Flecainide Paradoxically Activates Cardiac Ryanodine Receptor Channels under Low Activity Conditions: A Potential Pro-Arrhythmic Action.

Cardiac ryanodine receptor (RyR2) mutations are implicated in the potentially fatal catecholaminergic polymorphic ventricular tachycardia (CPVT) and in atrial fibrillation. CPVT has been successfully treated with flecainide monotherapy, with occasional notable exceptions. Reported actions of flecainide on cardiac sodium currents from mice carrying the pro-arrhythmic homozygotic RyR2-P2328S mutation prompted our explorations of the effects of flecainide on their RyR2 channels. Lipid bilayer electrophysiology techniques demonstrated a novel, paradoxical increase in RyR2 activity. Preceding flecainide exposure, channels were mildly activated by 1 mM luminal Ca2+ and 1 µM cytoplasmic Ca2+, with open probabilities (Po) of 0.03 ± 0.01 (wild type, WT) or 0.096 ± 0.024 (P2328S). Open probability (Po) increased within 0.5 to 3 min of exposure to 0.5 to 5.0 µM cytoplasmic flecainide, then declined with higher concentrations of flecainide. There were no such increases in a subset of high Po channels with Po ≥ 0.08, although Po then declined with ≥5 µM (WT) or ≥50 µM flecainide (P2328S). On average, channels with Po < 0.08 were significantly activated by 0.5 to 10 µM of flecainide (WT) or 0.5 to 50 µM of flecainide (P2328S). These results suggest that flecainide can bind to separate activation and inhibition sites on RyR2, with activation dominating in lower activity channels and inhibition dominating in more active channels.

Neutralizing the pathological effects of extracellular histones with small polyanions.

Extracellular histones in neutrophil extracellular traps (NETs) or in chromatin from injured tissues are highly pathological, particularly when liberated by DNases. We report the development of small polyanions (SPAs) (~0.9-1.4 kDa) that interact electrostatically with histones, neutralizing their pathological effects. In vitro, SPAs inhibited the cytotoxic, platelet-activating and erythrocyte-damaging effects of histones, mechanistic studies revealing that SPAs block disruption of lipid-bilayers by histones. In vivo, SPAs significantly inhibited sepsis, deep-vein thrombosis, and cardiac and tissue-flap models of ischemia-reperfusion injury (IRI), but appeared to differ in their capacity to neutralize NET-bound versus free histones. Analysis of sera from sepsis and cardiac IRI patients supported these differential findings. Further investigations revealed this effect was likely due to the ability of certain SPAs to displace histones from NETs, thus destabilising the structure. Finally, based on our work, a non-toxic SPA that inhibits both NET-bound and free histone mediated pathologies was identified for clinical development.

Ion channel gating in cardiac ryanodine receptors from the arrhythmic RyR2-P2328S mouse.

Mutations in the cardiac ryanodine receptor Ca2+ release channel (RyR2) can cause deadly ventricular arrhythmias and atrial fibrillation (AF). The RyR2-P2328S mutation produces catecholaminergic polymorphic ventricular tachycardia (CPVT) and AF in hearts from homozygous RyR2P2328S/P2328S (denoted RyR2S/S) mice. We have now examined P2328S RyR2 channels from RyR2S/S hearts. The activity of wild-type (WT) and P2328S RyR2 channels was similar at a cytoplasmic [Ca2+] of 1 mM, but P2328S RyR2 was significantly more active than WT at a cytoplasmic [Ca2+] of 1 µM. This was associated with a >10-fold shift in the half maximal activation concentration (AC50) for Ca2+ activation, from ∼3.5 µM Ca2+ in WT RyR2 to ∼320 nM in P2328S channels and an unexpected >1000-fold shift in the half maximal inhibitory concentration (IC50) for inactivation from ∼50 mM in WT channels to ≤7 µM in P2328S channels, which is into systolic [Ca2+] levels. Unexpectedly, the shift in Ca2+ activation was not associated with changes in sub-conductance activity, S2806 or S2814 phosphorylation or the level of FKBP12 (also known as FKBP1A) bound to the channels. The changes in channel activity seen with the P2328S mutation correlate with altered Ca2+ homeostasis in myocytes from RyR2S/S mice and the CPVT and AF phenotypes.This article has an associated First Person interview with the first author of the paper.

Association of FK506 binding proteins with RyR channels - effect of CLIC2 binding on sub-conductance opening and FKBP binding.

Ryanodine receptor (RyR) Ca2+ channels are central to striated muscle function and influence signalling in neurons and other cell types. Beneficially low RyR activity and maximum conductance opening may be stabilised when RyRs bind to FK506 binding proteins (FKBPs) and destabilised by FKBP dissociation, with submaximal opening during RyR hyperactivity associated with myopathies and neurological disorders. However, the correlation with submaximal opening is debated and quantitative evidence is lacking. Here, we have measured altered FKBP binding to RyRs and submaximal activity with addition of wild-type (WT) CLIC2, an inhibitory RyR ligand, or its H101Q mutant that hyperactivates RyRs, which probably causes cardiac and intellectual abnormalities. The proportion of sub-conductance opening increases with WT and H101Q CLIC2 and is correlated with reduced FKBP-RyR association. The sub-conductance opening reduces RyR currents in the presence of WT CLIC2. In contrast, sub-conductance openings contribute to excess RyR 'leak' with H101Q CLIC2. There are significant FKBP and RyR isoform-specific actions of CLIC2, rapamycin and FK506 on FKBP-RyR association. The results show that FKBPs do influence RyR gating and would contribute to excess Ca2+ release in this CLIC2 RyR channelopathy.

A new cytoplasmic interaction between junctin and ryanodine receptor Ca2+ release channels.

Junctin, a non-catalytic splice variant encoded by the aspartate-ß-hydroxylase (Asph) gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca(2+) store where it modifies Ca(2+) signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca(2+) release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca(2+) signalling, muscle dysfunction and cardiac arrhythmia. However, the nature of the molecular interaction between junctin and RyRs is largely unknown and was assumed to occur only in the SR lumen. We find that there is substantial binding of RyRs to full junctin, and the junctin luminal and, unexpectedly, cytoplasmic domains. Binding of these different junctin domains had distinct effects on RyR1 and RyR2 activity: full junctin in the luminal solution increased RyR channel activity by ∼threefold, the C-terminal luminal interaction inhibited RyR channel activity by ∼50%, and the N-terminal cytoplasmic binding produced an ∼fivefold increase in RyR activity. The cytoplasmic interaction between junctin and RyR is required for luminal binding to replicate the influence of full junctin on RyR1 and RyR2 activity. The C-terminal domain of junctin binds to residues including the S1-S2 linker of RyR1 and N-terminal domain of junctin binds between RyR1 residues 1078 and 2156.

Cardiac ryanodine receptor activation by a high Ca²âº store load is reversed in a reducing cytoplasmic redox environment.

Here, we report the impact of redox potential on isolated cardiac ryanodine receptor (RyR2) channel activity and its response to physiological changes in luminal [Ca(2+)]. Basal leak from the sarcoplasmic reticulum is required for normal Ca(2+) handling, but excess diastolic Ca(2+) leak attributed to oxidative stress is thought to lower the threshold of RyR2 for spontaneous sarcoplasmic reticulum Ca(2+) release, thus inducing arrhythmia in pathological situations. Therefore, we examined the RyR2 response to luminal [Ca(2+)] under reducing or oxidising cytoplasmic redox conditions. Unexpectedly, as luminal [Ca(2+)] increased from 0.1 to 1.5 mM, RyR2 activity declined when pretreated with cytoplasmic 1 mM DTT or buffered with GSH∶GSSG to a normal reduced cytoplasmic redox potential (-220 mV). Conversely, with 20 µM cytoplasmic 4,4'-DTDP or buffering of the redox potential to an oxidising value (-180 mV), RyR2 activity increased with increasing luminal [Ca(2+)]. The luminal redox potential was constant at -180 mV in each case. These responses to luminal [Ca(2+)] were maintained with cytoplasmic 2 mM Na2ATP or 5 mM MgATP (1 mM free Mg(2+)). Overall, the results suggest that the redox potential in the RyR2 junctional microdomain is normally more oxidised than that of the bulk cytoplasm.

Multiple actions of phi-LITX-Lw1a on ryanodine receptors reveal a functional link between scorpion DDH and ICK toxins.

We recently reported the isolation of a scorpion toxin named U1-liotoxin-Lw1a (U1-LITX-Lw1a) that adopts an unusual 3D fold termed the disulfide-directed hairpin (DDH) motif, which is the proposed evolutionary structural precursor of the three-disulfide-containing inhibitor cystine knot (ICK) motif found widely in animals and plants. Here we reveal that U1-LITX-Lw1a targets and activates the mammalian ryanodine receptor intracellular calcium release channel (RyR) with high (fM) potency and provides a functional link between DDH and ICK scorpion toxins. Moreover, U1-LITX-Lw1a, now described as ϕ-liotoxin-Lw1a (ϕ-LITX-Lw1a), has a similar mode of action on RyRs as scorpion calcines, although with significantly greater potency, inducing full channel openings at lower (fM) toxin concentrations whereas at higher pM concentrations increasing the frequency and duration of channel openings to a submaximal state. In addition, we show that the C-terminal residue of ϕ-LITX-Lw1a is crucial for the increase in full receptor openings but not for the increase in receptor subconductance opening, thereby supporting the two-binding-site hypothesis of scorpion toxins on RyRs. ϕ-LITX-Lw1a has potential both as a pharmacological tool and as a lead molecule for the treatment of human diseases that involve RyRs, such as malignant hyperthermia and polymorphic ventricular tachycardia.

An X-linked channelopathy with cardiomegaly due to a CLIC2 mutation enhancing ryanodine receptor channel activity.

Chloride intracellular channel 2 (CLIC2) protein is a member of the glutathione transferase class of proteins. Its' only known function is the regulation of ryanodine receptor (RyR) intracellular Ca(2+) release channels. These RyR proteins play a major role in the regulation of Ca(2+) signaling in many cells. Utilizing exome capture and deep sequencing of genes on the X-chromosome, we have identified a mutation in CLIC2 (c.303C>G, p.H101Q) which is associated with X-linked intellectual disability (ID), atrial fibrillation, cardiomegaly, congestive heart failure (CHF), some somatic features and seizures. Functional studies of the H101Q variant indicated that it stimulated rather than inhibited the action of RyR channels, with channels remaining open for longer times and potentially amplifying Ca(2+) signals dependent on RyR channel activity. The overly active RyRs in cardiac and skeletal muscle cells and neuronal cells would result in abnormal cardiac function and trigger post-synaptic pathways and neurotransmitter release. The presence of both cardiomegaly and CHF in the two affected males and atrial fibrillation in one are consistent with abnormal RyR2 channel function. Since the dysfunction of RyR2 channels in the brain via 'leaky mutations' can result in mild developmental delay and seizures, our data also suggest a vital role for the CLIC2 protein in maintaining normal cognitive function via its interaction with RyRs in the brain. Therefore, our patients appear to suffer from a new channelopathy comprised of ID, seizures and cardiac problems because of enhanced Ca(2+) release through RyRs in neuronal cells and cardiac muscle cells.

The ß(1a) subunit of the skeletal DHPR binds to skeletal RyR1 and activates the channel via its 35-residue C-terminal tail.

Although it has been suggested that the C-terminal tail of the ß(1a) subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca(2+) release in skeletal muscle by interacting with the skeletal ryanodine receptor (RyR1), a direct functional interaction between the two proteins has not been demonstrated previously. Such an interaction is reported here. A peptide with the sequence of the C-terminal 35 residues of ß(1a) bound to RyR1 in affinity chromatography. The full-length ß(1a) subunit and the C-terminal peptide increased [(3)H]ryanodine binding and RyR1 channel activity with an AC(50) of 450-600 pM under optimal conditions. The effect of the peptide was dependent on cytoplasmic Ca(2+), ATP, and Mg(2+) concentrations. There was no effect of the peptide when channel activity was very low as a result of Mg(2+) inhibition or addition of 100 nM Ca(2+) (without ATP). Maximum increases were seen with 1-10 µM Ca(2+), in the absence of Mg(2+) inhibition. A control peptide with the C-terminal 35 residues in a scrambled sequence did not bind to RyR1 or alter [(3)H]ryanodine binding or channel activity. This high-affinity in vitro functional interaction between the C-terminal 35 residues of the DHPR ß(1a) subunit and RyR1 may support an in vivo function of ß(1a) during voltage-activated Ca(2+) release.

The elusive role of the SPRY2 domain in RyR1.

The second of three SPRY domains (SPRY2, S1085 -V1208) located in the skeletal muscle ryanodine receptor (RyR1) is contained within regions of RyR1 that influence EC coupling and bind to imperatoxin A, a toxin probe of RyR1 channel gating. We examined the binding of the F loop (P1107-A1121) in SPRY2 to the ASI/basic region in RyR1 (T3471-G3500, containing both alternatively spliced (ASI) residues and neighboring basic amino acids). We then investigated the possible influence of this interaction on excitation contraction (EC) coupling. A peptide with the F loop sequence and an antibody to the SPRY2 domain each enhanced RyR1 activity at low concentrations and inhibited at higher concentrations. A peptide containing the ASI/basic sequence bound to SPRY2 and binding decreased ~10-fold following mutation or structural disruption of the basic residues. Binding was abolished by mutation of three critical acidic F loop residues. Together these results suggest that the ASI/basic and SPRY2 domains interact in an F loop regulatory module. Although a region that includes the SPRY2 domain influences EC coupling, as does the ASI/basic region, Ca2+ release during ligand- and depolarization-induced RyR1 activation were not altered by mutation of the three critical F loop residues following expression of mutant RyR1 in RyR1-null myotubes. Therefore the electrostatic regulatory interaction between the SPRY2 F loop residues (that bind to imperatoxin A) and the ASI/basic residues of RyR1 does not influence bi-directional DHPR-RyR1 signaling during skeletal EC coupling, possibly because the interaction is interrupted by the influence of factors present in intact muscle cells.

Junctin and triadin each activate skeletal ryanodine receptors but junctin alone mediates functional interactions with calsequestrin.

Normal Ca(2+) signalling in skeletal muscle depends on the membrane associated proteins triadin and junctin and their ability to mediate functional interactions between the Ca(2+) binding protein calsequestrin and the type 1 ryanodine receptor in the lumen of the sarcoplasmic reticulum. This important mechanism conserves intracellular Ca(2+) stores, but is poorly understood. Triadin and junctin share similar structures and are lumped together in models of interactions between skeletal muscle calsequestrin and ryanodine receptors, however their individual roles have not been examined at a molecular level. We show here that purified skeletal ryanodine receptors are similarly activated by purified triadin or purified junctin added to their luminal side, although a lack of competition indicated that the proteins act at independent sites. Surprisingly, triadin and junctin differed markedly in their ability to transmit information between skeletal calsequestrin and ryanodine receptors. Purified calsequestrin inhibited junctin/triadin-associated, or junctin-associated, ryanodine receptors and the calsequestrin re-associated channel complexes were further inhibited when luminal Ca(2+) fell from 1mM to

Dissection of the inhibition of cardiac ryanodine receptors by human glutathione transferase GSTM2-2.

The muscle specific glutathione transferase GSTM2-2 inhibits the activity of cardiac ryanodine receptor (RyR2) calcium release channels with high affinity and activates skeletal RyR (RyR1) channels with lower affinity. To determine which overall region of the GSTM2-2 molecule supports binding to RyR2, we examined the effects of truncating GSTM2-2 on its ability to alter Ca(2+) release from sarcoplasmic reticulum (SR) vesicles and RyR channel activity. The C-terminal half of GSTM2-2 which lacks the critical GSH binding site supported the inhibition of RyR2, but did not support activation of RyR1. Smaller fragments of GSTM2-2 indicated that the C-terminal helix 6 was crucial for the action of GSTM2-2 on RyR2. Only fragments containing the helix 6 sequence inhibited Ca(2+) release from cardiac SR. Single RyR2 channels were strongly inhibited by constructs containing the helix 6 sequence in combination with adjacent helices (helices 5-8 or 4-6). Fragments containing helices 5-6 or helix 6 sequences alone had less well-defined effects. Chemical cross-linking indicated that C-terminal helices 5-8 bound to RyR2, but not RyR1. Structural analysis with circular dichroism showed that the helical content was greater in the longer helix 6 containing constructs, while the helix 6 sequence alone had minimal helical structure. Therefore the active centre of GSTM2-2 for inhibition of cardiac RyR2 involves the helix 6 sequence and the helical nature of this region is essential for its efficacy. GSTM2-2 helices 5-8 may provide the basis for RyR2-specific compounds for experimental and therapeutic use.

Redox potential and the response of cardiac ryanodine receptors to CLIC-2, a member of the glutathione S-transferase structural family.

The type 2 chloride intracellular channel, CLIC-2, is a member of the glutathione S-transferase structural family and a suppressor of cardiac ryanodine receptor (RyR2) Ca2+ channels located in the membrane of the sarcoplasmic reticulum (SR). Modulators of RyR2 activity can alter cardiac contraction. Since both CLIC-2 and RyR2 are modified by redox reactions, we speculated that the action of CLIC-2 on RyR2 may depend on redox potential. We used a GSH:GSSG buffer system to produce mild changes in redox potential to influence redox sensors in RyR2 and CLIC-2. RyR2 activity was modified only when both luminal and cytoplasmic solutions contained the GSH:GSSG buffer and the effects were reversed by removing the buffer from one of the solutions. Channel activity increased with an oxidizing redox potential and decreased when the potential was more reducing. Addition of cytoplasmic CLIC-2 inhibited RyR2 with oxidizing redox potentials, but activated RyR2 under reducing conditions. The results suggested that both RyR2 and CLIC-2 contain redox sensors. Since cardiac ischemia involves a destructive Ca2+ overload that is partly due to oxidation-induced increase in RyR2 activity, we speculate that the properties of CLIC-2 place it in an ideal position to limit ischemia-induced cellular damage in cardiac muscle.

Muscle-specific GSTM2-2 on the luminal side of the sarcoplasmic reticulum modifies RyR ion channel activity.

We show that a glutathione transferase (GST) protein, which is recognised by an antibody against the muscle-specific human GSTM2-2 (hGSTM2-2), is associated with the lumen of the sarcoplasmic reticulum (SR) of cardiac muscle, but not skeletal muscle. We further show that hGSTM2-2 modifies both cardiac and skeletal ryanodine receptor (RyR) activity when it binds to the luminal domain of the RyR channel complex. The properties of hGSTM2-2 were compared with those of the calsequestrin (CSQ), a Ca(2+) binding protein also present in the lumen of the SR which, like GSTM2-2, contains a thioredoxin-fold structure and modifies RyR activity (Wei, L., Varsanyi, M., Dulhunty, A. F., Beard, N. A. (2006). The Biophysical Journal, 91, 1288-1301). The glutathione transferase activity of hGSTM2-2 is strong, while CSQ is essentially inactive. Conversely CSQ is a strong Ca(2+) binder, but hGSTM2-2 is not. The effects of luminal hGSTM2-2 on RyR activity differ from those of CSQ in that hGSTM2-2 activates RyRs by increasing their open probability and conductance and the effects are independent of luminal Ca(2+) concentration. The results suggest that GSTM2-2 can interact with specific luminal sites on the RyR complex and that the interaction is likely to be within the pore of the RyR channel. The differences between the effects of CSQ and hGSTM2-2 suggest that the thioredoxin fold is not a major determinant of the luminal actions of either protein. The results indicate that GSTM2-2 is a novel luminal regulator of the RyR channels in the heart.

The Mu class glutathione transferase is abundant in striated muscle and is an isoform-specific regulator of ryanodine receptor calcium channels.

Members of the glutathione transferase (GST) structural family are novel regulators of cardiac ryanodine receptor (RyR) calcium channels. We present the first detailed report of the effect of endogenous muscle GST on skeletal and cardiac RyRs. An Mu class glutathione transferase is specifically expressed in human muscle. An hGSTM2-2-like protein was isolated from rabbit skeletal muscle and sheep heart, at concentrations of approximately 17-93 microM. When added to the cytoplasmic side of RyRs, hGSTM2-2 and GST isolated from skeletal or cardiac muscle, modified channel activity in an RyR isoform-specific manner. High activity skeletal RyR1 channels were inactivated at positive potentials or activated at negative potentials by hGSTM2-2 (8-30 microM). Inactivation became faster as the positive voltage was increased. Channels recovered from inactivation when the voltage was reversed, but recovery times were significantly slowed in the presence of hGSTM2-2 and muscle GSTs. Low activity RyR1 channels were activated at both potentials. In contrast, hGSTM2-2 and GSTs isolated from muscle (1-30 microM) in the cytoplasmic solution, caused a voltage-independent inhibition of cardiac RyR2 channels. The results suggest that the major GST isoform expressed in muscle regulates Ca2+ signalling in skeletal and cardiac muscle and conserves Ca2+ stores in the sarcoplasmic reticulum.

Exclusion of linkage of the RYR1, CACNA1S, and ATP2A1 genes to recurrent exertional rhabdomyolysis in Thoroughbreds.

OBJECTIVE: To determine whether there was genetic linkage between the recurrent exertional rhabdomyolysis (RER) trait in Thoroughbred horse pedigrees and DNA markers in genes (the sarcoplasmic reticulum calcium release channel [RYR1] gene, the sarcoplasmic reticulum calcium ATPase [ATP2A1] gene, and the transverse tubule dihydropyridine receptor-voltage sensor [CACNA1S] gene) that are important in myoplasmic calcium regulation. ANIMALS: 34 horses in the University of Minnesota RER resource herd and 62 Thoroughbreds from 3 families of Thoroughbreds outside of the university in which RER-affected status was assigned after 2 or more episodes of ER had been observed. PROCEDURES: Microsatellite DNA markers from the RYR1, ATP2A1, and CACNA1S gene loci on equine chromosomes 10, 13, and 30 were identified. Genotypes were obtained for all horses in the 4 families affected by RER, and data were used to test for linkage of these 3 loci to the RER phenotype. RESULTS: Analysis of the RYR1, CACNA1S, and ATP2A1 microsatellites excluded a link between those markers and the RER trait. CONCLUSIONS AND CLINICAL RELEVANCE: It is likely that the heritable alterations in muscle contractility that are characteristic of RER are caused by a gene that is not yet known to cause related muscle disease in other species.

Effects of an alpha-helical ryanodine receptor C-terminal tail peptide on ryanodine receptor activity: modulation by Homer.

We have determined the structure of a domain peptide corresponding to the extreme 19 C-terminal residues of the ryanodine receptor Ca2+ release channel. We examined functional interactions between the peptide and the channel, in the absence and in the presence of the regulatory protein Homer. The peptide was partly alpha-helical and structurally homologous to the C-terminal end of the T1 domain of voltage-gated K+ channels. The peptide (0.1-10 microM) inhibited skeletal ryanodine receptor channels when the cytoplasmic Ca2+ concentration was 1 microM; but with 10 microM cytoplasmic Ca2+, skeletal ryanodine receptors were activated by < or = 1.0 microM peptide and inhibited by 10 microM peptide. Cardiac ryanodine receptors on the other hand were inhibited by all peptide concentrations, at both Ca2+ concentrations. When channels did open in the presence of the peptide, they were more likely to open to substate levels. The inhibition and increased fraction of openings to subconductance levels suggested that the domain peptide might destabilise inter-domain interactions that involve the C-terminal tail. We found that Homer 1b not only interacts with the channels, but reduces the inhibitory action of the C-terminal tail peptide, perhaps by stabilizing inter-domain interactions and preventing their disruption.

The cysteine-rich secretory protein domain of Tpx-1 is related to ion channel toxins and regulates ryanodine receptor Ca2+ signaling.

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 IC(50) between 0.5 and 1.0 microM and activated the skeletal RyR1 with an AC(50) between 1 and 10 microM 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 Ca(2+) fluxes observed during sperm capacitation.