concentration range, the macroscopic response is compose of single-molecule events (Fig. 1i).Stimulation by resact prouces a rapi rise of the concentration of cGMP 1 . To test the hypothesis that cGMP causes hyperpolarization, werecore the voltage responses evoke by release of cGMP from cagecompouns. cGMP an resact evoke signals of similar waveform, exceptthat the cGMP-inuce signal was faster (Fig. 2a). cAMP also initiatea rapi but smaller signal (Fig. 2b). The voltage responses prouce by cGMP were so rapi that a large part of the rising phase was hien in thetime winow between the peak of the flash an the opening of photoshut-ters ( ~ 12 ms) (see Supplementary Information, Fig. S2). We estimate ahalf-rise time of ≤ 6 ms, implying that the voltage response starte witha short elay, if any. The rapi kinetics of channel opening strongly ini-cates that the channel is gate irectly by cyclic nucleoties rather thanby phosphorylation via a cGMP-epenent kinase. The instantaneousopening both by cAMP an cGMP is a hallmark of cyclic-nucleotie-gate(CNG) channels 10 . Most CNG channels are non-selective cation chan-nels that, on opening, woul not hyperpolarize a cell. To rigorously testwhether a K + -selective channel is unerlying the cGMP-inuce hyper-polarization, we recore the voltage response in the presence of various[K + ] o . With increasing [K + ] o , the hyperpolarization became smaller anisplaye a Nernstian behaviour (Fig. 2c). Moreover, the sign of the resact-an cGMP-inuce voltage responses reverse at similar [K + ] o (compareFigs 1f an 2c). These results inicate that the same K + -selective channel isunerlying both the resact- an the cGMP-inuce hyperpolarization.Because both cAMP an cGMP can activate the channel, althoughwith ifferent sensitivity, the voltage response will epen on the intra-cellular ynamics of cyclic nucleoties. Comparison of the time course 01234 − 0.03 − 0.02 − − 0.03 − 0.02 −       ∆    R       ∆    R       ∆    R   n  o  r  m       ∆    R Time (s)Time (s)Time (s)Time (s) abdc − 0.03 − 0.02 − − 0.03 − 0.02 − [K + ] o (mM)   x  -   f  o   l   d   i  n  c  r  e  a  s  e   i  n  c   N   M   P  s Figure 2 The K + channel is directly gated by cyclic nucleotides.( a ) Comparison of the time course of Δ R signals stimulated by resact(25 pM, green) and by the release of cGMP from 30 µM DEACM-cagedcGMP (red). ( b ) Δ R signals elicited by cGMP (red) or cAMP (black);average of 15 recordings. Arrow indicates the time of the ultraviolet-lightflash. ( c ) Relationship between the changes in Δ R and the logarithm of[K + ] o . Bar at the lowest [K + ] o indicates s.d. ( n = 3); in all other cases, thes.d. lies within the data points. The dotted line indicates the [K + ] o of 76 mMat which Δ R reversed sign, corresponding to a V rest of –43.2 mV. Inset:cGMP-induced changes in Δ R at various [K + ] o (in mM): 9 (black), 19.5(red), 29.5 (green), 49.5 (dark blue) and 99.5 (light blue) — average ofsix recordings. ( d ) Comparison of the time course of normalized changesin Δ R (green), cGMP (red) and cAMP (black). For better comparison, thehyperpolarizing signal has been inverted. Changes in cyclic nucleotideswere measured with the quenched-flow technique 1 ; data represent means oftriplicates from one experiment. Sperm were stimulated with 25 nM resact. NaturePublishingGroup ©200 6  1152   NATURE CELL BIOLOGY   VOLUME 8 | NUMBER 10 | OCTOBER 2006 LETTERS of the changes in voltage an cyclic nucleoties emonstrate that thehyperpolarization an the increase of cGMP commence at the sametime an rose rapily to a maximum; in contrast, cAMP i not changesignificantly uring the first 200 ms (Fig. 2). Therefore, cGMP is thephysiological messenger that causes the hyperpolarization.In the Supplementary Information, we provie genetic an biochemi-cal evience for a flagellar channel protein that carries all the signaturemotifs of a K + -selective CNG channel (see Supplementary Information,Fig. S3a). The core omain consists of six transmembrane segments(S1–S6). The pore region carries the canonical GYGD motif of voltage-epenent K  v  channels 11 ; the sequence of the pore region is more similarto that of a K + -selective channel from the bacterium  Mesorhizobium loti  than to the pore of non-selective cation CNG channels or weakly K + -selective HCN channels (see Supplementary Information, Table 1). Thecarboxy-terminal omain shows characteristic sequence similarity withthe cyclic-nucleotie-bining omain of CNG channels an other cyclicnucleotie-bining proteins (see Supplementary Information, Fig. S3b).Proteomic analysis by mass spectrometry of membrane proteins frompurifie flagella confirme the presence of 20 pepties, preicte by the clone cDNA fragment (see Supplementary Information, Fig. S3a).Complete functional an structural characterization of the CNG channelwill require further investigation.How oes hyperpolarization stimulate Ca 2+ entry? Chemoattractantpepties evoke a host of cellular reactions 12,13 , many of which, in prin-ciple, coul be involve in Ca 2+ entry. To examine the activation mech-anism, we compare the time course of the voltage an Ca 2+ signals(Fig. 3). The Ca 2+ signal began to rise at or shortly after the peak of thehyperpolarization — that is, when V m starte to become more positiveagain. This result inicates that Ca 2+ enters the cell through voltage-activate Ca  v  channels that recover from inactivation an open againon subsequent epolarization. For signals of the same amplitue, the V m  at which Ca  v  channels opene was similar, regarless of whether Ca 2+  entry was prouce by resact or by the release of cGMP. For example,the resact- an cGMP-inuce Ca 2+ signals of Fig. 3b an c commenceat a V m of –61.1 mV an –60.3 mV, respectively.A istinctive feature that sets Ca  v  channels apart from each other isthe threshol (V th ) at which they open from a close state 14 . The mostnegative value of V th for the Ca 2+ entry was –71.9 ± 8 mV (Fig. 3). This value is iagnostic of low-voltage-activate (LVA) or T-type Ca  v  chan-nels (V th ≈ –70 mV)) 14,15 , inicating that the hyperpolarization removesinactivation from Ca  v  channels an thereby promotes Ca 2+ entry. A time-to-peak of 70–360 ms for the hyperpolarization (Fig. 1c) an a elay of ~200–600 ms for the Ca 2+ entry  1 are both consistent with a time con-stant of 110–450 ms for recovery from inactivation of T-type Ca  v  chan-nels 15 . We provie partial sequence information for a T-type Ca  v  channelfrom the sea urchin Strongylocentrotus purpuratus (see Supplementary Information, Fig. S3c) . At concentrations of resact ≥25 pM , Ca 2+ signals isplay a secon,slow kinetic component 1 . The late Ca 2+ entry procees in parallel withthe onset of a slow epolarization of the cell (Fig. 3e), inicating thatsperm become further epolarize by the late Ca 2+ entry. The slow epo-larization began at V m values ≥–40 mV (Fig. 3e, arrow). The simplestexplanation is that the late Ca 2+ entry occurs via high-voltage-activate(HVA) Ca  v  channels that open in this voltage range 14 . − 0.04 − (s)Time (s)Time (s)Time (s)0510 − 0.04 − − 50 − 60 − 70 − 80 − 90(8)(8)(7)(13)[Resact] (pM) − 0.030 − 0.0150.0000.0150.03001234 − 0.030 − 0.0150.0000.0150.030 adbec       ∆    R  a  n   d       ∆    F   n  o  r  m       ∆    R  a  n   d       ∆    F   n  o  r  m       ∆    R  a  n   d       ∆    F   n  o  r  m       ∆    R  a  n   d       ∆    F   n  o  r  m    V   t   h  r  e  s   h  o   l   d   (  m   V   ) Figure 3 Mechanism of Ca 2+ entry. ( a ) Superposition of voltage signals( Δ R, black) and Ca 2+ signals ( Δ F, red) evoked by 6.25 pM, 25 pM and125 pM of resact in sperm loaded with Fluo-4 or di-8-ANEPPS. The peakof Ca 2+ signals has been normalized to the peak of the respective voltagesignals. ( b ) Time course of voltage and Ca 2+ signals evoked by 25 pMresact (from a ). The dotted vertical line indicates the voltage V m at whichthe Ca 2+ signal commences to rise; V m = –61.6 mV. ( c ) Time course ofvoltage and Ca 2+ signals evoked by cGMP; arrow indicates the releaseof cGMP (flash); V m = –60.3 mV. ( d ) Dependence of V m of Ca v channelopening on the concentration of resact. The most negative value of V m  is the voltage threshold V th (–72.3 mV). The number of experiments aregiven in parentheses above the data points. The error bars represent s.d.( e ) Comparison of voltage and Ca 2+ signals on an extended time scale(125 pM resact). NaturePublishingGroup ©200 6  NATURE CELL BIOLOGY   VOLUME 8 | NUMBER 10 | OCTOBER 2006 1153 LETTERS Our results allow us to outline the complete cGMP signalling path-way that is involve in sperm chemotaxis (Fig. 4). When chemoat-tractants bin to a receptor-type guanylyl cyclase, the ensuing rapirise of the cGMP concentration 1 opens K + -selective CNG channelsan thereby hyperpolarizes the cell. The hyperpolarization removesinactivation from LVA Ca  v  channels that are inactivate at rest an acti- vates hyperpolarization-activate an cyclic-nucleotie-gate (HCN)channels. The repolarizing current that follows the hyperpolarizationis probably carrie by both HCN channels an Ca  v  channels. The early Ca 2+ influx via LVA Ca  v  channels controls the swimming behaviourof sperm 1,2 . The late Ca 2+ influx may occur via HVA Ca  v  channels. Arecent stuy has ientifie fragments of two istinct HVA Ca  v  chan-nels in testis of  S. purpuratus 16 . Alternatively, Ca 2+ entry through HCNchannels that are Ca 2+ permeable 17 shoul also be consiere.The ‘recovery-from-inactivation’ mechanism explains several featuresof the Ca 2+ signal. The elay of the Ca 2+ influx 1 is reaily accountefor by the kinetics of the hyperpolarization an the time it takes Ca  v   channels to recover from inactivation. The Ca 2+ signal saturates at lowconcentrations of resact 1 , probably because the hyperpolarization thatis prouce by ≥25 pM is sufficient to transfer all Ca  v  channels from theinactivate to the close state.Our results resolve long-staning controversies of chemoattractantsignalling in sperm. The persistent hyperpolarization that is observein swollen sperm reportely stimulates a rise in cAMP an pH i thateventually gate open the Ca 2+ entry channels 13,18–20 . However, cAMP isnot significantly altere uring the short hyperpolarization in intactsperm, supporting previous stuies that exclue cAMP as the trig-ger of the initial Ca 2+ influx 1,21 . In a similar vein, it is unlikely that thehyperpolarization promotes H + extrusion by voltage-activate Na + /H +  exchange 12  , because the resact-inuce change in pH i is slower an therelease of cGMP from cage compouns oes not evoke an increase of pH i22 but oes hyperpolarize sperm.The signalling pathways of sperm an ro photoreceptors 9 arestrikingly similar. Sperm an ros can etect single molecules anphotons, respectively. Sperm an ros both hyperpolarize on stimula-tion, although the mechanism of the hyperpolarization is ifferent:In sperm, a rise of cGMP opens K + -selective CNG channels, whereasin ros, a ecrease of cGMP closes non-selective cation CNG chan-nels. In this respect, cGMP signalling in sperm is more akin to ciliary photoreceptors of invertebrates that use a K + -selective CNG chan-nel for light signalling 23 . HCN channels in ros repolarize the cellquickly in bright light an prevent the voltage from reaching E K24 .HCN channels in sperm 25 are likely to serve a similar function: they resist hyperpolarization an allow sperm to encoe a wie range of chemoattractant concentrations.In ros, a single photon causes the hyrolysis of thousans of cGMPmolecules. Due to the slow rate of cGMP synthesis of receptor guanylylcyclases, the amplification in sperm is likely to be orers of magnitue cGMPcAMPGTPGCoutin‘Turn’Flagellar asymmetrypolarizationpolarizationpolarization + Stimulation − 42 mVTimeKCNGHCN & LVA Ca v HVA Ca v ab K +    K   C   N   G HyperRe-De-    H   C   N   L   V   A   C  a   v    H   V   A   C  a   v Na + Ca 2+ Ca 2+ [Ca 2 + ] i [Ca 2 + ] i Ligand    V  o   l   t  a  g  e Figure 4 Model of chemotactic signalling events in sperm. ( a ) Binding ofthe chemoattractant to a receptor guanylyl cyclase activates the synthesisof cGMP from GTP. Cyclic GMP opens K + -selective CNG channels,thereby causing hyperpolarization of the membrane. On hyperpolarization,hyperpolarization-activated and cyclic-nucleotide-gated (HCN) channelsand low-voltage-activated Ca v channels (LVA Ca v ) allow the influx of Na +  and Ca 2+ , respectively. Finally, the opening of high-voltage-activated Ca 2+  channels (HVA Ca v ) produce a sustained elevation of [Ca 2+ ] i . Ca 2+ ionsinteract by unknown mechanisms with the axoneme of the flagellum andcause an increase in the asymmetry of flagellar beat and, finally, a turn inthe swimming trajectory. ( b ) Contributions of the distinct ion channels to thevoltage response. NaturePublishingGroup ©200 6
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