Steady-state and time-resolved fluorescent methodologies to characterize the conformational landscape of the selectivity filter of K+ channels.

A variety of equilibrium and non-equilibrium methods have been used in a multidisciplinary approach to study the conformational landscape associated with the binding of different cations to the pore of potassium channels. These binding processes, and the conformational changes resulting therefrom, modulate the functional properties of such integral membrane properties, revealing these permeant and blocking cations as true effectors of such integral membrane proteins. KcsA, a prototypic K+ channel from Streptomyces lividans, has been extensively characterized in this regard. Here, we revise several fluorescence-based approaches to monitor cation binding under different experimental conditions in diluted samples, analyzing the advantages and disadvantages of each approach. These studies have contributed to explain the selectivity, conduction, and inactivation properties of K+ channels at the molecular level, together with the allosteric communication between the two gates that control the ion channel flux, and how they are modulated by lipids.

Anionic Phospholipids Shift the Conformational Equilibrium of the Selectivity Filter in the KcsA Channel to the Conductive Conformation: Predicted Consequences on Inactivation.

Here, we report an allosteric effect of an anionic phospholipid on a model K+ channel, KcsA. The anionic lipid in mixed detergent-lipid micelles specifically induces a change in the conformational equilibrium of the channel selectivity filter (SF) only when the channel inner gate is in the open state. Such change consists of increasing the affinity of the channel for K+, stabilizing a conductive-like form by maintaining a high ion occupancy in the SF. The process is highly specific in several aspects: First, lipid modifies the binding of K+, but not that of Na+, which remains unperturbed, ruling out a merely electrostatic phenomenon of cation attraction. Second, no lipid effects are observed when a zwitterionic lipid, instead of an anionic one, is present in the micelles. Lastly, the effects of the anionic lipid are only observed at pH 4.0, when the inner gate of KcsA is open. Moreover, the effect of the anionic lipid on K+ binding to the open channel closely emulates the K+ binding behaviour of the non-inactivating E71A and R64A mutant proteins. This suggests that the observed increase in K+ affinity caused by the bound anionic lipid should result in protecting the channel against inactivation.

Biomarkers of Fibrosis in Patients with COVID-19 One Year After Hospital Discharge: A Prospective Cohort Study.

Beyond the acute infection of coronavirus disease (COVID-19), concern has arisen about long-term effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The aim of our study was to analyze if there is any biomarker of fibrogenesis in patients with COVID-19 pneumonia capable of predicting post-COVID-19 pulmonary sequelae. We conducted a multicenter, prospective, observational cohort study of patients admitted to a hospital with bilateral COVID-19 pneumonia. We classified patients into two groups according to severity, and blood sampling to measure matrix metalloproteinase 1 (MMP-1), MMP-7, periostin, and VEGF and respiratory function tests and high-resolution computed tomography were performed at 2 and 12 months after hospital discharge. A total of 135 patients were evaluated at 12 months. Their median age was 61 (interquartile range, 19) years, and 58.5% were men. We found between-group differences in age, radiological involvement, length of hospital stay, and inflammatory laboratory parameters. Differences were found between 2 and 12 months in all functional tests, including improvements in predicted forced vital capacity (98.0% vs. 103.9%; P = 0.001) and DlCO <80% (60.9% vs. 39.7%; P = 0.001). At 12 months, 63% of patients had complete high-resolution computed tomography resolution, but fibrotic changes persisted in 29.4%. Biomarker analysis demonstrated differences at 2 months in periostin (0.8893 vs. 1.437 ng/ml; P < 0.001) and MMP-7 (8.7249 vs. 15.2181 ng/ml; P < 0.001). No differences were found at 12 months. In multivariable analysis, only 2-month periostin was associated with 12-month fibrotic changes (odds ratio, 1.0013; 95% confidence interval, 1.0006-1.00231; P = 0.003) and 12-month DlCO impairment (odds ratio, 1.0006; 95% confidence interval, 1.0000-1.0013; P = 0.047). Our data suggest that early periostin postdischarge could predict the presence of fibrotic pulmonary changes.

Benchmarking of stray neutron fields produced by synchrocyclotrons and synchrotrons in compact protontherapy centers (CPTC) using MCNP6 Monte Carlo code.

Proton therapy is an external radiotherapy using proton beams with energies between 70 and 230 MeV to treat some type of tumours with outstanding benefits, due to its energy transfer plot. There is a growing demand of facilities taking up small spaces and Compact Proton Therapy Centers (CPTC), with one or two treatment rooms, supposing the technical response of manufacturers to this request. A large amount of stray radiation is yielded in the interaction of proton beam used in therapy, neutrons mainly, hence, optimal design of shielding and verifications must be carried out in commissioning phases. Currently, almost 50 proton centers are under construction and start up in several countries, including ten in Spain. In the present work the effectiveness of shielding in two CPTC was verified with the Monte Carlo code MCNP6 by calculating the ambient dose equivalent, H*(10) due to secondary neutrons, outside the enclosures and walls of the center. The facilities modelled were the two centers currently operating in Spain, the first, since December 2019, with a superconductor synchrocyclotron, and the second, since March 2020, with a compact synchrotron. The geometry and materials are based on dimensions proposed a priori by the vendors, therefore, the paper is focused on check the suitability of the materials and thickness of the walls of the centers. Several models of the radiation sources were simulated, starting from a conservative assumptions, followed by more realistic scenarios. In all cases, the results reached for the ambient dose equivalent, H*(10), were below 1 mSv/year, which is the legal limit considered for the public in international references. Finally, considering that the recent ICRU Report 95 proposes changes in the operational quantities, the dose outside shieldingt has been evaluated in terms of the new next area surveillance quantity, H*, known as ambient dose, in the process of implementation.

Xenopus Oocytes as a Powerful Cellular Model to Study Foreign Fully-Processed Membrane Proteins.

The use of Xenopus oocytes in electrophysiological and biophysical research constitutes a long and successful story, providing major advances to the knowledge of the function and modulation of membrane proteins, mostly receptors, ion channels, and transporters. Earlier reports showed that these cells are capable of correctly expressing heterologous proteins after injecting the corresponding mRNA or cDNA. More recently, the Xenopus oocyte has become an outstanding host-cell model to carry out detailed studies on the function of fully-processed foreign membrane proteins after their microtransplantation to the oocyte. This review focused on the latter overall process of transplanting foreign membrane proteins to the oocyte after injecting plasma membranes or purified and reconstituted proteins. This experimental approach allows for the study of both the function of mature proteins, with their native stoichiometry and post-translational modifications, and their putative modulation by surrounding lipids, mostly when the protein is purified and reconstituted in lipid matrices of defined composition. Remarkably, this methodology enables functional microtransplantation to the oocyte of membrane receptors, ion channels, and transporters from different sources including human post-mortem tissue banks. Despite the large progress achieved over the last decades on the structure, function, and modulation of neuroreceptors and ion channels in healthy and pathological tissues, many unanswered questions remain and, most likely, Xenopus oocytes will continue to help provide valuable responses.

Lung function and radiological findings 1 year after COVID-19: a prospective follow-up.

BACKGROUND: The coronavirus disease (COVID-19) pandemic has already affected more than 400 million people, with increasing numbers of survivors. These data indicate that a myriad of people may be affected by pulmonary sequelae of the infection. The aim of this study was to evaluate pulmonary sequelae in patients with bilateral COVID-19 pneumonia according to severity 1 year after hospital discharge. METHODS: COVID-FIBROTIC is a multicenter prospective observational cohort study for admitted patients with bilateral COVID-19 pneumonia. Pulmonary functional outcomes and chest computed tomography sequelae were analyzed 12 months after hospital discharge and we classified patients into three groups according to severity. A post hoc analysis model was designed to establish how functional test changed between groups and over time. A multivariable logistic regression model was created to study prognostic factors for lung diffusion impairment and radiological fibrotic-like changes at 12 months. RESULTS: Among 488 hospitalized patients with COVID-19 pneumonia, 284 patients had completed the entire evaluation at 12 months. Median age was 60.5 ± 11.9 and 55.3% were men. We found between-group differences in male sex, length of hospital stay, radiological involvement and inflammatory laboratory parameters. The functional evaluation of pulmonary sequelae showed that severe patients had statistically worse levels of lung diffusion at 2 months but no between group differences were found in subsequent controls. At 12-month follow up, however, we found impaired lung diffusion in 39.8% unrelated to severity. Radiological fibrotic-like changes at 12 months were reported in 22.7% of patients (102/448), only associated with radiological involvement at admission (OR: 1.55, 95% CI 1.06-2.38; p = 0.02) and LDH (OR: 0.99, 95% CI 0.98-0.99; p = 0.046). CONCLUSION: Our data suggest that a significant percentage of individuals would develop pulmonary sequelae after COVID 19 pneumonia, regardless of severity of the acute process. Trial registration clinicaltrials.gov NCT04409275 (June 1, 2020).

Molecular Events behind the Selectivity and Inactivation Properties of Model NaK-Derived Ion Channels.

Y55W mutants of non-selective NaK and partly K+-selective NaK2K channels have been used to explore the conformational dynamics at the pore region of these channels as they interact with either Na+ or K+. A major conclusion is that these channels exhibit a remarkable pore conformational flexibility. Homo-FRET measurements reveal a large change in W55-W55 intersubunit distances, enabling the selectivity filter (SF) to admit different species, thus, favoring poor or no selectivity. Depending on the cation, these channels exhibit wide-open conformations of the SF in Na+, or tight induced-fit conformations in K+, most favored in the four binding sites containing NaK2K channels. Such conformational flexibility seems to arise from an altered pattern of restricting interactions between the SF and the protein scaffold behind it. Additionally, binding experiments provide clues to explain such poor selectivity. Compared to the K+-selective KcsA channel, these channels lack a high affinity K+ binding component and do not collapse in Na+. Thus, they cannot properly select K+ over competing cations, nor reject Na+ by collapsing, as K+-selective channels do. Finally, these channels do not show C-type inactivation, likely because their submillimolar K+ binding affinities prevent an efficient K+ loss from their SF, thus favoring permanently open channel states.

Persistent thrombocytopenia predicts poor long-term survival in patients with antiphospholipid syndrome: a 38-year follow-up study.

OBJECTIVES: To investigate the impact of thrombocytopenia on survival in patients with APS. METHODS: Thrombocytopenia and other predictors of outcome were retrospectively evaluated in an aPL-positive and APS cohort with 38-year follow-up (1980-2018). Thrombocytopenia was defined as <150 × 109 platelets/l. Hazard ratios (HR) of mortality were calculated using Cox-regression models. RESULTS: Among 114 patients, 64% had primary APS, 25% secondary APS and 10% asymptomatic aPL. Mean follow-up was 19 (range 5-38) years. ANA [hazard ratio (HR) 1.8, 95% CI 0.8, 3.6, P = 0.10], arterial thrombotic events (HR 7.0, 95% CI 1.4, 3.5, P = 0.016), myocardial infarction (HR 8.3, 95% CI 1.1, 59, P = 0.03), intracardiac thrombosis (HR 17, 95% CI 1, 279, P = 0.04) and thrombocytopenia (HR 2.9, 95% CI 1.4, 6.1, P = 0.004) were risk factors for all-cause mortality, but in multivariate analysis only thrombocytopenia (HR 2.7, 95% CI 1.3, 6.0, P = 0.01) remained significant. Persistent (HR 4.4, 95% CI 2.1, 9.2, P = 0.001) and low-moderate thrombocytopenia (HR 2.8, 95% CI 1.2, 6.4, P = 0.01) were associated with a significant increase in mortality compared with acute (HR 1.6, 95% CI 0.5, 5.3, P = 0.40) and severe (HR 2.1, 95% CI 0.5, 9.2, P = 0.30) forms. APS patients with vs without thrombocytopenia were more frequently male (58 vs 24%, P = 0.001) with arterial thrombosis (55 vs 32%, P = 0.04), LA positivity (100 vs 87%, P = 0.04), type I aPL profile (89% vs 71%, P = 0.05) and anticoagulant treatment (89 vs 63%, P = 0.01). Thrombosis caused 13% of deaths in thrombocytopenic patients and 1% in those without (P = 0.01). CONCLUSION: Thrombocytopenia is an aPL-related manifestation that identifies patients with severe disease phenotype and high thrombotic risk. Persistent low-moderate thrombocytopenia is associated with a reduced long-term survival.

Lung Function, Radiological Findings and Biomarkers of Fibrogenesis in a Cohort of COVID-19 Patients Six Months After Hospital Discharge.

INTRODUCTION: Impairment in pulmonary function tests and radiological abnormalities are a major concern in COVID-19 survivors. Our aim is to evaluate functional respiratory parameters, changes in chest CT, and correlation with peripheral blood biomarkers involved in lung fibrosis at two and six months after SARS-CoV-2 pneumonia. METHODS: COVID-FIBROTIC (clinicaltrials.gov NCT04409275) is a multicenter prospective observational cohort study aimed to evaluate discharged patients. Pulmonary function tests, circulating serum biomarkers, chest radiography and chest CT were performed at outpatient visits. RESULTS: In total, 313, aged 61.12 ± 12.26 years, out of 481 included patients were available. The proportion of patients with DLCO < 80% was 54.6% and 47% at 60 and 180 days. Associated factors with diffusion impairment at 6 months were female sex (OR: 2.97, 95%CI 1.74-5.06, p = 0.001), age (OR: 1.03, 95% CI: 1.01-1.05, p = 0.005), and peak RALE score (OR: 1.22, 95% CI 1.06-1.40, p = 0.005). Patients with altered lung diffusion showed higher levels of MMP-7 (11.54 ± 8.96 vs 6.71 ± 4.25, p = 0.001), and periostin (1.11 ± 0.07 vs 0.84 ± 0.40, p = 0.001). 226 patients underwent CT scan, of whom 149 (66%) had radiological sequelae of COVID-19. In severe patients, 68.35% had ground glass opacities and 38.46% had parenchymal bands. Early fibrotic changes were associated with higher levels of MMP7 (13.20 ± 9.20 vs 7.92 ± 6.32, p = 0.001), MMP1 (10.40 ± 8.21 vs 6.97 ± 8.89, p = 0.023), and periostin (1.36 ± 0.93 vs 0.87 ± 0.39, p = 0.001). CONCLUSION: Almost half of patients with moderate or severe COVID-19 pneumonia had impaired pulmonary diffusion six months after discharge. Severe patients showed fibrotic lesions in CT scan and elevated serum biomarkers involved in pulmonary fibrosis.

INTRODUCCIÓN: El deterioro de la función pulmonar en las pruebas correspondientes y las alteraciones radiológicas son las preocupaciones principales en los supervivientes de la COVID-19. Nuestro objetivo fue evaluar los parámetros de la función respiratoria, los cambios en la TC de tórax y la correlación con los biomarcadores en sangre periférica involucrados en la fibrosis pulmonar a los 2 y a los 6 meses tras la neumonía por SARS-CoV-2. MÉTODOS: El ensayo COVID-FIBROTIC (clinicaltrials.gov NCT04409275) es un estudio de cohortes multicéntrico, prospectivo y observacional cuyo objetivo fue evaluar los pacientes dados de alta. Se realizaron pruebas de función pulmonar, detección de biomarcadores en plasma circulante y radiografía y TC de tórax durante las visitas ambulatorias. RESULTADOS: En total 313 pacientes, de 61,12 ± 12,26 años, de los 481 incluidos estuvieron disponibles.La proporción de pacientes con DLCO < 80% fue del 54,6 y del 47% a los 60 y 180 días.Los factores que se asociaron a la alteración de la difusión a los 6 meses fueron el sexo femenino (OR: 2,97; IC del 95%: 1,74-5,06; p = 0,001), la edad (OR: 1,03; IC del 95%: 1,01-1,05; p = 0,005) y la puntuación RALE más alta (OR: 1,22; IC del 95%: 1,06-1,40; p = 0,005). Los pacientes con alteración de la difusión pulmonar mostraron niveles más altos de MMP-7 (11,54 ± 8,96 frente a 6,71 ± 4,25; p = 0,001) y periostina (1,11 ± 0.07 frente a 0,84 ± 0,40; p = 0,001). Se le realizó una TC a 226 pacientes de los cuales 149 (66%) presentaban secuelas radiológicas de la COVID-19. En los pacientes graves, el 68,35% mostraban opacidades en vidrio esmerilado y el 38,46%, bandas parenquimatosas. Los cambios fibróticos tempranos se asociaron a niveles más altos de MMP7 (13,20 ± 9,20 frente a 7,92 ± 6,32; p = 0,001), MMP1 (10,40 ± 8,21 frente a 6,97 ± 8,89; p = 0,023), y periostina (1,36 ± 0,93 frente a 0,87 ± 0,39; p = 0,001). CONCLUSIÓN: Casi la mitad de los pacientes con neumonía moderada o grave por COVID-19 presentaba alteración de la difusión pulmonar 6 meses después del alta. Los pacientes graves mostraban lesiones fibróticas en laTC y un aumento de los biomarcadores séricos relacionados con la fibrosis pulmonar.

Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements.

The allosteric coupling between activation and inactivation processes is a common feature observed in K+ channels. Particularly, in the prokaryotic KcsA channel the K+ conduction process is controlled by the inner gate, which is activated by acidic pH, and by the selectivity filter (SF) or outer gate, which can adopt non-conductive or conductive states. In a previous study, a single tryptophan mutant channel (W67 KcsA) enabled us to investigate the SF dynamics using time-resolved homo-Förster Resonance Energy Transfer (homo-FRET) measurements. Here, the conformational changes of both gates were simultaneously monitored after labelling the G116C position with tetramethylrhodamine (TMR) within a W67 KcsA background. At a high degree of protein labeling, fluorescence anisotropy measurements showed that the pH-induced KcsA gating elicited a variation in the homo-FRET efficiency among the conjugated TMR dyes (TMR homo-FRET), while the conformation of the SF was simultaneously tracked (W67 homo-FRET). The dependence of the activation pKa of the inner gate with the ion occupancy of the SF unequivocally confirmed the allosteric communication between the two gates of KcsA. This simple TMR homo-FRET based ratiometric assay can be easily extended to study the conformational dynamics associated with the gating of other ion channels and their modulation.

Peimine, an Anti-Inflammatory Compound from Chinese Herbal Extracts, Modulates Muscle-Type Nicotinic Receptors.

Fritillaria bulbs are used in Traditional Chinese Medicine to treat several illnesses. Peimine (Pm), an anti-inflammatory compound from Fritillaria, is known to inhibit some voltage-dependent ion channels and muscarinic receptors, but its interaction with ligand-gated ion channels remains unexplored. We have studied if Pm affects nicotinic acetylcholine receptors (nAChRs), since they play broad functional roles, both in the nervous system and non-neuronal tissues. Muscle-type nAChRs were incorporated to Xenopus oocytes and the action of Pm on the membrane currents elicited by ACh (IAChs) was assessed. Functional studies were combined with virtual docking and molecular dynamics assays. Co-application of ACh and Pm reversibly blocked IACh, with an IC50 in the low micromolar range. Pm inhibited nAChR by: (i) open-channel blockade, evidenced by the voltage-dependent inhibition of IAch, (ii) enhancement of nAChR desensitization, revealed by both an accelerated IACh decay and a decelerated IACh deactivation, and (iii) resting-nAChR blockade, deduced from the IACh inhibition elicited by Pm when applied before ACh superfusion. In good concordance, virtual docking and molecular dynamics assays demonstrated that Pm binds to different sites at the nAChR, mostly at the transmembrane domain. Thus, Pm from Fritillaria bulbs, considered therapeutic herbs, targets nAChRs with high affinity, which might account for its anti-inflammatory actions.

Neutron dosimetry and shielding verification in commissioning of Compact Proton Therapy Centers (CPTC) using MCNP6.2 Monte Carlo code.

Proton therapy (PT) is an external radiotherapy using proton beams with energies between 70 and 230 MeV to treat some type of tumours with outstanding benefits, due to its energy transfer plot. There is a growing demand of facilities taking up small spaces and Compact Proton Therapy Centers (CPTC), with one or two treatment rooms, supposing the technical response of manufacturers to this request. A large amount of stray radiation is produced in the interaction of protons used in therapy, neutrons mainly, hence, optimal design of shielding and verifications must be carried out in commissioning stages. Currently, almost 50 CPTC are under construction and start up in many countries, including several in Spain. In the present work, the effectiveness of shielding in a CPTC was verified with the Monte Carlo code MCNP6 by calculating the ambient dose equivalent, H*(10) due to secondary neutrons, outside the enclosures and walls of the center. The facility modelled was similar to one planned to start operating in 2019 in Spain, a CPTC, made up of a superconducting synchrocyclotron and one treatment room, with a configuration standard, shielding and width of barriers based on dimensions proposed a priori by the vendor. Therefore, the paper is focused in check the suitability of the materials and thickness of the walls of the center and develop the assessment of enclosures. Several models of the radiation sources and type of concrete in walls were simulated, starting from a conservative assumptions, followed by more realistic models. In all cases, the results were below 1 mSv/year, which is the international legal limit considered for the general public. This work is part of the project Contributions to Shielding and Dosimetry of Neutrons in Compact Proton Therapy Centers (CPTC).

Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsA.

Alkylammonium salts have been used extensively to study the structure and function of potassium channels. Here, we use the hydrophobic tetraoctylammonium (TOA+) to shed light on the structure of the inactivated state of KcsA, a tetrameric prokaryotic potassium channel that serves as a model to its homologous eukaryotic counterparts. By the combined use of a thermal denaturation assay and the analysis of homo-Förster resonance energy transfer in a mutant channel containing a single tryptophan (W67) per subunit, we found that TOA+ binds the channel cavity with high affinity, either with the inner gate open or closed. Moreover, TOA+ bound at the cavity allosterically shifts the equilibrium of the channel's selectivity filter conformation from conductive to an inactivated-like form. The inactivated TOA+-KcsA complex exhibits a loss in the affinity towards permeant K+ at pH 7.0, when the channel is in its closed state, but maintains the two sets of K+ binding sites and the W67-W67 intersubunit distances characteristic of the selectivity filter in the channel resting state. Thus, the TOA+-bound state differs clearly from the collapsed channel state described by X-ray crystallography and claimed to represent the inactivated form of KcsA.

On the Better Performance of Pianists with Motor Imagery-Based Brain-Computer Interface Systems.

Motor imagery (MI)-based brain-computer interface (BCI) systems detect electrical brain activity patterns through electroencephalogram (EEG) signals to forecast user intention while performing movement imagination tasks. As the microscopic details of individuals' brains are directly shaped by their rich experiences, musicians can develop certain neurological characteristics, such as improved brain plasticity, following extensive musical training. Specifically, the advanced bimanual motor coordination that pianists exhibit means that they may interact more effectively with BCI systems than their non-musically trained counterparts; this could lead to personalized BCI strategies according to the users' previously detected skills. This work assessed the performance of pianists as they interacted with an MI-based BCI system and compared it with that of a control group. The Common Spatial Patterns (CSP) and Linear Discriminant Analysis (LDA) machine learning algorithms were applied to the EEG signals for feature extraction and classification, respectively. The results revealed that the pianists achieved a higher level of BCI control by means of MI during the final trial (74.69%) compared to the control group (63.13%). The outcome indicates that musical training could enhance the performance of individuals using BCI systems.

Modulation of Function, Structure and Clustering of K+ Channels by Lipids: Lessons Learnt from KcsA.

KcsA, a prokaryote tetrameric potassium channel, was the first ion channel ever to be structurally solved at high resolution. This, along with the ease of its expression and purification, made KcsA an experimental system of choice to study structure-function relationships in ion channels. In fact, much of our current understanding on how the different channel families operate arises from earlier KcsA information. Being an integral membrane protein, KcsA is also an excellent model to study how lipid-protein and protein-protein interactions within membranes, modulate its activity and structure. In regard to the later, a variety of equilibrium and non-equilibrium methods have been used in a truly multidisciplinary effort to study the effects of lipids on the KcsA channel. Remarkably, both experimental and "in silico" data point to the relevance of specific lipid binding to two key arginine residues. These residues are at non-annular lipid binding sites on the protein and act as a common element to trigger many of the lipid effects on this channel. Thus, processes as different as the inactivation of channel currents or the assembly of clusters from individual KcsA channels, depend upon such lipid binding.

Mechanisms of Blockade of the Muscle-Type Nicotinic Receptor by Benzocaine, a Permanently Uncharged Local Anesthetic.

Most local anesthetics (LAs) are amine compounds bearing one or several phenolic rings. Many of them are protonated at physiological pH, but benzocaine (Bzc) is permanently uncharged, which is relevant because the effects of LAs on nicotinic acetylcholine (ACh) receptors (nAChRs) depend on their presence as uncharged or protonated species. The aims of this study were to assess the effects of Bzc on nAChRs and to correlate them with its binding to putative interacting sites on this receptor. nAChRs from Torpedo electroplaques were microtransplanted to Xenopus oocytes and currents elicited by ACh (IAChs), either alone or together with Bzc, were recorded at different potentials. Co-application of ACh with increasing concentrations of Bzc showed that Bzc reversibly blocked nAChRs. IACh inhibition by Bzc was voltage-independent, but the IACh rebound elicited when rinsing Bzc suggests an open-channel blockade. Besides, ACh and Bzc co-application enhanced nAChR desensitization. When Bzc was just pre-applied it also inhibited IACh, by blocking closed (resting) nAChRs. This blockade slowed down the kinetics of both the IACh activation and the recovery from blockade. The electrophysiological results indicate that Bzc effects on nAChRs are similar to those of 2,6-dimethylaniline, an analogue of the hydrophobic moiety of lidocaine. Furthermore, docking assays on models of the nAChR revealed that Bzc and DMA binding sites on nAChRs overlap fairly well. These results demonstrate that Bzc inhibits nAChRs by multiple mechanisms and contribute to better understanding both the modulation of nAChRs and how LAs elicit some of their clinical side effects. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Modulation of the potassium channel KcsA by anionic phospholipids: Role of arginines at the non-annular lipid binding sites.

The role of arginines R64 and R89 at non-annular lipid binding sites of KcsA, on the modulation of channel activity by anionic lipids has been investigated. In wild-type (WT) KcsA reconstituted into asolectin lipid membranes, addition of phosphatidic acid (PA) drastically reduces inactivation in macroscopic current recordings. Consistent to this, PA increases current amplitude, mean open time and open probability at the single channel level. Moreover, kinetic analysis reveals that addition of PA causes longer open channel lifetimes and decreased closing rate constants. Effects akin to those of PA on WT-KcsA are observed when R64 and/or R89 are mutated to alanine, regardless of the added anionic lipids. We interpret these results as a consequence of interactions between the arginines and the anionic PA bound to the non-annular sites. NMR data shows indeed that at least R64 is involved in binding PA. Moreover, molecular dynamics (MD) simulations predict that R64, R89 and surrounding residues such as T61, mediate persistent binding of PA to the non-annular sites. Channel inactivation depends on interactions within the inactivation triad (E71-D80-W67) behind the selectivity filter. Therefore, it is expected that such interactions are affected when PA binds the arginines at the non-annular sites. In support of this, MD simulations reveal that PA binding prevents interaction between R89 and D80, which seems critical to the effectiveness of the inactivation triad. This mechanism depends on the stability of the bound lipid, favoring anionic headgroups such as that of PA, which thrive on the positive charge of the arginines.

Conformational plasticity in the KcsA potassium channel pore helix revealed by homo-FRET studies.

Potassium channels selectivity filter (SF) conformation is modulated by several factors, including ion-protein and protein-protein interactions. Here, we investigate the SF dynamics of a single Trp mutant of the potassium channel KcsA (W67) using polarized time-resolved fluorescence measurements. For the first time, an analytical framework is reported to analyze the homo-Förster resonance energy transfer (homo-FRET) within a symmetric tetrameric protein with a square geometry. We found that in the closed state (pH 7), the W67-W67 intersubunit distances become shorter as the average ion occupancy of the SF increases according to cation type and concentration. The hypothesis that the inactivated SF at pH 4 is structurally similar to its collapsed state, detected at low K+, pH 7, was ruled out, emphasizing the critical role played by the S2 binding site in the inactivation process of KcsA. This homo-FRET approach provides complementary information to X-ray crystallography in which the protein conformational dynamics is usually compromised.

Accessibility of Cations to the Selectivity Filter of KcsA in the Inactivated State: An Equilibrium Binding Study.

Cation binding under equilibrium conditions has been used as a tool to explore the accessibility of permeant and nonpermeant cations to the selectivity filter in three different inactivated models of the potassium channel KcsA. The results show that the stack of ion binding sites (S1 to S4) in the inactivated filter models remain accessible to cations as they are in the resting channel state. The inactivated state of the selectivity filter is therefore "resting-like" under such equilibrium conditions. Nonetheless, quantitative differences in the apparent KD's of the binding processes reveal that the affinity for the binding of permeant cations to the inactivated channel models, mainly K⁺, decreases considerably with respect to the resting channel. This is likely to cause a loss of K⁺ from the inactivated filter and consequently, to promote nonconductive conformations. The most affected site by the affinity loss seems to be S4, which is interesting because S4 is the first site to accommodate K⁺ coming from the channel vestibule when K⁺ exits the cell. Moreover, binding of the nonpermeant species, Na⁺, is not substantially affected by inactivation, meaning that the inactivated channels are also less selective for permeant versus nonpermeant cations under equilibrium conditions.