Engineered nucleases have transformed biological research and offer great therapeutic potential by enabling the straightforward modification of desired genomic sequences. While many nuclease platforms have proven functional, all can produce unanticipated off-target lesions and have difficulty discriminating between homologous sequences, limiting their therapeutic application. Here we describe a multi-reporter selection system that allows the screening of large protein libraries to uncover variants able to discriminate between sequences with substantial homology. We have used this system to identify zinc-finger nucleases that exhibit high cleavage activity (up to 60% indels) at their targets within the CCR5 and HBB genes and strong discrimination against homologous sequences within CCR2 and HBD. An unbiased screen for off-target lesions using a novel set of CCR5-targeting nucleases confirms negligible CCR2 activity and demonstrates minimal off-target activity genome wide. This system offers a straightforward approach to generate nucleases that discriminate between similar targets and provide exceptional genome-wide specificity.

Signaling in the brain depends on rapid opening and closing of pentameric ligand-gated ion channels (pLGICs). These proteins are the targets of various clinical drugs and, defects in their function is linked to a variety of diseases including myasthenia, epilepsy and sleep-disorders. While recent high-resolution structures of prokaryotic and eukaryotic pLGICs have shed light on the molecular architecture of these proteins, describing their conformational dynamics in physiological lipids is essential for understanding their function. Here, we used site-directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy and functional channels reconstituted in liposomes to reveal ligand-induced structural changes in the extracellular domain (ECD) of GLIC. Proton-activation caused an inward motion of labeled sites at the top of β-strands (β1, 2, 5, 6, 8) towards the channel lumen, consistent with an agonist-induced inward tilting motion of the ECD. Similar proton-dependent GLIC ECD motions were detected in the presence of a non-activating (gating deficient) mutation, suggesting that the inward tilting of the ECD does not accompany channel opening but is associated with an agonist-induced closed pre-activated channel state. These findings provide new insights into the protein dynamics underlying pLGIC gating transitions.

Alteration of the electrical activity of the nervous system causes plasticity in neural circuits. Many of the changes occur at synapses. For example, neurotransmitter switching involves changes in the identity of presynaptic neurotransmitters and corresponding changes in postsynaptic transmitter receptors, thereby achieving a match between the transmitter and its cognate receptor. However, it is unknown whether changes in postsynaptic receptors can regulate presynaptic transmitters. Here we address this question at the developing neuromuscular junction. We find that blockade of endogenous postsynaptic acetylcholine receptors leads to loss of the cholinergic phenotype in motor neurons and the reappearance and stabilization of an earlier, developmentally transient glutamatergic phenotype. In addition, exogenous postsynaptic expression of GABAA receptors leads to the appearance and stabilization of an earlier, transient GABAergic motor neuron phenotype. Thus, acetylcholine receptors are necessary to stabilize acetylcholine as a transmitter, and GABAA receptors are sufficient to stabilize GABA as a transmitter. GARLH4 links the GABAA receptor to neuroligin, and Lrp4 links the acetylcholine receptor to dystroglycan through rapsyn and MUSK. Both neuroligin and dystroglycan bind to neurexin, which in turn binds to forms of the CASK transcription factor in motor neurons. Knock down of GARLH4 or Lrp4 postsynaptically or CASK presynaptically blocks stabilization of the GABAergic and cholinergic phenotypes. These results implicate transsynaptic bridges in establishing receptor-dependent stability of the cognate neurotransmitters. Our findings provide opportunities to investigate a role for dysfunctional transmitter receptors in neurological disorders that involve the loss of the presynaptic transmitter.

First synthesized in the 1950s, benzodiazepines are widely prescribed drugs that exert their anxiolytic, sedative and anticonvulsant actions by binding to GABA-A receptors, the main inhibitory ligand-gated ion channel in the brain. Scientists have long theorized that there exists an endogenous benzodiazepine, or endozepine, in the brain. While there is indirect evidence suggesting a peptide, the diazepam binding inhibitor, is capable of modulating the GABA-A receptor, direct evidence of the modulatory effects of the diazepam binding inhibitor is limited. Here we take a reductionist approach to understand how purified diazepam binding inhibitor interacts with and affects GABA-A receptor activity. We used two-electrode voltage clamp electrophysiology to study how the effects of diazepam binding inhibitor vary with GABA-A receptor subunit composition, and found that GABA-evoked currents from α3-containing GABA-A receptors are weakly inhibited by the diazepam binding inhibitor, while currents from α5-containing receptors are positively modulated. We also used in silico   protein-protein docking to visualize potential diazepam binding inhibitor/GABA-A receptor interactions that revealed diazepam binding inhibitor bound at the benzodiazepine α/γ binding site interface, which provides a structural framework for understanding diazepam binding inhibitor effects on GABA-A receptors. Our results provide novel insights into mechanisms underlying how the diazepam binding inhibitor modulates GABA-mediated inhibition in the brain.

We are exploring the effects of recently discovered (Götz, et al., (2011) Am. J. Hum. Genet. 88, 635) pathogenic mutations in human mitochondrial alanyl‐tRNA synthetase (mt AlaRS) on tRNA recognition and enzymatic function. These authors found an R592W mutation in multiple patients with severe infantile cardiomyopathy. R592 lies within the editing domain of mt AlaRS, but is distal to the aminoacyl editing active site. To characterize the editing function of mt AlaRS, we have constructed an editing‐defective C719A/Q622H mutant of the enzyme. While this editing defective mutant effectively mischarges human mt tRNAAla with serine, no accumulation of Ser‐tRNAAla is observed with wildtype or R592W mt AlaRS. We are currently investigating the hypothesis that, rather than disabling the enzyme's ability to hydrolyze misacylated tRNAs, the R592W mutation enables hydrolysis of the correctly acylated Ala‐tRNAAla product in the editing site of mt AlaRS.This work was supported by Carleton College and the Howard Hughes Medical Institute.

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