Germline de novo variant F747S extends the phenotypic spectrum of CACNA1D Ca2+ channelopathies.

Germline gain-of-function missense variants in the pore-forming Cav1.3 α1-subunit (CACNA1D gene) confer high risk for a severe neurodevelopmental disorder with or without endocrine symptoms. Here, we report a 4-week-old new-born with the novel de novo missense variant F747S with a so far not described prominent jittering phenotype in addition to symptoms previously reported for CACNA1D mutations including developmental delay, elevated aldosterone level and transient hypoglycemia. We confirmed the pathogenicity of this variant in whole-cell patch-clamp experiments with wild-type and F747S mutant channels heterologously expressed together with α2δ1 and cytosolic ß3 or membrane-bound ß2a subunits. Mutation F747S caused the quantitatively largest shift in the voltage dependence of activation (-28 mV) reported so far for CACNA1D germline mutations. It also shifted inactivation to more negative voltages, slowed the time course of current inactivation and slowed current deactivation upon repolarization with both co-expressed ß-subunits. In silico modelling and molecular docking, simulations revealed that this gain-of-function phenotype can be explained by formation of a novel inter-domain hydrogen bond between mutant residues S747 (IIS6) with N1145 (IIIS6) stabilizing selectively the activated open channel state. F747S displayed 2-6-fold increased sensitivity for the L-type Ca2+ channel blocker isradipine compared to wild type. Our data confirm the pathogenicity of the F747S variant with very strong gain-of-function gating changes, which may contribute to the novel jittering phenotype. Increased sensitivity for isradipine suggests this drug for potential symptomatic off-label treatment for carriers of this mutation.

Proteomic and toxicological analysis of the venom of Micrurus yatesi and its neutralization by an antivenom.

Coralsnakes belong to the family Elapidae and possess venoms which are lethal to humans and can be grouped based on the predominance of either three finger toxins (3FTxs) or phospholipases A2 (PLA2s). A proteomic and toxicological analysis of the venom of the coralsnake Micrurus yatesi was performed. This species, distributed in southeastern Costa Rica, was formerly considered a subspecies of M. alleni. Results showed that this venom is PLA2-rich, in contrast with the previously studied venom of Micrurus alleni. Toxicological evaluation of the venom, in accordance with proteomic data, revealed that it has a markedly higher in vitro PLA2 activity upon a synthetic substrate than M. alleni. The evaluation of in vivo myotoxicity in CD-1 mice using histological evaluation and plasma creatine kinase release also showed that M. yatesi venom caused muscle damage. A commercial equine antivenom prepared using the venom of Micrurus nigrocinctus displayed a similar recognition of the venoms of M. yatesi and M. nigrocinctus by enzyme immunoassay. This antivenom also immunorecognized the main fractions of the venom of M. yatesi and was able to neutralize its lethal effect in a murine model.

First look into the venom of Roatan Island's critically endangered coral snake Micrurus ruatanus: Proteomic characterization, toxicity, immunorecognition and neutralization by an antivenom.

A proteomic and toxicological study of the venom from one specimen of Micrurus ruatanus, a critically endangered coral snake species endemic to Roatan Island, Honduras, was carried out. Immunorecognition and neutralization of venom lethality by an anticoral antivenom was also evaluated. Forty peaks were collected from RP-HPLC fractionation of the venom. After SDS-PAGE analysis, fifty-eight bands were examined by MALDI-TOF/TOF mass spectrometry. Micrurus ruatanus venom displayed a three-finger toxin (3FTx)-rich venom phenotype, as well as a significant amount of phospholipases A2 (PLA2s). Various other proteins were identified, including Kunitz-type inhibitor proteins, L-amino acid oxidases, C-type lectin/lectin-like, metalloproteinases, serine proteinases, vespryn/ohanin, 5'-nucleotidases, glutathione peroxidases, and phosphodiesterases. Micrurus ruatanus venom displayed significant PLA2 activity in vitro and myotoxicity in vivo. The venom showed high lethal potency in mice, being one of the most lethal in Central America. The anticoral antivenom (SAC-ICP) produced by Instituto Clodomiro Picado neutralized the lethal activity of the venom. Major fractions with relevant lethal activity were also identified by a screening analysis. SIGNIFICANCE: The proteomic characterization, toxicity, immunorecognition and neutralization of Micrurus ruatanus venom have been determined for the first time. This coral snake is endemic to Roatan Island and contains a three-finger toxin-rich venom that displayed a potent lethal activity in mice. The anticoral antivenom produced by Instituto Clodomiro Picado neutralized the lethal activity of this venom in vivo, and therefore should be effective in the treatment of envenomings by this snake.