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Originally published In Press as doi:10.1074/jbc.M506849200 on December 9, 2005

J. Biol. Chem., Vol. 281, Issue 7, 4156-4163, February 17, 2006
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The Cysteine-rich Secretory Protein Domain of Tpx-1 Is Related to Ion Channel Toxins and Regulates Ryanodine Receptor Ca2+ Signaling*

Gerard M. Gibbs{ddagger}, Martin J. Scanlon§, James Swarbrick§, Suzanne Curtis, Esther Gallant, Angela F. Dulhunty, and Moira K. O'Bryan{ddagger}||1

From the {ddagger}Monash Institute of Medical Research, the ||Australian Research Council Centre of Excellence in Biotechnology and Development, and the §Department of Medicinal Chemistry, Monash University, Clayton, 3168 Melbourne, Victoria and John Curtain School of Medical Research, the Australian National University, Canberra, Australian Capital Territory 2601, Australia

Received for publication, June 23, 2005 , and in revised form, November 8, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cysteine-rich secretory proteins (Crisp) are predominantly found in the mammalian male reproductive tract as well as in the venom of reptiles. Crisps are two domain proteins with a structurally similar yet evolutionary diverse N-terminal domain and a characteristic cysteine-rich C-terminal domain, which we refer to as the Crisp domain. We presented the NMR solution structure of the Crisp domain of mouse Tpx-1, and we showed that it contains two subdomains, one of which has a similar fold to the ion channel regulators BgK and ShK. Furthermore, we have demonstrated for the first time that the ion channel regulatory activity of Crisp proteins is attributed to the Crisp domain. Specifically, the Tpx-1 Crisp domain inhibited cardiac ryanodine receptor (RyR) 2 with an IC50 between 0.5 and 1.0 µM and activated the skeletal RyR1 with an AC50 between 1 and 10 µM when added to the cytoplasmic domain of the receptor. This activity was nonvoltage-dependent and weakly voltage-dependent, respectively. Furthermore, the Tpx-1 Crisp domain activated both RyR forms at negative bilayer potentials and showed no effect at positive bilayer potentials when added to the luminal domain of the receptor. These data show that the Tpx-1 Crisp domain on its own can regulate ion channel activity and provide compelling evidence for a role for Tpx-1 in the regulation of Ca2+ fluxes observed during sperm capacitation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tpx-1 (testis specific protein-1) was originally identified in the mouse (1) and later found in the male reproductive tract of the human, guinea pig, rat, and horse (2–5). Tpx-1 is a member of the cysteine-rich secretory proteins (Crisp)2 that are in turn a subgroup of the CAP protein superfamily (abbreviated from Crisp, Antigen 5, and Pr-1 (6)). The CAP proteins each contain a structurally related and unique domain, the CAP domain (7–9), that at present has no clearly defined biological function, although their spatial and temporal expression suggests a function related to the regulation of the innate immune system (10, 11) and male reproductive function. The Crisp proteins have a characteristic C-terminal sequence containing 10 absolutely conserved cysteines. The Crisp domain is unique and is only observed in association with the CAP domain. Previously, there has been no biological activity attributed specifically to this domain.

In mammals, there are at least four Crisp proteins, Crisp-1, Tpx-1 (or Crisp-2), Crisp-3, and Crisp-4. Crisp-1 proteins are expressed predominantly in the epididymides where they coat the surface of sperm during epididymal maturation (12) and have been implicated in sperm oocyte binding and the regulation of capacitation (13–17). Tpx-1 is expressed only in the testis and localized to specific regions in the spermatozoa, notably the acrosome of the head, the outer dense fibers, and longitudinal columns of the fibrous sheath in the sperm tail and the connecting piece of the neck (3, 18). Transfection experiments have also suggested that Tpx-1 is involved in adhesion between germ cells and Sertoli cells within the seminiferous epithelium (19). Crisp-3 is expressed more widely, including the salivary gland, pancreas, prostate, and B-cells (12, 20–22). Crisp-4 proteins are expressed exclusively in the epididymal epithelium in an androgen-dependent manner (23).

No clear biological function has been attributed to any mammalian Crisp protein, and characterizations have historically focused on expression location and timing to infer function. Nonmammalian Crisp proteins have, however, provided preliminary biochemical data. XCrisp expression occurs exclusively in the hatching gland of Xenopus and is associated with cellular membranes (24). The XCrisp Crisp domain is indirectly required for the degradation of the vitelline envelope through activation of the degradation pathway. Helothermine, a Crisp protein from the venom of the Mexican beaded lizard, causes the reversible concentration-dependent blockage of voltage-gated Ca2+ and K+ channels and ryanodine receptors (RyR) (25–28). Helothermine is the only Crisp protein known to regulate RyRs. Crisp proteins have also been identified in the venom from a range of snakes (29–33), and many have been shown to have specific K+ or Ca2+ ion channel inhibition activities and to block depolarization-induced muscle contraction (reviewed in Ref. 34). PsTx is one of the more comprehensively characterized Crisp proteins and has been shown to block CNGA2 channels through interaction with regions in or near the channel pore (35). Investigations on venom Crisp proteins were performed using full-length native proteins containing both the CAP and Crisp domains. As such, it remains unclear whether the ion channel inhibition activity can be attributed to the CAP domain, the Crisp domain, or to both.

Recently the crystal structures of Stecrisp and Triflin, Crisps proteins from the venom of the snake Trimersurus stejnegeri and Trimersurus flavoviridis, respectively, were determined (9, 36). They showed that the CAP domain and the cysteine-rich Crisp domain were present as two discrete domains. The CAP domains had the same {alpha}-beta-{alpha} fold as other CAP proteins P14a, Ves V5, and GAPR-1 (7, 8, 37). The Crisp domains included a linker region, containing two crossed disulfide bridges, and a domain with structural homology to the BgK (38) and ShK (39) ion channel inhibitor toxins from sea anemones.

As a first step toward defining the in vivo function of Tpx-1, we have determined the structure of the Crisp domain of recombinant mouse Tpx-1 by using NMR, and based on its homology with several ion channel inhibitors and published reports of RyR in mammalian sperm (40–43), we tested its ability to regulate RyR activity. We show that the Tpx-1 Crisp domain can elicit the activation of RyR1 and the inhibition of RyR2 when added to the cytoplasmic domain of the receptor and the activation of both forms when added to the luminal domain of the receptor. These data show the first structural representation of a mammalian Crisp domain and its structural homology to vertebrate and invertebrate toxins. We also show, for the first time, direct evidence that the Crisp domain is responsible for the ion channel inhibition activity previously observed in full-length Crisp proteins of lizard and snake origin.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning Expression and Purification of 15N-Labeled Tpx-1 Crisp Domain—The cDNA fragment encoding the Crisp domain of mouse Tpx-1 (beginning at CASCP) was subcloned into pTriEx4 (Neo) (Novagen), and the recombinant protein containing an N-terminal His6 tag and a thrombin cleavage site was expressed in Escherichia coli ORIGAMI B(DE3) pLacI (Novagen), which facilitated the formation of disulfide bonds in the cytoplasm. 15N incorporation was carried out according to the method of Marley et al. (44) in M9 minimal medium. Briefly, cultures were incubated in LB medium at 37 °C until mid-log phase, and cells were subsequently washed twice in 15N M9 minimal medium, concentrated 4-fold, and equilibrated to 30 °C prior to the induction of protein expression with 0.1 mM isopropyl 1-thio-beta-D-galactopyranoside for 4 h. The His:Tpx-1 Crisp domain was purified using nickel-nitrilotriacetic acid-immobilized metal affinity chromatography affinity resin (Calbiochem). 49 mg of His:Tpx-1 was purified from 4 liters of the original culture volume. The His6 tag was proteolytically removed by thrombin cleavage, and the Tpx-1 Crisp domain was purified by nickel-nitrilotriacetic acid affinity chromatography (to remove the His6 tag) and semi-preparative reversed phase HPLC to separate folded and mis-folded forms. The Tpx-1 Crisp domain was at least 95% pure, and ~98% of the purified recombinant Tpx-1 Crisp domain contained 15N as determined by liquid chromatography-mass spectrometry. Unlabeled protein was prepared using the same method, except expression was carried out in LB medium.

Generation of Denatured Tpx-1 Crisp Domain for RyR Channel Control Experiments—Mis-folded Tpx-1 Crisp domain was reduced with 10 mM dithiothreitol (Sigma) overnight and subsequently alkylated with 30 mM iodoacetamide (Sigma) for 90 min in the dark. The completeness of the alkylation reaction was assessed by mass spectroscopy. Alkylated protein was purified by reversed phase HPLC and freeze-dried. Immediately prior to use, proteins were resuspended in the required buffer, denatured by heating at 50 °C for 10 min, followed by rapid cooling on ice.

NMR Spectroscopy—NMR spectra were acquired at 500 MHz and 25 °C on a Bruker DRX500 equipped with a triple resonance TXI cryoprobe. Three-dimensional 15N-TOCSY-HSQC and 15N-NOESY-HSQC (45) experiments were acquired on a sample of ~1 mM uniformly 15N-labeled Tpx-1 Crisp domain in 90% H2O, 10% D2O, pH 5.8. pH values were uncorrected meter readings at room temperature. The sample was lyophilized and resuspended in 2H2O prior to the acquisition of the following two-dimensional experiments: double quantum-filtered correlated spectroscopy (46), TOCSY (47), NOESY (48), and 13C-HSQC. Acquisition parameters for the various experiments are summarized in Table 1.


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  		TABLE 1
Acquisition parameters for NMR data

 
Spectra were processed using XWINNMR and were analyzed using the program SPARKY (49). Sequence specific resonance assignments were made using standard methods (50).

Structure Calculations—Initial rounds of structure calculation were performed using the CANDID module as implemented in CYANA 1.0.6 (51). The final round of structure calculations was performed using by XPLOR-NIH (52) using experimental NOE-derived distance constraints and C-{alpha} and C-beta chemical shifts supplemented with Ramachandran data base potentials that were turned on during the annealing protocol. Structures were calculated using a simulated annealing protocol in torsion angle space (53) and finally subjected to energy minimization.

RyR Channel Activity Measurements—All methods for RyR channel activity measurements, including the preparation of sarcoplasmic reticulum vesicles, single channel techniques, analysis of channel activity, and the statistical analysis were performed as described previously (54). Tpx-1 Crisp domain was added to the cis chamber containing the cytoplasmic domain of RyR at concentrations of 0.1, 1.0, 10, and 50 µM. Tpx-1 Crisp domain was added to the trans chamber containing the luminal domain of RyR at 10 µM. All channel recordings using denatured Tpx-1 Crisp domain were done using 10 µM protein.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
NMR Spectroscopy—1H and 15N resonances were assigned from NOESY-HSQC and TOCSY-HSQC experiments using standard procedures (50). A single peak was obtained for each 1H-15N pair in the HSQC experiment (Fig. 1) consistent with a single set of conformations in solution.


Figure 1
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  		FIGURE 1.
1H-15N HSQC spectrum of the Tpx-1 Crisp domain. Labels represent assignments for the cross-peaks, with the numbering referring to the position of the corresponding residue in mature Tpx-1. The side chain NH2 amide protons of the asparagine and glutamine residues are indicated with horizontal straight lines.

 
Assignment of Tpx-1 Crisp Domain Disulfide Bonds—The disulfide bond connectivity in the Tpx-1 Crisp domain was determined from analysis of the NOESY spectra, which were examined for the presence of CAH{alpha}–CBHbeta and CAHbeta–CBHbeta correlations (55, 56). The absence of such NOEs was used to exclude potential disulfide bonds. This analysis suggested the disulfide bonding arrangement in the recombinant Tpx-1 Crisp domain to be Cys189–Cys196, Cys192–Cys201, Cys205–Cys238, Cys214–Cys232, and Cys223–Cys236. No anti-diagnostic NOEs were observed between CAH{alpha} and CBHN, which indicated that all cysteines were disulfide-bonded (57). This analysis was supported by the recently reported crystal structure of a native Crisp domain from Stecrisp (9).

The Tpx-1 Crisp Domain Structure Determination—Final structure calculations using XPLOR-NIH generated 894 distance constraints, including 230 long range NOEs, 226 medium range NOEs, 251 short range NOEs, 187 intra-range NOEs, and 39 CBCA shifts. Following structural refinement, a family of 23 structures (from a total of 49) with the lowest energies and least residual violations of the experimental restraints were chosen to represent the structure of the Tpx-1 Crisp domain. A summary of the structural statistics for this family of structures is given in Table 2. The structures have no violations of distance or dihedral restraints greater than 0.2 Å or 5°, respectively. They have good covalent geometry and favorable nonbonded contacts. 75% of the backbone {phi}/{psi} angles are in the most favored region of the Ramachandran plot.


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  		TABLE 2
Structural statistics for the family of Tpx-1 Crisp domain structures

Values are given as mean ± S.D. Force constants for the calculation of square well potentials for the NOE and chemical shift restraints were 30 kcal mol–1 Å–2 and 0.5 kcal mol–1 ppm–2, respectively. The Lennard-Jones van der Waals energy was calculated with the CHARMm empirical energy function.

 
The positional root mean square deviations (r.m.s.d.) of the backbone heavy atoms and angular order parameters are shown in Fig. 2. From these data it is apparent that the Tpx-1 Crisp domain contains two distinct and folded subdomains; one includes two short beta-strands and the other contains three short{alpha}-helices (Fig. 3A). The subdomains are linked via a poorly ordered loop encompassing residues Asn203–Ser204, and Fig. 3B highlights the relative rotational freedom of each subdomain. Stereoviews of the family of structures superimposed over the heavy atoms of the individual subdomains are presented in Fig. 3, C and D. Mean pairwise r.m.s.d. for the two subdomains are 0.78 and 1.29 Å over the backbone and heavy atoms of Cys192–Thr202 and 0.31 and 0.74 Å over the backbone and heavy atoms of residues Asp206–Cys238. No NOEs are observed between the two domains, suggesting that there is little or no interaction between them in solution. Coordinates for the final family of structures of Tpx-1 Crisp domain structures have been deposited in the Protein Data Bank under the accession code 2A05.

Comparison of the solution structure of Tpx-1 Crisp with the crystal structure of Stecrisp revealed that the two proteins adopt a similar fold. The positional r.m.s.d. of backbone heavy atoms (N, C, C-{alpha}) of the Tpx-1 Crisp domain and Stecrisp is 1.4 Å over amino acids Cys192 to Thr202 and 1.2 Å over amino acids Ser206 to Cys238. Structural comparisons using the DALI server (58) showed the region from Ser204 to His243 of Tpx-1 Crisp to be homologous to the voltage-sensitive K+ ion channel toxins BgK (38) and ShK (39). The r.m.s.d. of the backbone C-{alpha} atoms was 2.1 Å over 31 residues for BgK and 1.9 Å over 30 residues for ShK. The structural similarity to K+ ion channel toxins and the sequence homology to Crisp proteins such as PsTx and helothermine (Fig. 4) with ion channel blocking activity (28, 35) were highly suggestive that the Crisp domain of Tpx-1 and other Crisp proteins were responsible for this activity. We undertook experiments to test this hypothesis.


Figure 2
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  		FIGURE 2.
Positional r.m.s.d. of backbone heavy atoms (A) and angular order parameters for the {phi}, {psi} and {chi}1 angles in the family of the Tpx-1 Crisp domain structures (B). The positional r.m.s.d. in A was calculated with a structure alignment over amino acids Cys192–Thr202 (solid line) and Asp206–Cys238 (dashed line). Sequence and secondary structure elements are shown at the top of the figure.

 
The Tpx-1 Crisp Domain Shows a Subtype-specific Regulation of the Cytoplasmic Domain of RyR—The Tpx-1 Crisp domain, at concentrations between 0.1 and 50 µM, was added to the cis solution bathing the cytoplasmic side of RyR channels in lipid bilayers. The activity of the channels changed rapidly in a RyR subtype-specific manner when the protein was added. Cardiac RyR2 channels were inhibited, whereas skeletal RyR1 channels were activated. The addition of 10 µM denatured Tpx-1 Crisp domain to the cytoplasmic domain of RyR1 and RyR2 had no effect on the mean current at positive or negative applied bilayer potentials (Table 3).


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  		TABLE 3
Relative mean current after addition of 10 µM Tpx-1 Crisp domain to the luminal and cytoplasmic domains of RyR subtypes

Values are the relative mean current ± S.E. (S.D. for experiments on the luminal domain of RyR using denatured protein). n = number of replicates, p = probability (2 sided Student's t test).

 
The data in Fig. 5A show the decline in activity of a single RyR2 channel after addition of 1 µM Crisp domain to the cytoplasmic domain of RyR2 and further inhibition when the concentration was increased to 10 µM. The inhibition, apparent in the 3-s segments of activity and in the all-points histograms obtained from 30-s recordings, occurred within the 15-s stirring period after the protein was added to the solution. The inhibition was rapidly reversible when the 10 µM protein was perfused from the chamber (Fig. 5A) but did not reverse when 50 µM protein was removed, presumably because washout of the protein was incomplete, and the remaining Crisp domain after perfusion was sufficient to inhibit the channels. Although occasional full conductance openings were observed, most openings in the presence of the Tpx-1 Crisp domain were brief and poorly resolved. The average data obtained at bilayer potentials of +40 and –40 mV (Fig. 5, B and C, respectively) show the following: (a) the effects of the Tpx-1 Crisp domain on RyR2 inhibition were not voltage-dependent, (b) the IC50 was between 0.5 and 1 µM, and (c) incomplete recovery after removal of higher concentrations of the protein.


Figure 3
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  		FIGURE 3.
Structure of the Tpx-1 Crisp domain. A, single low energy conformation of the Tpx-1 Crisp domain showing residues Cys189–His243. B, residues Cys189–His243 superimposed over backbone heavy atoms Asp206–Cys238 (the ICR) of the 23 energy minimized structures. C, stereoviews of residues Cys205–Cys238, superimposed over the heavy atoms of residues Asp206–Cys238. D, stereoviews of residues Cys189–Ser205, superimposed over the backbone heavy atoms of residues Cys192–Thr202. Backbone heavy atoms are shown in blue, cysteine side chain heavy atoms in yellow, and the remaining side chain heavy atoms in magenta.

 
The effect of the Tpx-1 Crisp domain on RyR2 gating was to decrease the duration of channel openings and increase the closed periods. In 5 of 11 experiments, single channel recordings showed that the mean open time with 10 µM Crisp domain fell from 3.8 ± 1.2 to 2.3 ± 0.4 ms, whereas the mean closed time increased from 125.6 ± 33.48 to 1177 ± 607.8 ms (Fig. 5).

In marked contrast to the inhibitory effect on cardiac RyR2 channels, the Crisp domain enhanced the activity of skeletal RyR1 channels when added to the cytoplasmic domain (Fig. 6). The recordings in Fig. 6 were obtained from a bilayer containing two RyR1 channels. Long openings from one channel were apparent with additional brief openings of a second channel sometimes summing with the first channel. After adding the Tpx-1 Crisp domain at 1 and 10 µM, the duration of the openings increased, and more summed events were seen (Fig. 6A). The activation occurred without any increase in the single channel conductance. Changes in activity of the brief 1.5-s segments of activity shown in the figure reflect the changes in mean current measured from 30-s recordings at positive and negative potentials (given to the right of each record). The activity returned to control levels after removal of both 10 and 50 µM of the Tpx-1 Crisp domain. The average data in Fig. 6, B and C, show the following: (a) the activation displayed a weak voltage dependence; (b) the AC50 (activation constant) was between 1 and 10 µM; and (c) the effects were readily reversible.


Figure 4
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  		FIGURE 4.
Sequence alignment of the Crisp domain of selected Crisp proteins. 100% conserved amino acids are shaded. Tpx-1 structural elements and cysteine bonding architecture are indicated at the top of the alignment. Numbering is based on the translated Tpx-1 sequence. Also indicated are the structural elements observed in the Stecrisp crystal structure. Regions corresponding to the Hinge and ICR in the Crisp domain are indicated at the bottom of the alignment.

 
The effect of the Tpx-1 Crisp domain on RyR1 gating was measured in 2 of the 15 experiments in which the opening of only one channel was observed. The range of mean open times increased from 2.7 ± 3.5 to 4.2 ± 7.3 ms, and the mean closed times fell from 156 ± 670 to 16 ± 198 ms. Similar increases in open durations and decreases in closed durations could be observed in all multiple channel recording like those in Fig. 6.

The Tpx-1 Crisp Domain Caused an Increase in Mean Current through RyR1 and RyR2 at Negative Bilayer Potentials When Added to the Luminal Domain—Table 3 shows that addition of 10 µM Tpx-1 Crisp domain to the trans solution bathing the luminal domain of RyR1 and RyR2 caused an increase in the mean channel current when a bilayer potential of –40 mV was applied. A change in mean current was observed within the 15-s mixing period and over several subsequent 30-s recording periods. Mean current through RyR2 was significantly greater than control current (p = 0.032), and there was a nonsignificant trend for increased mean current for RyR1 (p = 0.09). No change in mean channel current was observed on either channel with an applied bilayer potential of +40 mV. Recordings returned to control levels following perfusion. It was not possible to determine changes in mean open and closed times as no single channel recordings were observed.

The addition of 10 µM denatured control Tpx-1 Crisp domain to the luminal domain of RyR1 and RyR2 had no effect at positive or negative applied bilayer potentials (Table 3).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Ion channels play a critical role in maintaining cellular homeostasis, and endogenous and exogenous regulators of ion channels are therefore of significant interest from a biological and pharmacological perspective. Crisp proteins appear unique as they contain a conserved domain that regulates ion channel activity and are introduced exogenously in the venom from a range of species and are present endogenously in the male reproductive tract. In both of these environments they are highly abundant proteins (29, 59, 60).

We have determined the first NMR solution structure of a mammalian Crisp domain and have shown the following. 1) It has structural homology to the Crisp domain of Stecrisp (9) and to the voltage-sensitive K+ ion channel regulators BgK (38) and ShK (39) from sea anemones. 2) It can activate RyR1 and inhibit RyR2 channel openings when applied to the cytoplasmic domain of the receptor. 3) It can activate both RyRs when added to the luminal domain of the receptor at negative applied potentials. 4) This activity is specifically attributed to the tertiary structure of the protein domain as denatured protein had no effect. This is the first time the ion channel regulatory activity of a Crisp protein has been shown specifically to be associated with the Crisp domain, and Tpx-1 is the first soluble sperm protein shown to regulate RyR Ca2+ ion channel activity directly.

The Tpx-1 Crisp Domain Has Two Subdomains—Tpx-1 has two subdomains (Figs. 2 and 3) that are structurally similar to Stecrisp (9) (Fig. 7). The Tpx-1 Hinge encompasses amino acids Cys189 to Asn203, and the cysteine-rich domain encompasses C-terminal amino acids Ser204 to His243 (Fig. 4). Rather than the cysteine-rich domain, as used by Guo et al. (9), we suggest the use of the more descriptive term for the C-terminal subdomain based upon the functional data showing regulation of RyRs. Therefore, we propose naming this the ion channel regulator (ICR) domain.


Figure 5
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  		FIGURE 5.
The Tpx-1 Crisp domain inhibits cardiac RyR2 channels. A, 3-s recordings from a bilayer containing a single RyR channel at a bilayer potential of –40 mV are shown to the left, and all points histograms obtained from 30 s of activity under each condition are to the right. As labeled, activity is shown under control conditions, after addition of 1 and then 10 µM of the Tpx-1 Crisp domain and finally after perfusion of the Crisp domain from the cis chamber. B and C, the bins show average relative open probability (+S.E.) for activity at +40 and –40 mV, respectively, under control (con) conditions, in the presence of the indicated concentrations of the Crisp domain and after perfusion of 10 or 50 µM the Crisp domain from the cis solution. Data were obtained from 11 experiments, with at least four observations at each concentration of the Crisp domain. Asterisks indicate values significantly different from control.

 
The Hinge contains two short beta-strands that are stabilized by crossed disulfide bonds that form a beta-hairpin. The Stecrisp crystal structure showed the conserved LCTN motif in the Hinge to be in close proximity to a conserved hydrophobic core of the CAP domain with extensive hydrogen bonding resulting in a stable interaction (9). The absence of NOEs and structural restraint between the subdomains of the Tpx-1 Crisp domain suggests that within the full-length Crisp protein the ICR will freely rotate in solution relative to the Hinge and the CAP domain and implies two discrete activities for the CAP domain and the ICR.

The Tpx-1 ICR Is Homologous to Venom Toxins—The Tpx-1 ICR forms an extremely stable and compact hydrophobic core. It is stabilized by three disulfide bonds whose conserved architecture and similar spacing with Stecrisp (9) and a family of voltage-sensitive K+ channel blockers, notably ShK (39) and BgK (38), results in a similar domain fold. The similarity in function of Tpx-1 to ShK and BgK is consistent with this structural similarity. The primary sequence homology between Crisp domains from human, mouse, snake, and lizard origin suggest that each of these domains will have a similar domain structure and function (Fig. 4). Indeed, similar activities have been observed for a range of native full-length Crisp proteins of reptile origin (34); however, it is now with absolute confidence that this activity can be attributed to the Crisp domain. Although this investigation has not conclusively demonstrated that the ion channel regulatory activity is restricted to the ICR, the structural relationship to the ShK and BgK toxins suggest this is the most likely function. The ICR is encoded by a distinct exon and present only in a subpopulation of the CAP superfamily, suggesting it has been acquired during the course of evolution and encodes for a separate and discrete activity, which is consistent with protein structural data.

The Tpx-1 Crisp Domain Is a Conserved Structural Element Displaying a Complex Ion Channel Regulatory Activity toward the RyRs—As Tpx-1 is localized to intracellular compartments in mature spermatozoa prior to the acrosome reaction (18), we were concerned in this investigation with understanding Tpx-1 function in relation to the release of Ca2+ from intracellular stores in sperm. In addition, given that helothermine has been shown to inhibit skeletal and cardiac RyRs (28), we thought it prudent to begin our investigation on this Ca2+ ion channel.

Although snake venom Crisp proteins have complex pharmacological activities, opposing activities on ion channel subtypes by Crisp proteins have not been reported previously. Furthermore, despite testing a range of snake venom Crisps (reviewed in Ref. 34), only helothermine has been shown to regulate RyR. The RyR1 stimulatory and RyR2 inhibitory activity reported herein following addition of the Tpx-1 Crisp domain to the cytoplasmic domain of RyR is unique among the Crisp proteins. However, it is not unique for regulators of the RyR. Subtype-specific effects on RyR channels have been reported with several proteins, e.g. homer (61, 62), glutathione transferases (63), and peptides corresponding to parts of the dihydropyridine receptor loop that display altered RyR activity depending upon the cytoplasmic [Ca2+] (64).


Figure 6
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  		FIGURE 6.
The Tpx-1 Crisp domain activates skeletal RyR1 channels. A, recordings of 1.5 s of activity from a bilayer containing two RyR1 channels at a bilayer potential of +40 mV are shown to the left, and the mean current measured form from 30s of activity (at +40 and –40 mV) under each condition are to the right. As labeled, activity is shown under control conditions, after addition of 1 and then 10 µM Crisp and finally after perfusion of Crisp from the cis chamber. B and C, the bins show average relative open probability (Po)(+S.E.) for activity at +40 and –40 mV, respectively, under control (con) conditions, in the presence of the indicated concentrations of Crisp and after perfusion of 10 or 50 µM Crisp from the cis solution. Data were obtained from 15 experiments, with at least five observations at each concentration of Crisp. Asterisks indicate values significantly different from control. The dot over the perfusion data indicates a significant fall in relative Po after perfusion.

 
The opposing effects observed for the 72% identical RyR1 and RyR2 (65) suggest that the Tpx-1 Crisp domain binds to multiple regions of the ion channel on the cytoplasmic domain. The voltage dependence observed for the activation of RyR1 through the cytoplasmic domain suggested that binding occurred in a region that is close to the membrane and sensitive to changes in the membrane field, but that it does not occlude the channel pore and thus must interact with regulatory regions near the pore. The occasional full conductance opening and voltage-independent inhibition of RyR2 activity suggested that the Tpx-1 Crisp domain regulated ion channel gating through interaction with regulatory regions of the ion channel remote from the pore and not influenced by the membrane field, or that it formed moderate strength-reversible interactions within the pore that inhibited channel gating and blocked current flow through the channel while bound. When added to the luminal domain of both RyR1 and RyR2, Tpx-1 increased the mean current through the receptor at a negative applied bilayer potential, suggesting that binding occurred close to the membrane field at regulatory sites near the pore but that it did not occlude the pore. In this regard the Tpx-1 Crisp domain appears to function as a gating modifier of RyRs and to bind multiple sites on the receptor influenced by the membrane field.


Figure 7
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  		FIGURE 7.
Structural comparison of the Tpx-1 Crisp domain to the Stecrisp Crisp domain. Backbone heavy atoms of Tpx-1 Crisp domain (shown in red), from Cys192–His243 were overlaid with Stecrisp at residues Cys192–Thr202 (A) and residues Asp206–Cys238 (B). Numbering is relative to Tpx-1 translation.

 
The data show that the affinity of the Tpx-1 Crisp domain for the inhibition site on the cytoplasmic domain of RyR2 is greater than that for the activation site on the cytoplasmic domain of RyR1. In particular, 1) the concentration of the Tpx-1 Crisp domain required for inhibition of RyR2 was half that required for the activation of RyR1 and; 2) RyR2 inhibition could not be reversed at higher Tpx-1 Crisp domain concentrations following perfusion. The 1000–10,000-fold dilution of protein following perfusion (66), which would have left 5–50 nM of the Tpx-1 Crisp domain, was sufficient to maintain inhibition. A greater affinity of the Tpx-1 Crisp domain for the inhibition site on RyR2 is in contrast with that reported for helothermine (28), which had a greater affinity for the activation site on RyR1. Although reasons for these different affinities are not currently clear, it reminds us that Crisp proteins have subtle and complex regulatory activities, as highlighted by the 30-fold difference in activity on CNGA2 channels for the 97% identical PsTx and pseudecin (30).

Accumulating data on Crisp proteins here and elsewhere show that they regulate ion channel currents through interaction with the ion channel receptor in a highly sequence-specific manner. These data imply that Crisp proteins in the male reproductive tract have important role(s) in the regulation of sperm maturation processes involving ion flux such as Ca2+ signaling during sperm capacitation, the transition to hyperactivated motility, and/or fertilization.


   FOOTNOTES
 
* This work was supported by National Health and Medical Research Council Grant 334011 and Australian Research Council Grants CE0348239 (to M. K. O. B.) and DP0557780 (to A. F. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 2A05) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). Back

1 Recipient of a senior research fellowship from Monash University. To whom correspondence should be addressed: Monash Institute of Medical Research, Monash University, 27-31 Wright St., Clayton, 3168, Victoria, Australia. Tel.: 613-9594-7127; Fax: 613-9594-7114; E-mail: Moira.OBryan{at}med.monash.edu.au.

2 The abbreviations used are: Crisp, cysteine-rich secretory proteins; RyR, ryanodine receptor; r.m.s.d., root mean square deviations; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; HSQC, heteronuclear single quantum coherence; HPLC, high pressure liquid chromatography; ICR, ion channel regulator; TOCSY, total correlated spectroscopy. Back


   ACKNOWLEDGMENTS
 
We thank Stuart Thomson for the analysis of the recombinant Tpx-1 Crisp domain by liquid chromatography-mass spectrometry. We also thank Dane Culley for assistance with single channel recordings.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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