The culinary chemist: Out of the lab and into the tent.

In this Stories piece, Josh Smalley, a postdoctoral research associate at the University of Leicester and finalist of The Great British Bake Off, discusses the parallels between chemistry and baking, revealing how he successfully combines the two.

The PDE4 inhibitor apremilast modulates ethanol responses in Gabrb1-S409A knock-in mice via PKA-dependent and independent mechanisms.

We previously showed that the PDE4 inhibitor apremilast reduces ethanol consumption in mice by protein kinase A (PKA) and GABAergic mechanisms. Preventing PKA phosphorylation of GABAA ß3 subunits partially blocked apremilast-mediated decreases in drinking. Here, we produced Gabrb1-S409A mice to render GABAA ß1 subunits resistant to PKA-mediated phosphorylation. Mass spectrometry confirmed the presence of the S409A mutation and lack of changes in ß1 subunit expression or phosphorylation at other residues. ß1-S409A male and female mice did not differ from wild-type C57BL/6J mice in expression of Gabrb1, Gabrb2, or Gabrb3 subunits or in behavioral characteristics. Apremilast prolonged recovery from ethanol ataxia to a greater extent in Gabrb1-S409A mice but prolonged recovery from zolpidem and propofol to a similar extent in both genotypes. Apremilast shortened recovery from diazepam ataxia in wild-type but prolonged recovery in Gabrb1-S409A mice. In wild-type mice, the PKA inhibitor H89 prevented apremilast modulation of ataxia by ethanol and diazepam, but not by zolpidem. In Gabrb1-S409A mice, inhibiting PKA or EPAC2 (exchange protein directly activated by cAMP) partially reversed apremilast potentiation of ethanol, diazepam, and zolpidem ataxia. Apremilast prevented acute tolerance to ethanol ataxia in both genotypes, but there were no genotype differences in ethanol consumption before or after apremilast. In contrast to results in Gabrb3-S408A/S409A mice, PKA phosphorylation of ß1-containing GABAA receptors is not required for apremilast's effects on acute tolerance or on ethanol consumption but is required for its ability to decrease diazepam intoxication. Besides PKA we identified EPAC2 as an additional cAMP-dependent mechanism by which apremilast regulates responses to GABAergic drugs.

The brain-specific kinase LMTK3 regulates neuronal excitability by decreasing KCC2-dependent neuronal Cl- extrusion.

LMTK3 is a brain-specific transmembrane serine/threonine protein kinase that acts as a scaffold for protein phosphatase-1 (PP1). Although LMKT3 has been identified as a risk factor for autism and epilepsy, its physiological significance is unknown. Here, we demonstrate that LMTK3 copurifies and binds to KCC2, a neuron-specific K+/Cl- transporter. KCC2 activity is essential for Cl--mediated hyperpolarizing GABAAR receptor currents, the unitary events that underpin fast synaptic inhibition. LMTK3 acts to promote the association of KCC2 with PP1 to promote the dephosphorylation of S940 within its C-terminal cytoplasmic domain, a process the diminishes KCC2 activity. Accordingly, acute inhibition of LMTK3 increases KCC2 activity dependent upon S940 and increases neuronal Cl- extrusion. Consistent with this, LMTK3 inhibition reduced intrinsic neuronal excitability and the severity of seizure-like events in vitro. Thus, LMTK3 may have profound effects on neuronal excitability as an endogenous modulator of KCC2 activity.

MDM2 Antagonist Idasanutlin Reduces HDAC1/2 Abundance and Corepressor Partners but Not HDAC3.

Histone deacetylases 1-3 (HDAC1, HDAC2, and HDAC3) and their associated corepressor complexes play important roles in regulating chromatin structure and gene transcription. HDAC enzymes are also validated drug targets for oncology and offer promise toward new drugs for neurodegenerative diseases and cardiovascular diseases. We synthesized four novel heterobifunctional molecules designed to recruit the mouse double minute 2 homologue (MDM2) E3 ligase to degrade HDAC1-3 utilizing the MDM2 inhibitor idasanutlin, known as proteolysis targeting chimeras (PROTACs). Idasanutlin inhibits the MDM2-P53 protein-protein interaction and is in clinical trials. Although two MDM2-recruiting heterobifunctional molecules reduced HDAC1 and HDAC2 abundance with complete selectivity over HDAC3 and reduced HDAC1/2 corepressor components LSD1 and SIN3A, we were surprised to observe that idasanutlin alone was also capable of this effect. This finding suggests an association between the MDM2 E3 ligase and HDAC1/2 corepressor complexes, which could be important for designing future dual/bifunctional HDAC- and MDM2-targeting therapeutics, such as PROTACs.

Selection, Initiation, Continuation, and Efficacy of Reversible Contraception Among Enlisted U.S. Service Women in Their First Term of Service From 2012 to 2020.

BACKGROUND: Pregnancy is the second most common cause of limited duty days among active duty service members in the U.S. Military. Pregnancy accounts for 10% of all days on restricted duty, despite impacting a minority of active duty service members. One out of five service women will experience an unintended pregnancy every year despite the availability of no-cost contraception and reproductive healthcare. Young, single, junior enlisted service women experience the highest rate of unintentional pregnancy. Previous studies have demonstrated service branch-based variability in selection, initiation, and continuation of specific contraceptive methods related to service branch culture and access to contraception during basic training. It is unclear if these differences impact overall contraception use or fertility rates among junior enlisted service women in their first term of enlistment. This study examines rates of contraceptive selection, initiation, continuation, and efficacy among junior enlisted service women in their first 4-year enlistment period, and the service branch specific variability in these outcomes. METHODS: This study is a secondary analysis of Military Healthcare Data Repository records from women who began basic training between 2012 and 2020 and remained on active duty for at least 12 months. We used Kaplan-Meier analyses to examine the effect of age and military branch on contraceptive continuation and efficacy. We used binomial regression for interval censored data, to assess the association of service branch with rates of contraceptive initiation, contraception use, births, and childbirth-related duty restrictions. RESULTS: We identified 147,594 women who began basic training between 2012 and 2020. The mean age of these women at the beginning of basic training was 20.4 ± 3.1 years. Women in the marines and navy had higher contraceptive initiation rates than women in the army or air force. Among women initiating a contraceptive pill, patch, or ring (short-acting reversible contraception), 58.3% were still using some form of hormonal contraception 3 months later. Among women initiating depot-medroxyprogesterone (DMPA), 38.8% were still using any form of hormonal contraception 14 weeks later. Long-acting reversible contraceptive methods, such as intrauterine or subdermal contraceptives, had higher continuation rates and less service-based variability in continuation and failure rates than short-acting reversible contraception or depot-medroxyprogesterone. The proportion of days on any form of prescription contraception during the first 4 years on active duty varied from 23.3% in the army to 38.6% in the navy. The birth rate varied from 34.8 births/1,000 woman-years in the air force up to 62.7 births/1,000 woman-years in the army. Compared with women in the air force, women in the army experienced 2,191 additional days of postpartum leave and 13,908 days on deployment restrictions per 1,000 woman-years. DISCUSSION: Service branch specific variability in contraceptive use is associated with differences in days of pregnancy-related duty restrictions during first 4 years on active duty among junior enlisted females. Robust implementation of best practices in contraceptive care across the military health system to improve contraceptive initiation and continuation appears to offer an opportunity to improve military readiness and promote the health and well-being of active duty service women, particularly in the army.

PROTAC chemical probes for histone deacetylase enzymes.

Over the past three decades, we have witnessed the progression of small molecule chemical probes designed to inhibit the catalytic active site of histone deacetylase (HDAC) enzymes into FDA approved drugs. However, it is only in the past five years we have witnessed the emergence of proteolysis targeting chimeras (PROTACs) capable of promoting the proteasome mediated degradation of HDACs. This is a field still in its infancy, however given the current progress of PROTACs in clinical trials and the fact that FDA approved HDAC drugs are already in the clinic, there is significant potential in developing PROTACs to target HDACs as therapeutics. Beyond therapeutics, PROTACs also serve important applications as chemical probes to interrogate fundamental biology related to HDACs via their unique degradation mode of action. In this review, we highlight some of the key findings to date in the discovery of PROTACs targeting HDACs by HDAC class and HDAC isoenzyme, current gaps in PROTACs to target HDACs and future outlooks.

Brief report: Influence of mandatory contraceptive education emphasizing long-acting reversible methods on continuation rates among military recruits.

OBJECTIVES: Determine if the replacement of patient-initiated, individual contraceptive education with mandatory group contraceptive education, during US Navy basic training, was associated with decreased LARC continuation. STUDY DESIGN: Secondary analysis of administrative billing data from female military recruits who began basic training between September 2012 and February 2020. RESULTS: Servicewomen who started LARC method during rather than after basic training had higher continuation rates. Servicewomen who started training before the implementation of mandatory group education had higher IUD continuation than those trained after. CONCLUSIONS: Implementation of mandatory group contraceptive education during basic training was not associated with a decline in LARC continuation.

Direct activation of KCC2 arrests benzodiazepine refractory status epilepticus and limits the subsequent neuronal injury in mice.

Hyperpolarizing GABAAR currents, the unitary events that underlie synaptic inhibition, are dependent upon efficient Cl- extrusion, a process that is facilitated by the neuronal specific K+/Cl- co-transporter KCC2. Its activity is also a determinant of the anticonvulsant efficacy of the canonical GABAAR-positive allosteric: benzodiazepines (BDZs). Compromised KCC2 activity is implicated in the pathophysiology of status epilepticus (SE), a medical emergency that rapidly becomes refractory to BDZ (BDZ-RSE). Here, we have identified small molecules that directly bind to and activate KCC2, which leads to reduced neuronal Cl- accumulation and excitability. KCC2 activation does not induce any overt effects on behavior but prevents the development of and terminates ongoing BDZ-RSE. In addition, KCC2 activation reduces neuronal cell death following BDZ-RSE. Collectively, these findings demonstrate that KCC2 activation is a promising strategy to terminate BDZ-resistant seizures and limit the associated neuronal injury.

Spectrin-beta 2 facilitates the selective accumulation of GABAA receptors at somatodendritic synapses.

Fast synaptic inhibition is dependent on targeting specific GABAAR subtypes to dendritic and axon initial segment (AIS) synapses. Synaptic GABAARs are typically assembled from α1-3, ß and γ subunits. Here, we isolate distinct GABAARs from the brain and interrogate their composition using quantitative proteomics. We show that α2-containing receptors co-assemble with α1 subunits, whereas α1 receptors can form GABAARs with α1 as the sole α subunit. We demonstrate that α1 and α2 subunit-containing receptors co-purify with distinct spectrin isoforms; cytoskeletal proteins that link transmembrane proteins to the cytoskeleton. ß2-spectrin was preferentially associated with α1-containing GABAARs at dendritic synapses, while ß4-spectrin was associated with α2-containing GABAARs at AIS synapses. Ablating ß2-spectrin expression reduced dendritic and AIS synapses containing α1 but increased the number of synapses containing α2, which altered phasic inhibition. Thus, we demonstrate a role for spectrins in the synapse-specific targeting of GABAARs, determining the efficacy of fast neuronal inhibition.

Comprehensive Transcriptomic Analysis of Novel Class I HDAC Proteolysis Targeting Chimeras (PROTACs).

The class I histone deacetylase (HDAC) enzymes;HDAC1,2 and 3 form the catalytic engine of at least seven structurally distinct multiprotein complexes in cells. These molecular machines play a vital role in the regulation of chromatin accessibility and gene activity via the removal of acetyl moieties from lysine residues within histone tails. Their inhibition via small molecule inhibitors has beneficial effects in a number of disease types, including the clinical treatment of hematological cancers. We have previously reported a library of proteolysis targeting chimeras (PROTACs) incorporating a benzamide-based HDAC ligand (from CI-994), with an alkyl linker and ligand for the von Hippel-Lindau (VHL) E3 ubiquitin ligase that degrade HDAC1-3 at submicromolar concentrations. Here we report the addition of two novel PROTACs (JPS026 and JPS027), which utilize a ligand for the cellular inhibitor of apoptosis (IAP) family of E3 ligases. We found that both VHL (JPS004)- and IAP (JPS026)-based PROTACs degrade HDAC1-3 and induce histone acetylation to a similar degree. However, JPS026 is significantly more potent at inducing cell death in HCT116 cells than is JPS004. RNA sequencing analysis of PROTAC-treated HCT116 cells showed a distinct gene expression signature in which cell cycle and DNA replication machinery are repressed. Components of the mTORC1 and -2 complexes were also reduced, leading to an increase in FOXO3 and downstream target genes that regulate autophagy and apoptosis. In summary, a novel combination of HDAC and IAP ligands generates a PROTAC with a potent ability to stimulate apoptosis and differential gene expression in human cancer cells.

A 'click' chemistry approach to novel entinostat (MS-275) based class I histone deacetylase proteolysis targeting chimeras.

Click chemistry was utilised to prepare a library of PROTACs based on entinostat a class I histone deacetylase (HDAC) inhibitor in clinical trials. A novel PROTAC JMC-137 was identified as a HDAC1/2 and HDAC3 degrader in HCT116 cells. However, potency was compromised compared to previously identified class I HDAC PROTACs highlighting the importance in the choice of HDAC ligand, functional group for linker attachment and positioning in PROTAC design.

Analyzing the mechanisms that facilitate the subtype-specific assembly of γ-aminobutyric acid type A receptors.

Impaired inhibitory signaling underlies the pathophysiology of many neuropsychiatric and neurodevelopmental disorders including autism spectrum disorders and epilepsy. Neuronal inhibition is regulated by synaptic and extrasynaptic γ-aminobutyric acid type A receptors (GABA A Rs), which mediate phasic and tonic inhibition, respectively. These two GABA A R subtypes differ in their function, ligand sensitivity, and physiological properties. Importantly, they contain different α subunit isoforms: synaptic GABA A Rs contain the α1-3 subunits whereas extrasynaptic GABA A Rs contain the α4-6 subunits. While the subunit composition is critical for the distinct roles of synaptic and extrasynaptic GABA A R subtypes in inhibition, the molecular mechanism of the subtype-specific assembly has not been elucidated. To address this issue, we purified endogenous α1- and α4-containing GABA A Rs from adult murine forebrains and examined their subunit composition and interacting proteins using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and quantitative analysis. We found that the α1 and α4 subunits form separate populations of GABA A Rs and interact with distinct sets of binding proteins. We also discovered that the ß3 subunit, which co-purifies with both the α1 and α4 subunits, has different levels of phosphorylation on serines 408 and 409 (S408/9) between the two receptor subtypes. To understand the role S408/9 plays in the assembly of α1- and α4-containing GABA A Rs, we examined the effects of S408/9A (alanine) knock-in mutation on the subunit composition of the two receptor subtypes using LC-MS/MS and quantitative analysis. We discovered that the S408/9A mutation results in the formation of novel α1α4-containing GABA A Rs. Moreover, in S408/9A mutants, the plasma membrane expression of the α4 subunit is increased whereas its retention in the endoplasmic reticulum is reduced. These findings suggest that S408/9 play a critical role in determining the subtype-specific assembly of GABA A Rs, and thus the efficacy of neuronal inhibition.

Fit Transitioning: When Can Transgender Airmen Fitness Test in Their Affirmed Gender?

INTRODUCTION: Transgender individuals have served openly in the U.S. Military since 2016. Official policies for transgender servicemembers continue to evolve, including approaches to physical fitness testing of transgender servicemembers. There is a paucity of scientific data regarding the effects of gender affirming hormone therapy (GAHT) on athletic performance for the past 24 months of treatment. Identification of expected trends in performance during and after gender transition is essential to allow for the development of appropriate military policy regarding when to assess servicemembers' fitness by standards of their affirmed gender. MATERIALS AND METHODS: We identified Department of the Air Force transgender patients using the Transgender Health Medical Evaluation Unit database and recorded dates of GAHT initiation through a retrospective chart review. We recorded performance values for the Air Force physical fitness test components 1 year before and up to 4 years after GAHT initiation. Performance measures were maximum sit-ups in 1 minute, push-ups in 1 minute, and 1.5-mile run time. Pre- and post-GAHT scores were compared using one-sample T-test to mean scores of Air Force-wide cisgender averages to assess for significant difference between affirmed transgender and cisgender airmen. We then performed the two one-sided test (TOST) procedure for equivalence with upper and lower bounds set at 1 SD from the means for cisgender airmen. Finally, using Z-scores, average transgender group scores were assigned a percentile rank with their respect to affirmed gender throughout the transition process. RESULTS: Following initiation of GAHT, transgender males demonstrated statistically significant worse performance than cisgender males in all events until 3 years of GAHT. Their average scores would have attained a comfortable passing score within 1 year of GAHT. Transgender females' performance showed statistically significantly better performance than cisgender females until 2 years of GAHT in run times and 4 years in sit-up scores and remained superior in push-ups at the study's 4-year endpoint. TOST confirmed equivalence at all points where statistical difference was not demonstrated. Servicemembers approximate their pre-GAHT assigned gender percentile ranking in their affirmed gender in a manner consistent with hypothesis and TOST testing in the push-up event for both transgender males and females and in the 1.5-mile run event for transgender males. CONCLUSIONS: In a sample of Air Force adult transgender patients, athletic performance measures demonstrate variable rates of change depending on the patients' affirmed gender and differ by physical fitness test component. Based on this study, transgender females should begin to be assessed by the female standard no later than 2 years after starting GAHT, while transgender males could be assessed by their affirmed standard no earlier than 3 years after initiating GAHT.

Optimization of Class I Histone Deacetylase PROTACs Reveals that HDAC1/2 Degradation is Critical to Induce Apoptosis and Cell Arrest in Cancer Cells.

Class I histone deacetylase (HDAC) enzymes 1, 2, and 3 organize chromatin as the catalytic subunits within seven distinct multiprotein corepressor complexes and are established drug targets. We report optimization studies of benzamide-based Von Hippel-Lindau (VHL) E3-ligase proteolysis targeting chimeras (PROTACs) and for the first time describe transcriptome perturbations resulting from these degraders. By modifying the linker and VHL ligand, we identified PROTACs 7, 9, and 22 with submicromolar DC50 values for HDAC1 and/or HDAC3 in HCT116 cells. A hook effect was observed for HDAC3 that could be negated by modifying the position of attachment of the VHL ligand to the linker. The more potent HDAC1/2 degraders correlated with greater total differentially expressed genes and enhanced apoptosis in HCT116 cells. We demonstrate that HDAC1/2 degradation by PROTACs correlates with enhanced global gene expression and apoptosis, important for the development of more efficacious HDAC therapeutics with reduced side effects.

Improving Global Access to Transgender Health Care: Outcomes of a Telehealth Quality Improvement Study for the Air Force Transgender Program.

Purpose: To describe the development of the United States Air Force's (USAF) telehealth program from fall 2017 through fall of 2020 in response to the unique challenges associated with providing care for a global transgender military population. Methods: Telehealth visit completion rates were monitored at time of encounters and through electronic health record reports. Patient satisfaction data were obtained by immediate postvisit survey, across provider care received, logistics of setting up the appointment, and quality of the virtual health system connection. Patient cases highlighting opportunities for transgender telehealth were summarized. Results: Between September 9, 2019 and October 28, 2020, 99 telehealth encounters with video-to-video connection occurred. Twenty-three of the encounters were for gender-affirming hormone therapy, 17 for mental health visits, and 59 for speech therapy. Thirty-five surveys were collected from 20 patients. Overall patients were "satisfied" or "very satisfied" with providers' ability to manage their chief complaint through this modality (average 4.9 out of 5 on 1 to 5 scale with 1 being "very dissatisfied" and 5 being "very satisfied") and "strongly agree" that telehealth is an effective means to accomplish care (average score 4.8 on 1 to 5 scale with 1 being "strongly disagree" and 5 "strongly agree"). Services provided spanned 11 USAF bases worldwide. Conclusions: Telehealth is successful in ensuring ongoing transgender health care services for a global military population. The success of this program may have implications for future military and civilian endeavors to bridge care gaps for transgender patients in resource-poor or distant-site locations.

Delineation of the functional properties exhibited by the Zinc-Activated Channel (ZAC) and its high-frequency Thr128Ala variant (rs2257020) in Xenopus oocytes.

The signalling characteristics of the Zinc-Activated Channel (ZAC), a member of the Cys-loop receptor (CLR) superfamily, are presently poorly elucidated. The ZACN polymorphism c.454G>A encoding for the Thr128Ala variation in ZAC is found in extremely high allele frequencies across ethnicities. In this, the first study of ZAC in Xenopus oocytes by TEVC electrophysiology, ZACThr128 and ZACAla128 exhibited largely comparable pharmacological and signalling characteristics, but interestingly the Zn2+- and H+-evoked current amplitudes in ZACAla128-oocytes were dramatically smaller than those in ZACThr128-oocytes. While the variation thus appeared to impact cell surface expression and/or channel properties of ZAC, the similar expression properties exhibited by ZACThr128 and ZACAla128 in transfected mammalian cells indicated that their distinct functionalities could arise from the latter. In co-expression experiments, wild-type and variant ZAC subunits assembled efficiently into "heteromeric" complexes in HEK293 cells, while the concomitant presence of ZACAla128 in ZACThr128:ZACAla128-oocytes did not exert a dominant negative effect on agonist-evoked current amplitudes compared to those in ZACThr128-oocytes. Finally, the structural determinants of the functional importance of the 1-hydroxyethyl side-chain of Thr128 appeared to be subtle, as agonist-evoked current amplitudes in ZACSer128-, ZACVal128- and ZACIle128-oocytes also were substantially lower than those in ZACThr128-oocytes. In conclusion, the functional properties exhibited by ZAC in this work substantiate the notion of it being an atypical CLR. While the impact of the Thr128Ala variation on ZAC functionality in oocytes is striking, it remains to be investigated whether and to which extent this translates into an in vivo setting and thus could constitute a source of inter-individual variation in ZAC physiology.

KCC2 is required for the survival of mature neurons but not for their development.

The K+/Cl- cotransporter KCC2 (SLC12A5) allows mature neurons in the CNS to maintain low intracellular Cl- levels that are critical in mediating fast hyperpolarizing synaptic inhibition via type A γ-aminobutyric acid receptors (GABAARs). In accordance with this, compromised KCC2 activity results in seizures, but whether such deficits directly contribute to the subsequent changes in neuronal structure and viability that lead to epileptogenesis remains to be assessed. Canonical hyperpolarizing GABAAR currents develop postnatally, which reflect a progressive increase in KCC2 expression levels and activity. To investigate the role that KCC2 plays in regulating neuronal viability and architecture, we have conditionally ablated KCC2 expression in developing and mature neurons. Decreasing KCC2 expression in mature neurons resulted in the rapid activation of the extrinsic apoptotic pathway. Intriguingly, direct pharmacological inhibition of KCC2 in mature neurons was sufficient to rapidly induce apoptosis, an effect that was not abrogated via blockade of neuronal depolarization using tetrodotoxin (TTX). In contrast, ablating KCC2 expression in immature neurons had no discernable effects on their subsequent development, arborization, or dendritic structure. However, removing KCC2 in immature neurons was sufficient to ablate the subsequent postnatal development of hyperpolarizing GABAAR currents. Collectively, our results demonstrate that KCC2 plays a critical role in neuronal survival by limiting apoptosis, and mature neurons are highly sensitive to the loss of KCC2 function. In contrast, KCC2 appears to play a minimal role in mediating neuronal development or architecture.

Effect of gender affirming hormones on athletic performance in transwomen and transmen: implications for sporting organisations and legislators.

OBJECTIVE: To examine the effect of gender affirming hormones on athletic performance among transwomen and transmen. METHODS: We reviewed fitness test results and medical records of 29 transmen and 46 transwomen who started gender affirming hormones while in the United States Air Force. We compared pre- and post-hormone fitness test results of the transwomen and transmen with the average performance of all women and men under the age of 30 in the Air Force between 2004 and 2014. We also measured the rate of hormone associated changes in body composition and athletic performance. RESULTS: Participants were 26.2 years old (SD 5.5). Prior to gender affirming hormones, transwomen performed 31% more push-ups and 15% more sit-ups in 1 min and ran 1.5 miles 21% faster than their female counterparts. After 2 years of taking feminising hormones, the push-up and sit-up differences disappeared but transwomen were still 12% faster. Prior to gender affirming hormones, transmen performed 43% fewer push-ups and ran 1.5 miles 15% slower than their male counterparts. After 1 year of taking masculinising hormones, there was no longer a difference in push-ups or run times, and the number of sit-ups performed in 1 min by transmen exceeded the average performance of their male counterparts. SUMMARY: The 15-31% athletic advantage that transwomen displayed over their female counterparts prior to starting gender affirming hormones declined with feminising therapy. However, transwomen still had a 9% faster mean run speed after the 1 year period of testosterone suppression that is recommended by World Athletics for inclusion in women's events.

Isolation and Characterization of Multi-Protein Complexes Enriched in the K-Cl Co-transporter 2 From Brain Plasma Membranes.

Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanisms neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of K-Cl Co-transporter 2 (Kcc2) from the plasma membrane of murine forebrain. We resolved these using blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS and label-free quantification. Data are available via ProteomeXchange with identifier PXD021368. Purified Kcc2 migrated as distinct molecular species of 300, 600, and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N- and C-termini of Kcc2 was obtained. In total we identified 246 proteins significantly associated with Kcc2. The 300 kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting, immune related and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed localization of Kcc2. These included spectrins, C1qa/b/c and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5, Akap13, and Lmtk3. Finally, we used LC-MS/MS on the same purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity.