RESUMEN
BACKGROUND: Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. METHODS: Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. RESULTS: Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4-C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. CONCLUSIONS: This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.
RESUMEN
Familial hemiplegic migraine type 1 (FHM-1) is a monogenic form of migraine with aura that is characterized by recurrent attacks of a typical migraine headache with transient hemiparesis during the aura phase. In a subset of patients, additional symptoms such as epilepsy and cerebellar ataxia are part of the clinical phenotype. FHM-1 is caused by missense mutations in the CACNA1A gene that encodes the pore-forming subunit of Ca(V)2.1 voltage-gated Ca(2+) channels. Although the functional effects of an increasing number of FHM-1 mutations have been characterized, knowledge on the influence of most of these mutations on G protein regulation of channel function is lacking. Here, we explored the effects of G protein-dependent modulation on mutations W1684R and V1696I which cause FHM-1 with and without cerebellar ataxia, respectively. Both mutations were introduced into the human Ca(V)2.1α(1) subunit and their functional consequences investigated after heterologous expression in human embryonic kidney 293 (HEK-293) cells using patch-clamp recordings. When co-expressed along with the human µ-opioid receptor, application of the agonist [d-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) inhibited currents through both wild-type (WT) and mutant Ca(V)2.1 channels, which is consistent with the known modulation of these channels by G protein-coupled receptors. Prepulse facilitation, which is a way to characterize the relief of direct voltage-dependent G protein regulation, was reduced by both FHM-1 mutations. Moreover, the kinetic analysis of the onset and decay of facilitation showed that the W1684R and V1696I mutations affect the apparent dissociation and reassociation rates of the Gßγ dimer from the channel complex, suggesting that the G protein-Ca(2+) channel affinity may be altered by the mutations. These biophysical studies may shed new light on the pathophysiology underlying FHM-1.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Ataxia Cerebelosa/genética , Ataxia Cerebelosa/metabolismo , Proteínas de Unión al GTP/metabolismo , Trastornos Migrañosos/genética , Trastornos Migrañosos/metabolismo , Animales , Canales de Calcio Tipo N/genética , Línea Celular , Proteínas de Unión al GTP/genética , Estudio de Asociación del Genoma Completo , Genotipo , Células HEK293 , Humanos , Activación del Canal Iónico , Ratones , Mutación , Ratas , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , TransfecciónRESUMEN
By mediating depolarization-induced Ca(2+) influx, high-voltage-activated Ca(2+) channels control a variety of cellular events. These heteromultimeric proteins are composed of an ion-conducting (alpha(1)) and three auxiliary (alpha(2)delta, beta and gamma) subunits. The alpha(2)delta subunit enhances the trafficking of the channel complex to the cell surface and increases channel open probability. To exert these effects, alpha(2)delta must undergo important post-translational modifications, including a proteolytic cleavage that separates the extracellular alpha(2) from its transmembrane delta domain. After this proteolysis both domains remain linked by disulfide bonds. In spite of its central role in determining the final conformation of the fully mature alpha(2)delta, almost nothing is known about the physiological implications of this structural modification. In the current report, by using site-directed mutagenesis, the proteolytic site of alpha(2)delta was mapped to amino acid residues Arg-941 and Val-946. Substitution of these residues renders the protein insensitive to proteolytic cleavage as evidenced by the lack of molecular weight shift upon treatment with a disulfide-reducing agent. Interestingly, these mutations significantly decreased whole-cell patch-clamp currents without affecting the voltage dependence or kinetics of the channels, suggesting a reduction in the number of channels targeted to the plasma membrane.
Asunto(s)
Canales de Calcio/química , Canales de Calcio/metabolismo , Secuencias de Aminoácidos , Canales de Calcio/genética , Línea Celular Transformada , Estimulación Eléctrica/métodos , Expresión Génica , Humanos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Mutagénesis/fisiología , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína , Relación Estructura-Actividad , TransfecciónRESUMEN
An increase in intracellular Ca2+ due to voltage-gated Ca2+ (CaV) channel opening represents an important trigger for a number of second-messenger-mediated effects ranging from neurotransmitter release to gene activation. Ca2+ entry occurs through the principal pore-forming protein but several ancillary subunits are known to more precisely tune ion influx. Among them, the CaVbeta subunits are perhaps the most important, given that they largely influence the biophysical and pharmacological properties of the channel. Notably, several functional features may be associated with specific structural regions of the CaVbeta subunits emphasizing the relevance of intramolecular domains in the physiology of these proteins. In the current report, we show that CaVbeta3 contains two PEST motifs and undergoes Ca2+ -dependent degradation which can be prevented by the specific calpain inhibitor calpeptin. Using mutant constructs lacking the PEST motifs, we present evidence that they are necessary for the cleavage of CaVbeta3 by calpain. Furthermore, the deletion of the PEST sequences did not affect the binding of CaVbeta3 to the ion-conducting CaV2.2 subunit and, when expressed in human embryonic kidney-293 cells, the PEST motif-deleted CaVbeta3 significantly increased whole-cell current density and retarded channel inactivation. Consistent with this observation, calpeptin treatment of human embryonic kidney-293 cells expressing wild-type CaVbeta3 resulted in an increase in current amplitude. Together, these findings suggest that calpain-mediated CaVbeta3 proteolysis may be an essential process for Ca2+ channel functional regulation.