Dopaminergic error signals retune to social feedback during courtship.

Hunger, thirst, loneliness and ambition determine the reward value of food, water, social interaction and performance outcome1. Dopamine neurons respond to rewards meeting these diverse needs2-8, but it remains unclear how behaviour and dopamine signals change as priorities change with new opportunities in the environment. One possibility is that dopamine signals for distinct drives are routed to distinct dopamine pathways9,10. Another possibility is that dopamine signals in a given pathway are dynamically tuned to rewards set by the current priority. Here we used electrophysiology and fibre photometry to test how dopamine signals associated with quenching thirst, singing a good song and courting a mate change as male zebra finches (Taeniopygia guttata) were provided with opportunities to retrieve water, evaluate song performance or court a female. When alone, water reward signals were observed in two mesostriatal pathways but singing-related performance error signals were routed to Area X, a striatal nucleus specialized for singing. When courting a female, water seeking was reduced and dopamine responses to both water and song performance outcomes diminished. Instead, dopamine signals in Area X were driven by female calls timed with the courtship song. Thus the dopamine system handled coexisting drives by routing vocal performance and social feedback signals to a striatal area for communication and by flexibly re-tuning to rewards set by the prioritized drive.

Beyond Erectile Dysfunction: cGMP-Specific Phosphodiesterase 5 Inhibitors for Other Clinical Disorders.

Cyclic guanosine monophosphate (cGMP), an important intracellular second messenger, mediates cellular functional responses in all vital organs. Phosphodiesterase 5 (PDE5) is one of the 11 members of the cyclic nucleotide phosphodiesterase (PDE) family that specifically targets cGMP generated by nitric oxide-driven activation of the soluble guanylyl cyclase. PDE5 inhibitors, including sildenafil and tadalafil, are widely used for the treatment of erectile dysfunction, pulmonary arterial hypertension, and certain urological disorders. Preclinical studies have shown promising effects of PDE5 inhibitors in the treatment of myocardial infarction, cardiac hypertrophy, heart failure, cancer and anticancer-drug-associated cardiotoxicity, diabetes, Duchenne muscular dystrophy, Alzheimer's disease, and other aging-related conditions. Many clinical trials with PDE5 inhibitors have focused on the potential cardiovascular, anticancer, and neurological benefits. In this review, we provide an overview of the current state of knowledge on PDE5 inhibitors and their potential therapeutic indications for various clinical disorders beyond erectile dysfunction.

Improved efficiency using sequential automated immunoassays for syphilis screening in blood donors.

Using sequential immunoassays for the screening of blood donors is well described for viral serology testing but not for the screening of syphilis. In this study, we report the evaluation results and 2-year sequential testing data using two highly sensitive automated serology assays, the Alinity s Syphilis chemiluminescent immunoassay for screening, with all repeatedly reactive samples then tested on the Elecsys Syphilis electrochemiluminescence immunoassay. We screened 1,767,782 blood donor samples between 7 July 2021 and 6 July 2023 and found the Alinity false-positive rate to be low at 0.08% (1,456/1,767,782). The common false-positive rate between the two assays was also low (3.83%, 58/1,514). Concordantly reactive samples were further tested using a Treponema pallidum particle agglutination test, a rapid plasma reagin test, and a fluorescent treponemal antibody absorption test. There were 262/1,376 concordantly reactive Alinity and Elecsys blood donor samples with reactivity on one or more of the confirmatory tests. A total of 26/1,376 donors had a current syphilis infection, 152/1,376 reported a past history of syphilis and had been treated, and 84/1,376 did not report a past history of syphilis. We suggest that future studies could explore the use of sequential immunoassays to aid in the serodiagnosis for syphilis. IMPORTANCE: The serodiagnosis for syphilis usually follows two methodologies-a "traditional" algorithm using a non-treponemal test followed by confirmation using a treponemal test, or a "reverse" algorithm using a treponemal test followed by a non-treponemal test. There are limited reports in the literature of using a modified reverse algorithm (treponemal test followed by a second treponemal test), and to the best of knowledge, there are currently no published articles using two highly sensitive automated immunoassays to aid the serodiagnosis of syphilis. In addition, the Treponema pallidum particle agglutination (TPPA) assay is commonly used as a confirmatory test for the diagnosis of syphilis. With the withdrawal of the TPPA assay from Australia and presumably from the global market also, alternative testing algorithms are now required. This study provides proof of concept for using sequential immunoassays in the diagnosis of syphilis.

A comparative dose-escalation analysis for reirradiated cancer patients with and without appropriate dose mapping.

This study aims to compare dose escalation between two groups of reirradiated cancer patients, one with the previous contour and radiotherapy plan available on the treatment planning system and the other without. First group is identified as DICOM-group, while the other one is called non-DICOM group. The current study included 89 patients, 57 in the DICOM, and 32 in the non-DICOM group, who received reirradiation for recurrent or second primary tumours between 2019 and 2021. For the DICOM group, doses to 0.2cc volume for spine, brainstem, and optic apparatus from first radiation were converted into structures and transferred to reirradiation CT using deformable registration. First, one radiotherapy plan was created using the doctor prescribed dose (baseline prescription RxD_B); further an escalated dose (RxD_E) plan, taking into account all the dose volume parameters from previous radiation, was created only for DICOM group. In non-DICOM group patients were planned only for RxD_B. The maximum accepted dose escalation was 21 Gy. Radiotherapy prescription dose during earlier (first) treatment in DICOM and non-DICOM groups were 61 ± 5.6 Gy and 30-66 Gy, respectively. DICOM and non-DICOM groups had nearly identical baseline doses: 52.5 ± 10.7 Gy and 50.6 ± 6.9 Gy (difference 1.9 ± 12.7 Gy). Dose escalation was possible for 51 out of 57 patients in the DICOM-group. Average escalated dose in DICOM-group was 59.2 ± 6.2 Gy, with an incremental dose of 6.7 ± 12.4 Gy from the baseline prescription. No dose escalation was opted for in the non-DICOM group due to the unavailability of dose volume information from previous radiation. Reirradiation for head and neck cases allowed for a moderate to high dose escalation, facilitated by the presence of pertinent DICOM information from the initial radiotherapy.

Seed-Induced Living Two-Dimensional (2D) Supramolecular Polymerization in Water: Implications on Protein Adsorption and Enzyme Inhibition.

In biological systems, programmable supramolecular frameworks characterized by coordinated directional non-covalent interactions are widespread. However, only a small number of reports involve pure water-based dynamic supramolecular assembly of artificial π-amphiphiles, primarily due to the formidable challenge of counteracting the strong hydrophobic dominance of the π-surface in water, leading to undesired kinetic traps. This study reveals the pathway complexity in hydrogen-bonding-mediated supramolecular polymerization of an amide-functionalized naphthalene monoimide (NMI) building block with a hydrophilic oligo-oxyethylene (OE) wedge. O-NMI-2 initially produced entropically driven, collapsed spherical particles in water (Agg-1); however, over a span of 72 h, these metastable Agg-1 gradually transformed into two-dimensional (2D) nanosheets (Agg-2), favoured by both entropy and enthalpy contributions. The intricate self-assembly pathways in O-NMI-2 enable us to explore seed-induced living supramolecular polymerization (LSP) in water for controlled synthesis of monolayered 2D assemblies. Furthermore, we demonstrated the nonspecific surface adsorption of a model enzyme, serine protease α-Chymotrypsin (α-ChT), and consequently the enzyme activity, which could be regulated by controlling the morphological transformation of O-NMI-2 from Agg-1 to Agg-2. We delve into the thermodynamic aspects of such shape-dependent protein-surface interactions and unravel the impact of seed-induced LSP on temporally controlling the catalytic activity of α-ChT.

Partial agonism in heteromeric GLUK2/GLUK5 kainate receptor.

Kainate receptors are a subtype of ionotropic glutamate receptors that form transmembrane channels upon binding glutamate. Here, we have investigated the mechanism of partial agonism in heteromeric GluK2/K5 receptors, where the GluK2 and GluK5 subunits have distinct agonist binding profiles. Using single-molecule Förster resonance energy transfer, we found that at the bi-lobed agonist-binding domain, the partial agonist AMPA-bound receptor occupied intermediate cleft closure conformational states at the GluK2 cleft, compared to the more open cleft conformations in apo form and more closed cleft conformations in the full agonist glutamate-bound form. In contrast, there is no significant difference in cleft closure states at the GluK5 agonist-binding domain between the partial agonist AMPA- and full agonist glutamate-bound states. Additionally, unlike the glutamate-bound state, the dimer interface at the agonist-binding domain is not decoupled in the AMPA-bound state. Our findings suggest that partial agonism observed with AMPA binding is mediated primarily due to differences in the GluK2 subunit, highlighting the distinct contributions of the subunits towards activation.

Pathway Complexity in Supramolecular Copolymerization and Blocky Star Copolymers by a Hetero-Seeding Effect.

This study unravels the intricate kinetic and thermodynamic pathways involved in the supramolecular copolymerization of the two chiral dipolar naphthalene monoimide (NMI) building blocks (O-NMI and S-NMI), differing merely by a single heteroatom (oxygen vs sulfur). O-NMI exhibits distinct supramolecular polymerization features as compared to S-NMI in terms of its pathway complexity, hierarchical organization, and chiroptical properties. Two distinct self-assembly pathways in O-NMI occur due to the interplay between the competing dipolar interactions among the NMI chromophores and amide-amide hydrogen (H)-bonding that engenders distinct nanotapes and helical fibers, from its antiparallel and parallel stacking modes, respectively. In contrast, the propensity of S-NMI to form only a stable spherical assembly is ascribed to its much stronger amide-amide H-bonding, which outperforms other competing interactions. Under the thermodynamic route, an equimolar mixture of the two monomers generates a temporally controlled chiral statistical supramolecular copolymer that autocatalytically evolves from an initially formed metastable spherical heterostructure. In contrast, the sequence-controlled addition of the two monomers leads to the kinetically driven hetero-seeded block copolymerization. The ability to trap O-NMI in a metastable state allows its secondary nucleation from the surface of the thermodynamically stable S-NMI spherical "seed", which leads to the core-multiarmed "star" copolymer with reversibly and temporally controllable length of the growing O-NMI "arms" from the S-NMI "core". Unlike the one-dimensional self-assembly of O-NMI and its random co-assembly with S-NMI, which are both chiral, unprecedentedly, the preferred helical bias of the nucleating O-NMI fibers is completely inhibited by the absence of stereoregularity of the S-NMI "seed" in the "star" topology.

In Vivo Behavior of Ultrasmall Spherical Nucleic Acids.

The biological properties of spherical nucleic acids (SNAs) are largely independent of nanoparticle core identity but significantly affected by oligonucleotide surface density. Additionally, the payload-to-carrier (i.e., DNA-to-nanoparticle) mass ratio of SNAs is inversely proportional to core size. While SNAs with many core types and sizes have been developed, all in vivo analyses of SNA behavior have been limited to cores >10 nm in diameter. However, "ultrasmall" nanoparticle constructs (<10 nm diameter) can exhibit increased payload-to-carrier ratios, reduced liver accumulation, renal clearance, and enhanced tumor infiltration. Therefore, we hypothesized that SNAs with ultrasmall cores exhibit SNA-like properties, but with in vivo behavior akin to traditional ultrasmall nanoparticles. To investigate, we compared the behavior of SNAs with 1.4-nm Au102 nanocluster cores (AuNC-SNAs) and SNAs with 10-nm gold nanoparticle cores (AuNP-SNAs). Significantly, AuNC-SNAs possess SNA-like properties (e.g., high cellular uptake, low cytotoxicity) but show distinct in vivo behavior. When intravenously injected in mice, AuNC-SNAs display prolonged blood circulation, lower liver accumulation, and higher tumor accumulation than AuNP-SNAs. Thus, SNA-like properties persist at the sub-10-nm length scale and oligonucleotide arrangement and surface density are responsible for the biological properties of SNAs. This work has implications for the design of new nanocarriers for therapeutic applications.

A Versatile Step-Growth Polymerization Route to Functional Polyesters from an Activated Diester Monomer.

This work describes a versatile and efficient condensation polymerization route to aliphatic polyesters by organo-catalyzed (4-dimethylaminopyridine) transesterification reactions between an activated pentafluorophenyl-diester of adipic acid and structurally different diols. By introducing "monofunctional impurity" or "stoichiometric imbalance," this methodology can afford well-defined end-functionalized polyesters with predictable molecular weights and narrow dispersity under mild conditions without any necessity for the removal of the byproducts to accelerate the polymerization reaction, which remains a major challenge in conventional polyester synthesis with non-activated diesters. Wide substrate scope with structurally different monomers and the synthesis of block copolymers by chain extension following either ring-opening polymerization or controlled radical polymerization have been successfully demonstrated. Some of the polyesters synthesized by this newly introduced approach show high thermal stability, crystallinity, and enzymatic degradation in aqueous environments.

Treating diabetes with combination of phosphodiesterase 5 inhibitors and hydroxychloroquine-a possible prevention strategy for COVID-19?

Type 2 diabetes (T2D) is one of the major risk factors for developing cardiovascular disease and the resultant devastating morbidity and mortality. The key features of T2D are hyperglycemia, hyperlipidemia, insulin resistance, and impaired insulin secretion. Patients with diabetes and myocardial infarction have worse prognosis than those without T2D. Moreover, obesity and T2D are recognized risk factors in developing severe form of COVID-19 with higher mortality rate. The current lines of drug therapy are insufficient to control T2D and its serious cardiovascular complications. Phosphodiesterase 5 (PDE5) is a cGMP specific enzyme, which is the target of erectile dysfunction drugs including sildenafil, vardenafil, and tadalafil. Cardioprotective effects of PDE5 inhibitors against ischemia/reperfusion (I/R) injury were reported in normal and diabetic animals. Hydroxychloroquine (HCQ) is a widely used antimalarial and anti-inflammatory drug and its hyperglycemia-controlling effect in diabetic patients is also under investigation. This review provides our perspective of a potential use of combination therapy of PDE5 inhibitor with HCQ to reduce cardiovascular risk factors and myocardial I/R injury in T2D. We previously observed that diabetic mice treated with tadalafil and HCQ had significantly reduced fasting blood glucose and lipid levels, increased plasma insulin and insulin-like growth factor-1 levels, and improved insulin sensitivity, along with smaller myocardial infarct size following I/R. The combination treatment activated Akt/mTOR cellular survival pathway, which was likely responsible for the salutary effects. Therefore, pretreatment with PDE5 inhibitor and HCQ may be a potentially useful therapy not only for controlling T2D but also reducing the rate and severity of COVID-19 infection in the vulnerable population of diabetics.

Emissive Substoichiometric Covalent Organic Frameworks for Water Sensing and Harvesting.

Emissive covalent organic frameworks (COFs) have recently emerged as next-generation porous materials with attractive properties such as tunable topology, porosity, and inherent photoluminescence. Among the different types of COFs, substoichiometric frameworks (so-called Type III COFs) are especially attractive due to the possibility of not only generating unusual topology and complex pore architectures but also facilitating the introduction of well-defined functional groups at precise locations for desired functions. Herein, the first example of a highly emissive (PLQY 6.8%) substoichiometric 2D-COF (COF-SMU-1) featuring free uncondensed aldehyde groups is reported. In particular, COF-SMU-1 features a dual-pore architecture with an overall bex net topology, tunable emission in various organic solvents, and distinct colorimetric changes in the presence of water. To gain further insights into its photoluminescence properties, the charge transfer, excimer emission, and excited state exciton dynamics of COF-SMU-1 are investigated using femtosecond transient absorption spectroscopy in different organic solvents. Additionally, highly enhanced atmospheric water-harvesting properties of COF-SMU-1 are revealed using FT-IR and water sorption studies.The findings will not only lead to in-depth understanding of structure-property relationships in emissive COFs but also open new opportunities for designing COFs for potential applications in solid-state lighting and water harvesting.

mTOR Signaling: New Insights into Cancer, Cardiovascular Diseases, Diabetes and Aging.

The mechanistic/mammalian target of rapamycin (mTOR), a member of the phosphoinositide 3-kinase (PI3K) related kinase family, integrates intracellular and environmental cues that coordinate a diverse set of cellular/tissue functions, such as cell growth, proliferation, metabolism, autophagy, apoptosis, longevity, protein/lipid/nucleotide synthesis, and tissue regeneration and repair [...].

mTORC1 and SGLT2 Inhibitors-A Therapeutic Perspective for Diabetic Cardiomyopathy.

Diabetic cardiomyopathy is a critical diabetes-mediated co-morbidity characterized by cardiac dysfunction and heart failure, without predisposing hypertensive or atherosclerotic conditions. Metabolic insulin resistance, promoting hyperglycemia and hyperlipidemia, is the primary cause of diabetes-related disorders, but ambiguous tissue-specific insulin sensitivity has shed light on the importance of identifying a unified target paradigm for both the glycemic and non-glycemic context of type 2 diabetes (T2D). Several studies have indicated hyperactivation of the mammalian target of rapamycin (mTOR), specifically complex 1 (mTORC1), as a critical mediator of T2D pathophysiology by promoting insulin resistance, hyperlipidemia, inflammation, vasoconstriction, and stress. Moreover, mTORC1 inhibitors like rapamycin and their analogs have shown significant benefits in diabetes and related cardiac dysfunction. Recently, FDA-approved anti-hyperglycemic sodium-glucose co-transporter 2 inhibitors (SGLT2is) have gained therapeutic popularity for T2D and diabetic cardiomyopathy, even acknowledging the absence of SGLT2 channels in the heart. Recent studies have proposed SGLT2-independent drug mechanisms to ascertain their cardioprotective benefits by regulating sodium homeostasis and mimicking energy deprivation. In this review, we systematically discuss the role of mTORC1 as a unified, eminent target to treat T2D-mediated cardiac dysfunction and scrutinize whether SGLT2is can target mTORC1 signaling to benefit patients with diabetic cardiomyopathy. Further studies are warranted to establish the underlying cardioprotective mechanisms of SGLT2is under diabetic conditions, with selective inhibition of cardiac mTORC1 but the concomitant activation of mTORC2 (mTOR complex 2) signaling.

Single-Dose Treatment with Rapamycin Preserves Post-Ischemic Cardiac Function through Attenuation of Fibrosis and Inflammation in Diabetic Rabbit.

Robust activation of mTOR (mammalian target of rapamycin) signaling in diabetes exacerbates myocardial injury following lethal ischemia due to accelerated cardiomyocyte death with cardiac remodeling and inflammatory responses. We examined the effect of rapamycin (RAPA, mTOR inhibitor) on cardiac remodeling and inflammation following myocardial ischemia/reperfusion (I/R) injury in diabetic rabbits. Diabetic rabbits (DM) were subjected to 45 min of ischemia and 10 days of reperfusion by inflating/deflating a previously implanted hydraulic balloon occluder. RAPA (0.25 mg/kg, i.v.) or DMSO (vehicle) was infused 5 min before the onset of reperfusion. Post-I/R left ventricular (LV) function was assessed by echocardiography and fibrosis was evaluated by picrosirius red staining. Treatment with RAPA preserved LV ejection fraction and reduced fibrosis. Immunoblot and real-time PCR revealed that RAPA treatment inhibited several fibrosis markers (TGF-ß, Galectin-3, MYH, p-SMAD). Furthermore, immunofluorescence staining revealed the attenuation of post-I/R NLRP3-inflammasome formation with RAPA treatment as shown by reduced aggregation of apoptosis speck-like protein with a caspase recruitment domain and active-form of caspase-1 in cardiomyocytes. In conclusion, our study suggests that acute reperfusion therapy with RAPA may be a viable strategy to preserve cardiac function with the alleviation of adverse post-infarct myocardial remodeling and inflammation in diabetic patients.

Cascade Energy Transfer and White-Light Emission in Chirality-Controlled Crystallization-Driven Two-Dimensional Co-assemblies from Donor and Acceptor Dye-Conjugated Polylactides.

Achieving predictable and programmable two-dimensional (2D) structures with specific functions from exclusively organic soft materials remains a scientific challenge. This article unravels stereocomplex crystallization-driven self-assembly as a facile method for producing thermally robust discrete 2D-platelets of diamond shape from biodegradable semicrystalline polylactide (PLA) scaffolds. The method involves co-assembling two PLA stereoisomers, namely, PY-PDLA and NMI-PLLA, which form stereocomplex (SC)-crystals in isopropanol. By conjugating a well-known Förster resonance energy transfer (FRET) donor and acceptor dye, namely, pyrene (PY) and naphthalene monoimide (NMI), respectively, to the chain termini of these two interacting stereoisomers, a thermally robust FRET process can be stimulated from the 2D array of the co-assembled dyes on the thermally resilient SC-PLA crystal surfaces. Uniquely, by decorating the surface of the SC-PLA crystals with an externally immobilized guest dye, Rhodamine-B, similar diamond-shaped structures could be produced that exhibit pure white-light emission through a surface-induced two-step cascade energy transfer process. The FRET response in these systems displays remarkable dependence on the intrinsic crystalline packing, which could be modulated by the chirality of the co-assembling PLA chains. This is supported by comparing the properties of similar 2D platelets generated from two homochiral PLLAs (PY-PLLA and NMI-PLLA) labeled with the same FRET pair.

Crystal Structure and Optical Properties of a Chiral Mixed Thiolate/Stibine-Protected Au18 Cluster.

We report the first example of a chiral mixed thiolate/stibine-protected gold cluster, formulated as Au18(S-Adm)8(SbPh3)4Br2 (where S-Adm = 1-adamantanethiolate). Single crystal X-ray crystallography reveals the origin of chirality in the cluster to be the introduction of the rotating arrangement of Au2(S-Adm)3 and Au(S-Adm)2 staple motifs on an achiral Au13 core and the subsequent capping of the remaining gold atoms by SbPh3 and Br- ligands. Interestingly, the structure and properties of this new Au18 cluster are found to be different from other reported achiral Au18 clusters and the only other stibine-protected [Au13(SbPh3)8Cl4]+ cluster. Detailed analyses on the geometric and electronic structures of the new cluster are carried out to gain insights into its optical properties as well as reactivity and stability of such mixed monolayer-protected clusters.

Songbird subthalamic neurons project to dopaminergic midbrain and exhibit singing-related activity.

Skill learning requires motor output to be evaluated against internal performance benchmarks. In songbirds, ventral tegmental area (VTA) dopamine neurons (DA) signal performance errors important for learning, but it remains unclear which brain regions project to VTA and how these inputs may contribute to DA error signaling. Here, we find that the songbird subthalamic nucleus (STN) projects to VTA and that STN microstimulation can excite VTA neurons. We also discover that STN receives inputs from motor cortical, auditory cortical, and ventral pallidal brain regions previously implicated in song evaluation. In the first neural recordings from songbird STN, we discover that the activity of most STN neurons is associated with body movements and not singing, but a small fraction of neurons exhibits precise song timing and performance error signals. Our results place the STN in a pathway important for song learning, but not song production, and expand the territories of songbird brain potentially associated with song learning.NEW & NOTEWORTHY Songbird subthalamic (STN) neurons exhibit singing-related signals and are interconnected with the motor cortical nucleus, auditory pallium, ventral pallidum, and ventral tegmental area, areas important for song generation and learning.

Crystallization-Driven Controlled Two-Dimensional (2D) Assemblies from Chromophore-Appended Poly(L-lactide)s: Highly Efficient Energy Transfer on a 2D Surface.

A rational approach towards precision two-dimensional (2D) assemblies by crystallization-driven self-assembly (CDSA) of poly(L-lactides) (PLLAs), end-capped with dipolar dyes like merocyanine (MC) or naphthalene monoimide (NMI) and hydrophobic pyrene (PY) or benzene (Bn) is described. PLLA chains crystallize into diamond-shaped platelets in isopropanol, which forces the terminal dyes to assemble into a 2D array on the platelet surface by either dipolar interactions or π-stacking and exhibit tunable emission. Dipolar dyes play a critical role in imparting colloidal stability and structural uniformity to the 2D crystals, which is partly compromised for hydrophobic ones. Co-crystallization between NMI- and PY-labeled PLLAs yields similar diamond-shaped co-platelets with highly efficient (≈80 %) Förster Resonance Energy Transfer on the 2D surface. Further, the "living" CDSA method confers enlarged, segmented block co-platelets using one of the homopolymers as "seed" and the other as "unimer".

Effectiveness of dual-detergent strategy using Triton X-100 in membrane protein purification.

Membrane solubilization by detergents is a critical step for successful membrane protein purification. Alkyl maltoside detergents such as DDM and DM are very expensive and are commonly used to produce most of the high-quality proteins in stable and functional form. Recently, dual-detergent strategy using inexpensive detergents for membrane solubilization step has been shown to be highly effective in purifying different classes of membrane proteins in a cost-effective manner. In this work, we have monitored the effectiveness of 'dual-detergent strategy' towards successful purification of the isolated voltage sensing domain (VSD) of KvAP and the inward rectifying K+ channel, KirBac1.1. We demonstrate that the inexpensive detergent Triton X-100 extracts the activated conformation of the KvAP-VSD well without compromising the structural integrity of the sensor, and also retains its proper structural dynamics. Importantly, the cost associated with solubilizing the KvAP sensor can be reduced by ∼2000 fold. To the best of our knowledge, our results constitute the first report characterizing the purification of KvAP voltage sensor using an inexpensive detergent. However, the dual-detergent strategy using Triton X-100 for membrane solubilization is not effective for the purification of inward rectifying K+ channel, KirBac1.1 even in presence of high salt concentration during solubilization. We propose that the dual-detergent strategy will be useful for extracting stable and functional proteins that are both DDM- and DM-extractable, but will be ineffective if the protein is only DM-extractable. The relevance of the effectiveness of dual-detergent strategy with respect to the hydrophobic thickness of proteins is discussed.

Structural Dynamics of the Paddle Motif Loop in the Activated Conformation of KvAP Voltage Sensor.

Voltage-dependent potassium (Kv) channels play a fundamental role in neuronal and cardiac excitability and are potential therapeutic targets. They assemble as tetramers with a centrally located pore domain surrounded by a voltage-sensing domain (VSD), which is critical for sensing transmembrane potential and subsequent gating. Although the sensor is supposed to be in "Up" conformation in both n-octylglucoside (OG) micelles and phospholipid membranes in the absence of membrane potential, toxins that bind VSD and modulate the gating behavior of Kv channels exhibit dramatic affinity differences in these membrane-mimetic systems. In this study, we have monitored the structural dynamics of the S3b-S4 loop of the paddle motif in activated conformation of KvAP-VSD by site-directed fluorescence approaches, using the environment-sensitive fluorescent probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl-ethylenediamine (NBD). Emission maximum of NBD-labeled loop region of KvAP-VSD (residues 110-117) suggests a significant change in the polarity of local environment in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) membranes compared to OG micelles. This indicates that S3b-S4 loop residues might be partitioning to membrane interface, which is supported by an overall increased mean fluorescence lifetimes and significantly reduced water accessibility in membranes. Further, the magnitude of red edge excitation shift (REES) supports the presence of restricted/bound water molecules in the loop region of the VSD in micelles and membranes. Quantitative analysis of REES data using Gaussian probability distribution function clearly indicates that the sensor loop has fewer discrete equilibrium conformational states when reconstituted in membranes. Interestingly, this reduced molecular heterogeneity is consistent with the site-specific NBD polarization results, which suggest that the membrane environment offers a relaxed/dynamic organization for most of the S3b-S4 loop residues of the sensor. Overall, our results are relevant for understanding toxin-VSD interaction and gating mechanisms of Kv channels in membranes.