Mutation of a single residue promotes gating of vertebrate and invertebrate two-pore domain potassium channels.
Ben Soussia I, El Mouridi S, Kang D, Leclercq-Blondel A, Khoubza L, Tardy P, Zariohi N, Gendrel M, Lesage F, Kim EJ, Bichet D, Andrini O, Boulin T. Nature Communications (2019) Feb 15;10(1):787.
Mutations that modulate the activity of ion channels are essential tools to understand the biophysical determinants that control their gating. Here, we reveal the conserved role played by a single amino acid position (TM2.6) located in the second transmembrane domain of two-pore domain potassium (K2P) channels. Mutations of TM2.6 to aspartate or asparagine increase channel activity for all vertebrate K2P channels. Using two-electrode voltage-clamp and single-channel recording techniques, we find that mutation of TM2.6 promotes channel gating via the selectivity filter gate and increases single channel open probability. Furthermore, channel gating can be progressively tuned by using different amino acid substitutions. Finally, we show that the role of TM2.6 was conserved during evolution by rationally designing gain-of-function mutations in four Caenorhabditis elegans K2P channels using CRISPR/Cas9 gene editing. This study thus describes a simple and powerful strategy to systematically manipulate the activity of an entire family of potassium channels.
Reliable CRISPR/Cas9 Genome Engineering in Caenorhabditis elegans Using a Single Efficient sgRNA and an Easily Recognizable Phenotype.
El Mouridi S, Lecroisey C, Tardy P, Mercier M, Leclercq-Blondel A, Zariohi N, Boulin T. G3 (Bethesda) (2017) May 5;7(5):1429-1437.
CRISPR/Cas9 genome engineering strategies allow the directed modification of the Caenorhabditis elegans genome to introduce point mutations, generate knock-out mutants, and insert coding sequences for epitope or fluorescent tags. Three practical aspects, however, complicate such experiments. First, the efficiency and specificity of single-guide RNAs (sgRNA) cannot be reliably predicted. Second, the detection of animals carrying genome edits can be challenging in the absence of clearly visible or selectable phenotypes. Third, the sgRNA target site must be inactivated after editing to avoid further double-strand break events. We describe here a strategy that addresses these complications by transplanting the protospacer of a highly efficient sgRNA into a gene of interest to render it amenable to genome engineering. This sgRNA targeting the dpy-10 gene generates genome edits at comparatively high frequency. We demonstrate that the transplanted protospacer is cleaved at the same time as the dpy-10 gene. Our strategy generates scarless genome edits because it no longer requires the introduction of mutations in endogenous sgRNA target sites. Modified progeny can be easily identified in the F1 generation, which drastically reduces the number of animals to be tested by PCR or phenotypic analysis. Using this strategy, we reliably generated precise deletion mutants, transcriptional reporters, and translational fusions with epitope tags and fluorescent reporter genes. In particular, we report here the first use of the new red fluorescent protein mScarlet in a multicellular organism. wrmScarlet, a C. elegans-optimized version, dramatically surpassed TagRFP-T by showing an eightfold increase in fluorescence in a direct comparison.
Microtubule severing by the katanin complex is activated by PPFR-1-dependent MEI-1 dephosphorylation.
Gomes JE, Tavernier N, Richaudeau B, Formstecher E, Boulin T, Mains PE, Dumont J, Pintard L. Journal of Cell Biology (2013) Aug 5;202(3):431-9.
Biosynthesis of ionotropic acetylcholine receptors requires the evolutionarily conserved ER membrane complex.
Richard M, Boulin T, Robert VJ, Richmond JE, Bessereau JL. PNAS (2013) Mar ;110(11):E1055-63.
Positive modulation of a Cys-loop acetylcholine receptor by an auxiliary transmembrane subunit.
Boulin T, Rapti G, Briseño-Roa L, Stigloher C, Richmond JE, Paoletti P, Bessereau JL. Nature Neuroscience (2012) Oct ;15(10):1374-81.
Functional reconstitution of Haemonchus contortus acetylcholine receptors in Xenopus oocytes provides mechanistic insights into levamisole resistance.
Boulin T*, Fauvin A*, Charvet C, Cortet J, Cabaret J, Bessereau JL, Neveu C. British Journal of Pharmacology (2011) Nov ;164(5):1421-32 ; Epub ahead of print Apr 12
A neuronal acetylcholine receptor regulates the balance of muscle excitation and inhibition in Caenorhabditis elegans.
Jospin M, Qi YB, Stawicki TM, Boulin T, Schuske KR, Horvitz HR, Bessereau JL, Jorgensen EM, Jin Y. PLoS Biology (2009) Dec ;7(12):e1000265
The Small, Secreted Immunoglobulin Protein ZIG-3 Maintains Axon Position in Caenorhabditis elegans.
Benard C, Tjoe N, Boulin T, Recio J, Hobert O. Genetics (2009) Nov ;183(3):917-27.
Eight genes are required for functional reconstitution of the C. elegans levamisole-sensitive acetylcholine receptor.
Boulin T*, Gielen M*, Richmond J, Williams DC, Paoletti P and Bessereau JL. PNAS (2008) 105(47):18590-18595.
A novel Eph receptor-interacting IgSF protein provides C. elegans motoneurons with midline guidepost function.
Boulin T, Pocock R, Hobert O. Current Biology (2006) 16(19):1871-83.
Comment by Quinn CC, Wadsworth, WG. Current Biology, 2006 ; 16(22):R954-5.
Developmental regulation of whole cell capacitance and membrane current in identified interneurons in C. elegans.
Faumont S, Boulin T, Hobert O, Lockery SR. J. Neurophysiology (2006) 95(6):3665-73.
Characterization of Mos1 Mediated Mutagenesis in C. elegans : A Method for the Rapid Identification of Mutated Genes.
Williams DC, Boulin T, Ruaud AF, Jorgensen EM, Bessereau JL. Genetics (2005) 169(3):1779-85.
Differential functions of the C. elegans FGF receptor in axon outgrowth and maintenance of axon position.
Bülow H*, Boulin T*, Hobert O. Neuron (2004) 42(3):367-74.
Identification of spatial and temporal cues that regulate postembryonic expression of axon maintenance factors in the C. elegans ventral nerve cord.
Aurelio O, Boulin T, Hobert O. Development (2003) 130(3):599-610.
From genes to function : the C. elegans genetic toolbox.
Boulin T, Hobert O Wiley Interdisciplinary Reviews : Developmental Biology (2011) Nov ;1(1):114-137.
This review aims to provide an overview of the technologies which make the nematode
Caenorhabditis elegans an attractive genetic model system. We describe transgenesis
techniques and forward and reverse genetic approaches to isolate mutants and clone genes.
In addition, we discuss the new possibilities offered by genome engineering strategies and
next-generation genome analysis tools.
Neuroinformatics for C. elegans: Relating Mind and Body in Wormbase.
Chen N, Lee RYN, Altun ZF, Boulin T, Sternberg PW, and Stein LD.
Neuroscience Databases: A Practical Guide. Edited by Rolf Kötter. Kluwer Academic Publishers. October 2002.