A lipid site shapes the agonist response of a pentameric ligand-gated ion channel.

Phospholipids are key components of cellular membranes and are emerging as important functional regulators of different membrane proteins, including pentameric ligand-gated ion channels (pLGICs). Here, we take advantage of the prokaryote channel ELIC (Erwinia ligand-gated ion channel) as a model to understand the determinants of phospholipid interactions in this family of receptors. A high-resolution structure of ELIC in a lipid-bound state reveals a phospholipid site at the lower half of pore-forming transmembrane helices M1 and M4 and at a nearby site for neurosteroids, cholesterol or general anesthetics. This site is shaped by an M4-helix kink and a Trp-Arg-Pro triad that is highly conserved in eukaryote GABAA/C and glycine receptors. A combined approach reveals that M4 is intrinsically flexible and that M4 deletions or disruptions of the lipid-binding site accelerate desensitization in ELIC, suggesting that lipid interactions shape the agonist response. Our data offer a structural context for understanding lipid modulation in pLGICs.

Off-Label Use of Clozapine in Children and Adolescents-A Literature Review.

BACKGROUND: Clozapine is a second-generation antipsychotic typically used for refractory schizophrenia or otherwise psychotic pathology. There are no FDA or manufacturer guidelines for use of clozapine in pediatric population. We investigated the current state of research concerning the use of clozapine in pediatric patients. AREAS OF UNCERTAINTY: We describe consistent calls for more research into the long-term and short-term effects of clozapine use in a young patient population. Despite the strongly supported efficacy, questions concerning clear indications for use and risk-benefit analysis persist. We acknowledge that a more comprehensive meta-analysis would greatly benefit the field. However, this is the first article of its kind for clozapine in recent history, and therefore, serves as a focus and reference point for future, more in-depth analyses. DATA SOURCES: We conducted a search of PubMed, ClinicalKey, PsycINFO, and MEDLINE databases. Keywords used included, in varying combinations: clozapine, off-label, indications, children and adolescent, pediatric, behavioral, suicidality, psychosis, early and very-early onset schizophrenia, side-effect profile, and long-term use. Further criteria and selection are described in Methods below. RESULTS: We describe the documented efficacy of clozapine for the management of refractory psychotic and nonpsychotic symptoms in the pediatric population. The authors highlight the risk of unmanaged early-onset schizophrenia, aggressive or suicidal behavior, and severe nonpsychotic pathology. Unfortunately, these studies are generally small. There is little consistency in when clozapine is prescribed, how long it is administered, and how long patients are followed. Despite the lack of FDA and manufacturer guidelines, clozapine continues to be used for the benefit of young patients. CONCLUSIONS: Indications for prescription of clozapine should be revisited, given the data presented in this manuscript of a low risk-benefit ratio for properly chosen patients. Larger studies should be conducted to provide more statistical power and determine clear guidelines for use, risk of side effects, and long-term adverse events that may arise.

A cost-effective protocol for the over-expression and purification of fully-functional and more stable Erwinia chrysanthemi ligand-gated ion channel.

The Erwinia chrysanthemi ligand-gated ion channel, ELIC, is considered an excellent structural and functional surrogate for the whole pentameric ligand-gated ion channel family. Despite its simplicity, ELIC is structurally capable of undergoing ligand-dependent activation and a concomitant desensitization process. To determine at the molecular level the structural changes underlying ELIC's function, it is desirable to produce large quantities of protein. This protein should be properly folded, fully-functional and amenable to structural determinations. In the current paper, we report a completely new protocol for the expression and purification of milligram quantities of fully-functional, more stable and crystallizable ELIC. The use of an autoinduction media and inexpensive detergents during ELIC extraction, in addition to the high-quality and large quantity of the purified channel, are the highlights of this improved biochemical protocol.

Brain Tumor Biobank Development for Precision Medicine: Role of the Neurosurgeon.

Neuro-oncology biobanks are critical for the implementation of a precision medicine program. In this perspective, we review our first year experience of a brain tumor biobank with integrated next generation sequencing. From our experience, we describe the critical role of the neurosurgeon in diagnosis, research, and precision medicine efforts. In the first year of implementation of the biobank, 117 patients (Female: 62; Male: 55) had 125 brain tumor surgeries. 75% of patients had tumors biobanked, and 16% were of minority race/ethnicity. Tumors biobanked were as follows: diffuse gliomas (45%), brain metastases (29%), meningioma (21%), and other (5%). Among biobanked patients, 100% also had next generation sequencing. Eleven patients qualified for targeted therapy based on identification of actionable gene mutations. One patient with a hereditary cancer predisposition syndrome was also identified. An iterative quality improvement process was implemented to streamline the workflow between the operating room, pathology, and the research laboratory. Dedicated tumor bank personnel in the department of neurosurgery greatly improved standard operating procedure. Intraoperative selection and processing of tumor tissue by the neurosurgeon was integral to increasing success with cell culture assays. Currently, our institutional protocol integrates standard histopathological diagnosis, next generation sequencing, and functional assays on surgical specimens to develop precision medicine protocols for our patients. This perspective reviews the critical role of neurosurgeons in brain tumor biobank implementation and success as well as future directions for enhancing precision medicine efforts.

CW-EPR Spectroscopy and Site-Directed Spin Labeling to Study the Structural Dynamics of Ion Channels.

Continuous-wave electron paramagnetic resonance spectroscopy (CW-EPR) and site-directed spin labeling (SDSL) are proven experimental approaches to assess the structural dynamics of proteins in general (Hubbell et al., Curr Opin Struct Biol 8(5):649-656, 1998; Kazmier et al., Curr Opin Struct Biol 45:100-108, 2016; Perozo et al., Science 285(5424):73-78, 1999). These techniques have been particularly effective assessing the structure of integral membrane proteins embedded in a lipid bilayer (Cortes et al., J Gen Physiol 117(2):165-180, 2001; Cuello et al., Science 306(5695):491-495, 2004; Dalmas et al., Structure 18(7):868-878, 2010; Li et al., Proc Natl Acad Sci U S A 112(44):E5926-5935, 2015; Perozo et al., J Gen Physiol 118(2):193-206, 2001), as well as determining the conformational changes associated with their biological function (Kazmier et al., Curr Opin Struct Biol 45:100-108, 2016; Perozo et al., Science 285(5424):73-78, 1999; Arrigoni et al., Cell 164(5):922-936, 2016; Dalmas et al., Nat Commun 5:3590, 2014; Dong et al., Science 308(5724):1023-1028, 2005; Farrens et al., Science 274(5288):768-770, 1996; Perozo et al., Nat Struct Biol 5(6):459-469, 1998; Perozo et al., Nature 418(6901):942-948, 2002). In this chapter, we described a practical guide for the spin-labeling, liposome reconstitution, and CW-EPR measurements of the prototypical bacterial K+ channel, KcsA.