A dyf-7 haplotype causes sensory neuron defects and is associated with macrocyclic lactone resistance worldwide in the nematode parasite Haemonchus contortus.

Heavy reliance on macrocyclic lactones to treat parasitic nematodes has resulted in the evolution of widespread drug resistance that threatens human and animal health. Management strategies have been proposed that would slow the rise of resistance, however testing these strategies has been hampered by the lack of identified strong-effect resistance markers in parasites. We show that the Caenorhabditis elegans gene Cel_dyf-7, necessary for amphid sensory neuron development, also confers macrocyclic lactone sensitivity. In the sheep parasite Haemonchus contortus: (i) strains selected for macrocyclic lactone resistance were enriched in a Hco_dyf-7 haplotype that was rare in the drug-naïve population, (ii) the resistant haplotype correlated with the sensory neuron defects, and (iii) the resistant haplotype was associated with decreased Hco_dyf-7 expression. Resistant field isolates of H. contortus from five continents were enriched for the resistant haplotype, demonstrating the relevance of the Hco_dyf-7 haplotype to practise and indicating that it is a locus of strong effect. Hemizygosity resulting from sex linkage of dyf-7 likely contributes to the rise of resistance in treated populations.

Src regulates membrane trafficking of the Kv3.1b channel.

The Kv3.1 channel plays a crucial role in regulating the high-frequency firing properties of neurons. Here, we determined whether Src regulates the subcellular distributions of the Kv3.1b channel. Co-expression of active Src induced a dramatic redistribution of Kv3.1b to the endoplasmic reticulum. Furthermore, co-expression of the Kv3.1b channel with active Src induced a remarkable decrease in the pool of Kv3.1b at the cell surface. Moreover, the co-expression of active Src results in a significant decrease in the peak current densities of the Kv3.1b channel, and a substantial alteration in the voltage dependence of its steady-state inactivation. Taken together, these results indicate that Src kinase may play an important role in regulating membrane trafficking of Kv3.1b channels.

Mass spectrometric analysis of novel phosphorylation sites in the TRPC4ß channel.

RATIONALE: The transient receptor potential canonical (TRPC) channel 4ß is a non-selective cation channel that is regulated by intracellular Ca(2+) and G protein-coupled receptors. Tyrosine phosphorylation of TRPC4ß is important in mediating the activity and membrane expression of this channel protein. However, studies of TRPC4ß Ser/Thr phosphorylation are lacking. METHODS: To investigate the phosphorylation sites involved in regulating the diverse functions of TRPC4ß in mammalian cells, we used nano-liquid chromatography/tandem mass spectrometry to identify key phosphorylation sites in TRPC4ß that was immunopurified from HEK293 cells with monoclonal anti-TRPC4ß antibody. RESULTS: We identified four phosphorylation sites in the C-terminus of TRPC4ß, none of which had been previously reported. Our data show that TRPC4ß in mammalian cells is highly phosphorylated under basal conditions at multiple sites, and that a mass spectrometric proteomic technique combined with antibody-based affinity purification is an effective approach to define the phosphorylation sites of TRPC4ß channels in mammalian cells. CONCLUSIONS: These novel phosphorylation sites on TRPC4ß may play a potential role in the phosphorylation-mediated regulation of TRPC4ß channel activity and function in mammalian cells.

Dynamic modulation of the kv2.1 channel by SRC-dependent tyrosine phosphorylation.

The voltage-gated K(+) channel Kv2.1 is expressed as a highly phosphorylated protein in most central neurons, where it plays a key role in regulating neuronal membrane excitability. Previous studies have shown that Kv2.1 channel activity is upregulated by Src-mediated phosphorylation through an unknown mechanism. However, a systematic analysis of the molecular mechanism of Kv2.1 channel phosphorylation by Src is lacking. Here, we show that tyrosine phosphorylation by Src plays a fundamental role in regulating Kv2.1-mediated K(+) current enhancement. We found that the level of expression of the Kv2.1 protein is increased by Src kinase. Using mass spectrometric proteomic techniques, we identified two novel phosphotyrosine sites, Y686 and Y810, in the cytoplasmic domains of Kv2.1. We found that Src-dependent phosphorylation at these sites affects Kv2.1 through distinct regulatory mechanisms. Whereas phosphorylation at Y686 regulates Kv2.1 activity similarly to the known site Y124, phosphorylation at Y810 plays a significant role in regulating the intracellular trafficking of Kv2.1 channels. Our results show that these two novel tyrosine phosphorylation sites of Kv2.1 are crucial to regulating diverse aspects of Kv2.1 channel function and provide novel insights into molecular mechanisms for the regulation of Src-dependent modulation of Kv2.1 channels.