Selenomethionine mis-incorporation and redox-dependent voltage-gated sodium channel gain of function.

Selenomethionine (SeMet) readily replaces methionine (Met) residues in proteins during translation. Long-term dietary SeMet intake results in the accumulation of the amino acid in tissue proteins. Despite the high rates of SeMet incorporation in proteins and its stronger susceptibility to oxidation compared to Met, little is known about the effect of SeMet mis-incorporation on electrical excitability and ion channels. Fast inactivation of voltage-gated sodium (NaV ) channels is essential for exact action potential shaping with even minute impairment of inactivation resulting in a plethora of adverse phenotypes. Met oxidation of the NaV channel inactivation motif (Ile-Phe-Met) and further Met residues causes a marked loss of inactivation. Here, we examined the impact of SeMet mis-incorporation on the function of NaV channels. While extensive SeMet incorporation into recombinant rat NaV 1.4 channels preserved their normal function, it greatly sensitized the channels to mild oxidative stress, resulting in loss of inactivation and diminished maximal current, both reversible by dithiothreitol-induced reduction. SeMet incorporation similarly affected human NaV 1.4, NaV 1.2, NaV 1.5, and NaV 1.7. In mouse dorsal root ganglia (DRG) neurons, 1 day of SeMet exposure exacerbated the oxidation-mediated broadening of action potentials. SeMet-treated DRGs also exhibited a stronger increase in the persistent NaV current in response to oxidation. SeMet incorporation in NaV proteins coinciding with oxidative insults may therefore result in hyperexcitability pathologies, such as cardiac arrhythmias and neuropathies, like congenital NaV channel gain-of-function mutations.

Selenomethionine incorporation in proteins of individual mammalian cells determined with a genetically encoded fluorescent sensor.

Selenomethionine (SeMet) randomly replaces methionine (Met) in protein translation. Because of strongly differing redox properties of SeMet and Met, SeMet mis-incorporation may have detrimental effects on protein function, possibly compromising the use of nutritional SeMet supplementation as an anti-oxidant. Studying the functional impact of SeMet in proteins on a cellular level is hampered by the lack of accurate and efficient methods for estimating the SeMet incorporation level in individual viable cells. Here we introduce and apply a method to measure the extent of SeMet incorporation in cellular proteins by utilizing a genetically encoded fluorescent methionine oxidation probe. Supplementation of SeMet in mammalian culture medium resulted in >84% incorporation of SeMet, and SeMet labeling as low as 5% was readily measured. Kinetics and extent of SeMet incorporation on the single-cell level under live-cell imaging conditions provided direct access to protein turn-over kinetics and SeMet redox properties in a cellular context. The method is furthermore suited for experiments utilizing high-throughput fluorescence microplate readers or fluorescence-activated cell sorting (FACS) analysis.

A GFP-based ratiometric sensor for cellular methionine oxidation.

Methionine oxidation is a reversible post-translational protein modification, affecting protein function, and implicated in aging and degenerative diseases. The detection of accumulating methionine oxidation in living cells or organisms, however, has not been achieved. Here we introduce a genetically encoded probe for methionine oxidation (GEPMO), based on the super-folder green fluorescent protein (sfGFP), as a specific, versatile, and integrating sensor for methionine oxidation. Placed at amino-acid position 147 in an otherwise methionine-less sfGFP, the oxidation of this specific methionine to methionine sulfoxide results in a ratiometric fluorescence change when excited with ∼400 and ∼470 nm light. The strength and homogeneity of the sensor expression is suited for live-cell imaging as well as fluorescence-activated cell sorting (FACS) experiments using standard laser wavelengths (405/488 nm). Expressed in mammalian cells and also in S. cerevisiae, the sensor protein faithfully reports on the status of methionine oxidation in an integrating manner. Variants targeted to membranes and the mitochondria provide subcellular resolution of methionine oxidation, e.g. reporting on site-specific oxidation by illumination of endogenous protoporphyrin IX.

Structure-activity relationship study of spider polyamine toxins as inhibitors of ionotropic glutamate receptors.

The spider polyamine toxins Joro spider toxin-3 (JSTX-3) and Nephila polyamine toxins-1 and -8 (NPTX-1 and NPTX-8) are isolated from the venom of the orb-weaver spider Nephila clavata (Joro spider). They share a high degree of structural resemblance, their aromatic head groups being the only difference, and were recently found to be very potent open-channel blockers of ionotropic glutamate (iGlu) receptors. In this study we designed and synthesized a collection of 24 analogues of these toxins using a recently developed solid-phase synthetic methodology. Systematic variation in two regions of the toxins and subsequent evaluation of biological activity at AMPA and NMDA subtypes of iGlu receptors provided succinct information on structure-activity relationships. In particular, one set of analogues were found to display exquisite selectivity and potency for AMPA receptors relative to the natural products. Thus, this systematic SAR study has provided new pharmacological tools for studies of iGlu receptors.