The Cysteine-rich Secretory Protein Domain of Tpx-1 Is Related to Ion Channel Toxins and Regulates Ryanodine Receptor Ca2+ Signaling

Abstract

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 M and activated the skeletal RyR1 with an AC 50 between 1 and 10 M 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.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)(8)(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...

Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function

“…The slow return to control current levels indicated a moderate-to-high affinity with a receptor. These data were consistent with previous results for CRISP2 inhibition of ryanodine receptor-2 (10). No inhibition was evident in control experiments performed with 1 μM reduced, alkylated, and heat-inactivated CRISP4 CRISP domain, indicating that inhibition required natively folded CRISP4.…”

“…Reptile CRISPs can regulate a range of ion channels including voltage-gated K + (K V ) and Ca 2+ (Ca V ) channels, cyclic-nucleotide gated A channels and ryanodine receptors (12,13). Consistent with this, mouse CRISP2 is capable of altering ryanodine receptor function in vitro (10).…”

“…CRISPs are vertebrate-specific, contain a C-terminal CRISP domain (10,11), and are found primarily in the mammalian male reproductive tract and in reptile venom (9,12). Reptile CRISPs can regulate a range of ion channels including voltage-gated K + (K V ) and Ca 2+ (Ca V ) channels, cyclic-nucleotide gated A channels and ryanodine receptors (12,13).…”

Allurin: Exploring the Activity of a Frog Sperm Chemoattractant in Mammals

“…4.1 , full-length Crisp proteins contain the PR domain-Hinge domain combination just discussed as well as the ion channel regulatory (ICR) domain. The ICR domain, as previously mentioned, has been demonstrated to modulate the actions of voltage-dependent potassium channels, voltage-dependent calcium channels, and ryanodine receptors (Gibbs et al 2006 ;Brown et al 1999 ;Morrissette et al 1995 ;Nobile et al 1994Nobile et al , 1996. These properties target channels in muscle and nerve during the actions of Crisp protein toxins found in snakes and lizards.…”

“…Next is the Hinge domain (yellow), the smallest of the three, named for the fact that it links the fi rst and third domain. Last is the ion channel regulatory (ICR) domain at the C-terminal (magenta), named for its ability to block or modulate a variety of voltage-dependent potassium and calcium channels as well as ryanodine receptors (Gibbs et al 2006 ). The ICR domain is similar in tertiary structure to ion channelblocking peptides from sea anenomes (Pennington et al 1999 ;Alessandri-Haber et al 1999 ;Cotton et al 1997 ).…”